glucose-6-phosphatase (nmol/min/ mg protein);

## tyrosine aminotranferase (nmol/min/mg protein); \$ µg/g tissue

Table 3. Biochemical effects of acephate (140mg/kg b.w.) in rats


Data analyzed by ANOVA followed by Tukey Test (n=6)

\* µg/dl; \*\* mg/g tissue ; # tyrosine aminotranferase (nmol/min/mg protein); ## µg/g tissue

#### Table 4. Biochemical effects of monocrotophos (1.8mg/kg b.w.) in rats

Indeed, role of adrenals in glucotoxicity of OPI has been explored earlier. Matin et al., (1989) earlier demonstrated that single dose diazinon (OPI) caused hyperglycemia and induction of liver gluconeogenesis enzymes in normal rats while these changes did not manifest in adrenalectomized rats, indicating the involvement of adrenals in the glucotoxicity of diazinon. Our attempts to study the adrenal and glycemic effects of acephate and monocrotophos revealed that two compounds, which exhibit anticholinesterase property, elicited similar effects. Thus, the effects raised question whether the adrenal and glycemic effects are mediated through the anticholinesterase property of OPI. To address the question, we studied the extent of AChE inhibition elicited by monocrotophos at 2 and 4h

Organophosphorus Insecticides and Glucose Homeostasis 73

Fig. 5. Protective effects of atropine (ATR) and methyl atropine (MATR) against stressogenic

Acetylcholine exerts strong influence on functioning of hypothalamus-pituitary-adrenal (HPA) axis. Acetylcholine has been found to increase corticotrophin releasing hormone (CRH) activity of hypothalamus *in vitro* as measured by effect on corticosteroidogenesis, an effect that was antagonized by atropine (Bradbury et al., 1974). ACh has also been shown to increase secretion of immunoreactive CRH from hypothalamus *in vitro* (Calogero et al., 1988), an effect that was antagonized by ACh receptor antagonists, atropine (muscarinic) and hexamethonium (nicotinic). Given the importance of ACh in excitation of HPA axis, assessment of cholinergic stress in activation of HPA axis in monocrotophos treated rats becomes important. The importance of ACh in functioning of HPA axis is further exemplified by the fact that muscarinic receptor agonists such as carbachol (Bugajski et al., 2002) and arecoline (Calogero et al., 1989) were found to increase ACTH and corticosterone *in vivo*. More importantly, the agonist induced increase in ACTH and corticosterone was antagonized by atropine (Bugajski et al., 2002), suggesting role for muscarinic ACh receptor in regulation of HPA axis. Role of anticholinesterase properties of organophosphate compounds in activation of HPA axis is demonstrated by studies showing elimination of stressogenic activity of cyclohexyl methyl phosphonofluoridate (as measured by plasma corticosterone and liver tyrosine aminotransferase activity) by HI-6, a cholinesterase reactivator that sufficiently reactivated inhibited AChE in brain and diaphragm (Kassa & Bajgar, 1995) and protection offered by atropine against diisopropylfluorophosphate induced increase in corticosterone levels (Smallridge et al., 1991). These studies clearly show the influence of ACh and involvement of muscarinic receptors in functioning of HPA axis. From our data on influence of cholinergic antagonists on stressogenic and hyperglycemic potential of monocrotophos, it could be hypothesized that muscarinic cholinergic stress

and hyperglycemic potential of monocrotophos (Mono) *(*Joshi and Rajini, 2010*).*

after administration. Influence of cholinergic antagonists was investigated at 2h after administration on stressogenic (hypercorticosteronemia and induction of liver TAT activity) and hyperglycemic potential of monocrotophos. For the purpose of mechanistic investigations, we employed two muscarinic cholinergic antagonists- atropine sulphate, a general ACh receptor antagonist that can pass through blood brain barrier (BBB) (Guarini et al., 2004) and methyl atropine nitrate, which is a peripherally active antagonist that does not pass through blood BBB (Pavlov et al., 2006). Both antagonists were administered at 30mols/kg b.w 3-5 min before monocrotophos (1/10 LD50).

Monocrotophos elicited signicant inhibition of AChE activity (>50%) in brain, adrenals and liver at both 2 and 4 h after exposure (**Fig. 5A**). Of the organs studied, maximum inhibition of AChE activity was evident in brain (84 and 78% at 2 and 4 h respectively) while the enzyme activity in adrenals was inhibited to 32 and 34% of control activity at 2 and 4 h after exposure respectively. Similarly, monocrotophos administration reduced liver AChE activities to 47 and 46% of control at 2 and 4 h after exposure respectively. More importantly, we did not observe any spontaneous reactivation of inhibited AChE activity at 4h after administration, which is an important feature of the enzymes' behavior (Reiner and Aldridge, 1967; Reiner, 1971). This indicates that, while hyperglycemic potential of monocrotophos in rats may be a result of its anticholinesterase potency, the reversibility of hyperglycemia is not a consequence of spontaneous reactivation of the enzyme. Reversibility of hyperglycemia may hence be a consequence of counter-regulatory mechanism as reflected by glycogen deposition at 4h after administration. Increase in glycogen content upon 4h exposure is a clear indication of mobilization of glucose into glycogen synthesis pathway as a measure to overcome hyperglycemia.

We also observed that both cholinergic antagonists were potent in offering protection against stressogenic and hyperglycemic potential of monocrotophos. Administration of monocrotophos elicited significant hyperglycemia (103%) (**Fig. 5B)**. Pre- treatment of rats with atropine sulfate (106.04 ±1.83 compared to 191.82 ±7.59 mg/dl of monocrotophos alone) and atropine methyl nitrate (123.49 ±4.12 compared to 191.82 ±7.59 mg/dl of monocrotophos alone) offered signicant protection against hyperglycemia induced by monocrotophos. It has been earlier demonstrated that diazinon-induced hyperglycemia was mediated by AChE inhibition, as revealed by protective effects of pralidoxime (AChE reactivator) (Seifert, 2001). Monocrotophos-induced hypercorticosteronemia (112%) was effectively prevented by cholinergic antagonists (**Fig. 5C)**. Pre-treatment of rats with atropine sulfate (33.98 ±2.89 compared to 76.63 ±1.76 µg/dl of monocrotophos alone) and atropine methyl nitrate (44.67 ±1.64 compared to 76.63 ±1.76 µg/dl of monocrotophos alone) offered signicant protection against hypercorticosteronemia induced by monocrotophos. Monocrotophos induced a marked increase in the TAT activity in liver (107%) (**Fig. 5D**). Pre-treatment of rats with atropine sulfate (20.42 ±1.70 compared to 33.38 ±1.09 nmol/min/mg protein) and atropine methyl nitrate (22.39 ±0.79 compared to 33.38 ±1.09 nmol/min/mg protein) offered signicant protection against induction of TAT activity. These results clearly indicated that both physiological stress (hypercorticosteronemia and induction of liver TAT activity) and hyperglycemia manifest as a consequence of peripheral muscarinic cholinergic stimulation. Corticosterone exerts hyperglycemic action by up-regulation of gluconeogenesis machinery. Hence, hypercorticosteronemia and induction of liver TAT (gluconeogenesis enzyme) activity accompanying hyperglycemia raises a question whether hypercorticosteronemia is responsible hyperglycemia in monocrotophos-treated rats.

after administration. Influence of cholinergic antagonists was investigated at 2h after administration on stressogenic (hypercorticosteronemia and induction of liver TAT activity) and hyperglycemic potential of monocrotophos. For the purpose of mechanistic investigations, we employed two muscarinic cholinergic antagonists- atropine sulphate, a general ACh receptor antagonist that can pass through blood brain barrier (BBB) (Guarini et al., 2004) and methyl atropine nitrate, which is a peripherally active antagonist that does not pass through blood BBB (Pavlov et al., 2006). Both antagonists were administered at

Monocrotophos elicited signicant inhibition of AChE activity (>50%) in brain, adrenals and liver at both 2 and 4 h after exposure (**Fig. 5A**). Of the organs studied, maximum inhibition of AChE activity was evident in brain (84 and 78% at 2 and 4 h respectively) while the enzyme activity in adrenals was inhibited to 32 and 34% of control activity at 2 and 4 h after exposure respectively. Similarly, monocrotophos administration reduced liver AChE activities to 47 and 46% of control at 2 and 4 h after exposure respectively. More importantly, we did not observe any spontaneous reactivation of inhibited AChE activity at 4h after administration, which is an important feature of the enzymes' behavior (Reiner and Aldridge, 1967; Reiner, 1971). This indicates that, while hyperglycemic potential of monocrotophos in rats may be a result of its anticholinesterase potency, the reversibility of hyperglycemia is not a consequence of spontaneous reactivation of the enzyme. Reversibility of hyperglycemia may hence be a consequence of counter-regulatory mechanism as reflected by glycogen deposition at 4h after administration. Increase in glycogen content upon 4h exposure is a clear indication of mobilization of glucose into

We also observed that both cholinergic antagonists were potent in offering protection against stressogenic and hyperglycemic potential of monocrotophos. Administration of monocrotophos elicited significant hyperglycemia (103%) (**Fig. 5B)**. Pre- treatment of rats with atropine sulfate (106.04 ±1.83 compared to 191.82 ±7.59 mg/dl of monocrotophos alone) and atropine methyl nitrate (123.49 ±4.12 compared to 191.82 ±7.59 mg/dl of monocrotophos alone) offered signicant protection against hyperglycemia induced by monocrotophos. It has been earlier demonstrated that diazinon-induced hyperglycemia was mediated by AChE inhibition, as revealed by protective effects of pralidoxime (AChE reactivator) (Seifert, 2001). Monocrotophos-induced hypercorticosteronemia (112%) was effectively prevented by cholinergic antagonists (**Fig. 5C)**. Pre-treatment of rats with atropine sulfate (33.98 ±2.89 compared to 76.63 ±1.76 µg/dl of monocrotophos alone) and atropine methyl nitrate (44.67 ±1.64 compared to 76.63 ±1.76 µg/dl of monocrotophos alone) offered signicant protection against hypercorticosteronemia induced by monocrotophos. Monocrotophos induced a marked increase in the TAT activity in liver (107%) (**Fig. 5D**). Pre-treatment of rats with atropine sulfate (20.42 ±1.70 compared to 33.38 ±1.09 nmol/min/mg protein) and atropine methyl nitrate (22.39 ±0.79 compared to 33.38 ±1.09 nmol/min/mg protein) offered signicant protection against induction of TAT activity. These results clearly indicated that both physiological stress (hypercorticosteronemia and induction of liver TAT activity) and hyperglycemia manifest as a consequence of peripheral muscarinic cholinergic stimulation. Corticosterone exerts hyperglycemic action by up-regulation of gluconeogenesis machinery. Hence, hypercorticosteronemia and induction of liver TAT (gluconeogenesis enzyme) activity accompanying hyperglycemia raises a question whether hypercorticosteronemia is

30mols/kg b.w 3-5 min before monocrotophos (1/10 LD50).

glycogen synthesis pathway as a measure to overcome hyperglycemia.

responsible hyperglycemia in monocrotophos-treated rats.

Fig. 5. Protective effects of atropine (ATR) and methyl atropine (MATR) against stressogenic and hyperglycemic potential of monocrotophos (Mono) *(*Joshi and Rajini, 2010*).*

Acetylcholine exerts strong influence on functioning of hypothalamus-pituitary-adrenal (HPA) axis. Acetylcholine has been found to increase corticotrophin releasing hormone (CRH) activity of hypothalamus *in vitro* as measured by effect on corticosteroidogenesis, an effect that was antagonized by atropine (Bradbury et al., 1974). ACh has also been shown to increase secretion of immunoreactive CRH from hypothalamus *in vitro* (Calogero et al., 1988), an effect that was antagonized by ACh receptor antagonists, atropine (muscarinic) and hexamethonium (nicotinic). Given the importance of ACh in excitation of HPA axis, assessment of cholinergic stress in activation of HPA axis in monocrotophos treated rats becomes important. The importance of ACh in functioning of HPA axis is further exemplified by the fact that muscarinic receptor agonists such as carbachol (Bugajski et al., 2002) and arecoline (Calogero et al., 1989) were found to increase ACTH and corticosterone *in vivo*. More importantly, the agonist induced increase in ACTH and corticosterone was antagonized by atropine (Bugajski et al., 2002), suggesting role for muscarinic ACh receptor in regulation of HPA axis. Role of anticholinesterase properties of organophosphate compounds in activation of HPA axis is demonstrated by studies showing elimination of stressogenic activity of cyclohexyl methyl phosphonofluoridate (as measured by plasma corticosterone and liver tyrosine aminotransferase activity) by HI-6, a cholinesterase reactivator that sufficiently reactivated inhibited AChE in brain and diaphragm (Kassa & Bajgar, 1995) and protection offered by atropine against diisopropylfluorophosphate induced increase in corticosterone levels (Smallridge et al., 1991). These studies clearly show the influence of ACh and involvement of muscarinic receptors in functioning of HPA axis. From our data on influence of cholinergic antagonists on stressogenic and hyperglycemic potential of monocrotophos, it could be hypothesized that muscarinic cholinergic stress

Organophosphorus Insecticides and Glucose Homeostasis 75

multiple administrations is a well documented phenomenon (Brodeur and DuBois, 1964; McPhillips, 1969; Sterri et al., 1980). Tolerance to the elevation of plasma corticosterone by DFP was reported to develop during repeated administration (Kokka et al., 1985). Several studies suggest that cholinergic receptors could be involved in onset of tolerance to OPI, which may be mediated by events such as down regulation of muscarinic receptors (Costa et al., 1982a&b). Tolerance to the toxic effects of dilsulfoton during multiple exposures has been attributed to reduced muscarinic receptor binding in tissues of rats tolerant to the insecticide (Schwab et al., 1981). Blunted responses observed by us in case of repeated administration of monocrotophos may be attributed to tolerance mechanisms such as down regulation of muscarinic receptors. One mechanism that may be responsible for development of resistance is increased blood insulin levels. Comparison of effects of acute and repeated doses of monocrotophos on plasma insulin levels, however, needs to be done. Such a hyperinsulinemic response has been reported in case of exposure to malathion. While malathion caused increase in blood glucose and insulin levels after single exposure and continued dietary administration for 4 weeks, the degree of hyperinsulinemia was markedly greater in dietary group (Panahi et al., 2006). Thus, repeated administration of organophosphorus insecticides leads to blunting of responses. Although blunted, these responses still represent a great threat to euglycemic balance. This is particularly true in the case of constant state of hypercorticosteronemia. This has propensity to affect skeletal muscle glucose metabolism and long-term impairments in such mechanisms may lead to

Based on our comprehensive studies described above, we have proposed a scheme on the mechanism/s through which OPI might regulate/ disrupt glucose homeostasis (**Fig. 7**). Oxidative stress in pancreatic milieu and glucose intolerance, up regulated gluconeogenesis machinery and hyperglycemia are critical factors in diabetes etiology. With the ability to induce the above-mentioned dysregulations, OPI may have far reaching consequences on diabetic outcomes. This may be a more pertinent issue in the present times since diabetes is fast emerging as a major wide spread disorder that threatens human life. With this realization, our laboratory has also committed to investigate if OPI act as

We observed that dichlorvos (DDVP) treated rats showed higher (22%) levels of blood glucose compared to normal control rats while as expected, rats injected with the diabetogenic agent, Streptozotocin (STZ) alone also showed elevated (37%) level of blood glucose. However, blood glucose levels of DDVP pre-treated rats administered STZ showed relatively higher blood glucose level compared to all the groups. Liver glycogen levels were significantly lower in rats administered either DDVP (18%) or STZ (19%) alone while, rats administered DDVP followed by STZ revealed further lower levels of glycogen (46 %) **(Table 5)**. Further, we also observed that DDVP pre-treatment resulted in more sever oxidative stress in STZ treated rats. ROS levels were significantly elevated in STZ (40%) and DDVP (55%) groups compared to 'untreated control' group. However, ROS levels were markedly higher (81.23 ± 6.52 pmole DCF/min/mg protein) in 'DDVP+STZ' group of rats. Pancreas of rats administered with either DDVP or STZ alone showed marginally higher levels of the lipid peroxides compared to that in 'untreated controls' while, the levels of lipid peroxides generated in pancreas of 'DDVP+STZ' rats showed

predisposing/aggravating factors for onset or progression of diabetic condition.

long lasting dysregulation in glucose homeostasis.

**3.3 OPI act as pre-disposing factors for onset of diabetes?** 

triggers hypercorticosteronemia, which may lead to induction of liver gluconeogenesis and hyperglycemia. However, experiments conducted with glucocorticoid receptor and adrenergic receptor antagonists revealed that hyperglycemia in mediated by adrenergic mechanisms while hypercorticosteronemia leads to only induction of liver TAT activity (data not shown). Further, we observed that monocrotophos-induced hyperglycemia was completely abolished by a gluconeogenesis inhibitor (data not shown). This establishes that physiological stress and hyperglycemia manifest in monocrotophos treated rats as independent consequence of peripheral cholinergic stress.

We further compared the effects of monocrotophos on adrenal functions and glycemic control in rats following single and repeated doses. Comparison was made between the effects of a single dose (measured 2h after administration) and that of 5 or 10 doses (one dose per day, measured 2h after last dose). In both cases, the oral dose of 1.8mg/kg b.w. was employed for the purpose of comparison. Interestingly, we observed that effects single dose of monocrotophos on adrenal functions and glycemic control was more severe than that of repeated doses. Single dose of monocrotophos elicited hypercorticosteronemia (114%) with concomitant decrease in adrenal cholesterol (33%). These adrenal effects of single dose were accompanied with hyperglycemia (109%) and induction of liver tyrosine aminotransferase activity (113%). However, repeated administration of monocrotophos for 5 or 10days resulted in blunting of responses. In case of repeated exposure, increase in corticosterone was 76 and 67% respectively in 5 and 10d exposure groups with 18 and 13% decrease in adrenal cholesterol. Similarly repeated administration elicited marginal increase in blood glucose (39 and 32%) and induction of liver TAT activity (56 and 61%) (**Fig. 6**).

Fig. 6. Adrenal and glycemic effects of monocrotophos.

The above data clearly shows that repeated administration results in blunting of responses. This indicates that multiple administrations are associated with onset of some sort of resistance to the action of OPI. Development of tolerance to cholinesterase inhibitors during

triggers hypercorticosteronemia, which may lead to induction of liver gluconeogenesis and hyperglycemia. However, experiments conducted with glucocorticoid receptor and adrenergic receptor antagonists revealed that hyperglycemia in mediated by adrenergic mechanisms while hypercorticosteronemia leads to only induction of liver TAT activity (data not shown). Further, we observed that monocrotophos-induced hyperglycemia was completely abolished by a gluconeogenesis inhibitor (data not shown). This establishes that physiological stress and hyperglycemia manifest in monocrotophos treated rats as

We further compared the effects of monocrotophos on adrenal functions and glycemic control in rats following single and repeated doses. Comparison was made between the effects of a single dose (measured 2h after administration) and that of 5 or 10 doses (one dose per day, measured 2h after last dose). In both cases, the oral dose of 1.8mg/kg b.w. was employed for the purpose of comparison. Interestingly, we observed that effects single dose of monocrotophos on adrenal functions and glycemic control was more severe than that of repeated doses. Single dose of monocrotophos elicited hypercorticosteronemia (114%) with concomitant decrease in adrenal cholesterol (33%). These adrenal effects of single dose were accompanied with hyperglycemia (109%) and induction of liver tyrosine aminotransferase activity (113%). However, repeated administration of monocrotophos for 5 or 10days resulted in blunting of responses. In case of repeated exposure, increase in corticosterone was 76 and 67% respectively in 5 and 10d exposure groups with 18 and 13% decrease in adrenal cholesterol. Similarly repeated administration elicited marginal increase in blood

glucose (39 and 32%) and induction of liver TAT activity (56 and 61%) (**Fig. 6**).

independent consequence of peripheral cholinergic stress.

Fig. 6. Adrenal and glycemic effects of monocrotophos.

The above data clearly shows that repeated administration results in blunting of responses. This indicates that multiple administrations are associated with onset of some sort of resistance to the action of OPI. Development of tolerance to cholinesterase inhibitors during multiple administrations is a well documented phenomenon (Brodeur and DuBois, 1964; McPhillips, 1969; Sterri et al., 1980). Tolerance to the elevation of plasma corticosterone by DFP was reported to develop during repeated administration (Kokka et al., 1985). Several studies suggest that cholinergic receptors could be involved in onset of tolerance to OPI, which may be mediated by events such as down regulation of muscarinic receptors (Costa et al., 1982a&b). Tolerance to the toxic effects of dilsulfoton during multiple exposures has been attributed to reduced muscarinic receptor binding in tissues of rats tolerant to the insecticide (Schwab et al., 1981). Blunted responses observed by us in case of repeated administration of monocrotophos may be attributed to tolerance mechanisms such as down regulation of muscarinic receptors. One mechanism that may be responsible for development of resistance is increased blood insulin levels. Comparison of effects of acute and repeated doses of monocrotophos on plasma insulin levels, however, needs to be done. Such a hyperinsulinemic response has been reported in case of exposure to malathion. While malathion caused increase in blood glucose and insulin levels after single exposure and continued dietary administration for 4 weeks, the degree of hyperinsulinemia was markedly greater in dietary group (Panahi et al., 2006). Thus, repeated administration of organophosphorus insecticides leads to blunting of responses. Although blunted, these responses still represent a great threat to euglycemic balance. This is particularly true in the case of constant state of hypercorticosteronemia. This has propensity to affect skeletal muscle glucose metabolism and long-term impairments in such mechanisms may lead to long lasting dysregulation in glucose homeostasis.

#### **3.3 OPI act as pre-disposing factors for onset of diabetes?**

Based on our comprehensive studies described above, we have proposed a scheme on the mechanism/s through which OPI might regulate/ disrupt glucose homeostasis (**Fig. 7**). Oxidative stress in pancreatic milieu and glucose intolerance, up regulated gluconeogenesis machinery and hyperglycemia are critical factors in diabetes etiology. With the ability to induce the above-mentioned dysregulations, OPI may have far reaching consequences on diabetic outcomes. This may be a more pertinent issue in the present times since diabetes is fast emerging as a major wide spread disorder that threatens human life. With this realization, our laboratory has also committed to investigate if OPI act as predisposing/aggravating factors for onset or progression of diabetic condition.

We observed that dichlorvos (DDVP) treated rats showed higher (22%) levels of blood glucose compared to normal control rats while as expected, rats injected with the diabetogenic agent, Streptozotocin (STZ) alone also showed elevated (37%) level of blood glucose. However, blood glucose levels of DDVP pre-treated rats administered STZ showed relatively higher blood glucose level compared to all the groups. Liver glycogen levels were significantly lower in rats administered either DDVP (18%) or STZ (19%) alone while, rats administered DDVP followed by STZ revealed further lower levels of glycogen (46 %) **(Table 5)**. Further, we also observed that DDVP pre-treatment resulted in more sever oxidative stress in STZ treated rats. ROS levels were significantly elevated in STZ (40%) and DDVP (55%) groups compared to 'untreated control' group. However, ROS levels were markedly higher (81.23 ± 6.52 pmole DCF/min/mg protein) in 'DDVP+STZ' group of rats. Pancreas of rats administered with either DDVP or STZ alone showed marginally higher levels of the lipid peroxides compared to that in 'untreated controls' while, the levels of lipid peroxides generated in pancreas of 'DDVP+STZ' rats showed

Organophosphorus Insecticides and Glucose Homeostasis 77

1mg/dl; 2mg/g tissue; Values are mean SEM (n=6); Mean in the same column with different

Table 5. Blood glucose and liver glycogen levels in rats treated with DDVP ± STZ (i.p , 25

Values are mean SEM (n=6); Mean in the same column with different superscript differ significantly

Table 6. Oxidative stress parameters in pancreas of rats treated with DDVP ± STZ (i.p , 25

To address this question, we investigated the diabetic outcomes in rats experimentally rendered diabetic and post-treated with monocrotophos. Rats were rendered diabetic with an acute dose (60 mg/kg b.w, i.p) of streptozotocin. Monocrotophos was orally administered at a sublethal dose (1/20 LD50, 0.9 mg /kg b.w./d, 5 days) to both normal and diabetic rats. We observed that monocrotophos *per se* moderately increased (25%) the blood glucose levels in normal rats, but significantly aggravated the hyperglycemic outcome in diabetic rats (56% above diabetic rats). We observed the typical lipid profile alterations among diabetic rats characterized by increase in total cholesterol and triglycerides (TG) in serum. While monocrotophos did not impact lipid profile *per se*, its interaction with diabetic component resulted in severe alterations lipid profile that reflected in phenomenal increase in serum triglyceride. Such augmented impairments may have high bearing on cardiovascular health since the cardiovascular risk index was alarmingly high among diabetic rats treated with monocrotophos (**Fig. 8**). Further, monocrotophos resulted in higher degree of hepatic and renal toxicity as reflected by alterations in serum transaminase

Further, it is now established that oxidative stress in an important consequence of diabetic condition and plays as an important pathophysiological factor in progression of diabetic complications (Maritim, 2003). We observed that STZ-induced diabetes was associated with increased lipid peroxidation (122%), depletion of reduced glutathione (10%) and alterations in activities of two important antioxidant enzymes (superoxide dismutase and catalase) in kidney. Monocrotophos further deteriorated oxidative impairments in kidney as evidenced by further increase in lipid peroxidation (170%) and depletion of reduced glutathione (18%) content. Diabetes was associated with marginal decrease in superoxide dismutase activity in

kidney, which was further reduced by monocrotophos treatment (35%) (**Fig. 9**).

superscript differ significantly (*p<0.05*)

1pmol DCF/min/mg protein; 2nmol/g tissue; 3mg/g tissue

activities and blood urea nitrogen values respectively (**Table 7)**.

**3.4 Do OPI aggravate diabetic outcomes?** 

mg/kg b.w).

(*p<0.05*)

mg/kg b.w).

Group Blood glucose1 Liver glycogen2 CONTROL 113.53a ± 2.31 41.55c ± 2.01 DDVP 138.37b ± 4.17 34.20b ± 1.42 STZ 155.03c ± 5.09 33.34b ± 2.23 DDVP+STZ 188.99d ± 4.44 22.62a ± 3.52

Group ROS1 TBARS2 GSH3 Untreated control 38.76a ± 4.04 242.76a ± 19.18 1.07c ± 0.03 DDVP 60.18b ± 4.59 294.94a ± 10.65 0.83b ± 0.01 STZ 54.27b ± 2.89 283.63a ± 7.27 0.78b ± 0.02 DDVP+STZ 81.23c ± 6.52 389.38b ± 38.47 0.67a ± 0.02

significant increase (110%) compared to all other groups. Pancreatic reduced glutathione level in 'DDVP+STZ' rats was significantly lower (37%) while, rats administered with DDVP or STZ alone also had significantly lower levels of GSH, although to a lesser extent (**Table 6**). These results clearly demonstrate that OPI act as pre-disposing factor for diabetes as reflected by higher degree of glucotoxicity of STZ (subdiabetogenic dose) in DDVP treated rats.

Fig. 7. Proposed scheme for OPI-induced alterations in glucose homeostasis.

Generally, an acute intraperitoneal dose of 40–60 mg/kg b.w is employed to induce significant hyperglycemia in rats. For the present study, we employed a lower dose of 25 mg/kg b.w ('sub-diabetogenic dose') in order to examine if pre-treatment with DDVP renders these rats more susceptible to hyperglycemia. Experimental regime began with two groups with 12 rats each-control and DDVP-treated group. The DDVP-treated group animals were orally administered daily DDVP at 20mg/kg b.w/d (corresponding to 1/5 of LD50 value: 100 mg/kg b.w, determined in a preliminary study) for 10 d. After 10 days, rats of the control group were further divided into two sub groups of six animals each ; the first sub group served as control ('untreated control'), while the second sub group of rats was intraperitoneally injected streptozotocin (STZ, 25 mg/kg b.w.) ('STZ'). The group of rats administered with DDVP was also divided into two sub groups; the first sub group of rats served as DDVP control ('DDVP'), while the second sub group of rats was injected with streptozotocin (i.p, 25mg/kg b.w.) ('DDVP+STZ').

significant increase (110%) compared to all other groups. Pancreatic reduced glutathione level in 'DDVP+STZ' rats was significantly lower (37%) while, rats administered with DDVP or STZ alone also had significantly lower levels of GSH, although to a lesser extent (**Table 6**). These results clearly demonstrate that OPI act as pre-disposing factor for diabetes as reflected by higher degree of glucotoxicity of STZ (subdiabetogenic dose) in

Fig. 7. Proposed scheme for OPI-induced alterations in glucose homeostasis.

streptozotocin (i.p, 25mg/kg b.w.) ('DDVP+STZ').

Generally, an acute intraperitoneal dose of 40–60 mg/kg b.w is employed to induce significant hyperglycemia in rats. For the present study, we employed a lower dose of 25 mg/kg b.w ('sub-diabetogenic dose') in order to examine if pre-treatment with DDVP renders these rats more susceptible to hyperglycemia. Experimental regime began with two groups with 12 rats each-control and DDVP-treated group. The DDVP-treated group animals were orally administered daily DDVP at 20mg/kg b.w/d (corresponding to 1/5 of LD50 value: 100 mg/kg b.w, determined in a preliminary study) for 10 d. After 10 days, rats of the control group were further divided into two sub groups of six animals each ; the first sub group served as control ('untreated control'), while the second sub group of rats was intraperitoneally injected streptozotocin (STZ, 25 mg/kg b.w.) ('STZ'). The group of rats administered with DDVP was also divided into two sub groups; the first sub group of rats served as DDVP control ('DDVP'), while the second sub group of rats was injected with

DDVP treated rats.


1mg/dl; 2mg/g tissue; Values are mean SEM (n=6); Mean in the same column with different superscript differ significantly (*p<0.05*)



1pmol DCF/min/mg protein; 2nmol/g tissue; 3mg/g tissue

Values are mean SEM (n=6); Mean in the same column with different superscript differ significantly (*p<0.05*)

Table 6. Oxidative stress parameters in pancreas of rats treated with DDVP ± STZ (i.p , 25 mg/kg b.w).

#### **3.4 Do OPI aggravate diabetic outcomes?**

To address this question, we investigated the diabetic outcomes in rats experimentally rendered diabetic and post-treated with monocrotophos. Rats were rendered diabetic with an acute dose (60 mg/kg b.w, i.p) of streptozotocin. Monocrotophos was orally administered at a sublethal dose (1/20 LD50, 0.9 mg /kg b.w./d, 5 days) to both normal and diabetic rats. We observed that monocrotophos *per se* moderately increased (25%) the blood glucose levels in normal rats, but significantly aggravated the hyperglycemic outcome in diabetic rats (56% above diabetic rats). We observed the typical lipid profile alterations among diabetic rats characterized by increase in total cholesterol and triglycerides (TG) in serum. While monocrotophos did not impact lipid profile *per se*, its interaction with diabetic component resulted in severe alterations lipid profile that reflected in phenomenal increase in serum triglyceride. Such augmented impairments may have high bearing on cardiovascular health since the cardiovascular risk index was alarmingly high among diabetic rats treated with monocrotophos (**Fig. 8**). Further, monocrotophos resulted in higher degree of hepatic and renal toxicity as reflected by alterations in serum transaminase activities and blood urea nitrogen values respectively (**Table 7)**.

Further, it is now established that oxidative stress in an important consequence of diabetic condition and plays as an important pathophysiological factor in progression of diabetic complications (Maritim, 2003). We observed that STZ-induced diabetes was associated with increased lipid peroxidation (122%), depletion of reduced glutathione (10%) and alterations in activities of two important antioxidant enzymes (superoxide dismutase and catalase) in kidney. Monocrotophos further deteriorated oxidative impairments in kidney as evidenced by further increase in lipid peroxidation (170%) and depletion of reduced glutathione (18%) content. Diabetes was associated with marginal decrease in superoxide dismutase activity in kidney, which was further reduced by monocrotophos treatment (35%) (**Fig. 9**).

Organophosphorus Insecticides and Glucose Homeostasis 79

which *per se* did not interfere with lipid profile in rats, yet causing augmentation of alteration in lipid profile in diabetic rats. However, other studies have clearly demonstrated that several OPI cause alterations in lipid profile, particularly hypertriglyceridemia (Ryhänen et al, 1984; Ibrahim & El-Gamal, 2003; Rezg et al., 2010). Dyslipidemia or lipid abnormalities play an important role in the progression of diabetes (Goldberg, 2001) and these are characterized by lipid derangements including hypertriglyceridemia, low highdensity cholesterol (HDL-C), and a high concentration of small dense low-density lipoprotein (LDL) particles. Further, a state of elevated hypertriglyceridemia is commonly associated with insulin resistance and represents a valuable clinical marker of the metabolic syndrome (Grundy et al., 1999). Propensity of OPI to induce hypertriglyceridemia coupled with their permissive effects of gluconeogenesis in liver creates a serious threat to glucose

Fig. 9. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on oxidative balance in kidney of control and diabetic rats (Begum and Rajini, 2011b).

Given the status of OPI as environmental pollutant with residues being detected in biosphere around, which are now being shown to make it into human body, it is almost certain that OPI will interact with etiological factors of diabetes at toxicologically significant levels. Interaction of living system with OPI may have severe two-way impact on glycemic control. As documented facilitators of hepatic glucose output via glycogenolysis and gluconeogenesis, OPI are most likely to elicit hyperglycaemic responses in humans during exposure. Further, OPI may also affect the responsiveness to human system to insulin via

homeostasis.

**4. Conclusion** 

Fig. 8. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on cardiovascular index in control and diabetic rats (Begum and Rajini, 2011a).


Data analyzed by Tukey's HSD test; Mean SEM (n=4)

TC: Total cholesterol; HDL-C: High-density lipoprotein ; TG: Triglyceride; BUN: Blood Urea Nitrogen; SC: Serum creatinine

Table 7. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on blood glucose, lipid profile and hepatic and renal damage markers in serum in control and diabetic rat.

Our work on interaction of OPI with diabetic component clearly shows that OPI can act as both predisposing and aggravating factors for diabetes. The inference becomes an important consideration to be made as the modern world is facing an escalating situation of alarming increase in the incidence of diabetes. Our study employed a low dose of monocrotophos,

Fig. 8. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on

126.91± 8.9

41.92 ± 1.9

32.68 ± 1.7

78.12 ± 6.9

51.88 ± 8.2

0.72 ± 0.1

72.24 ± 0.7

156.00 ± 18.7

(Begum and Rajini, 2011a)

TC: Total cholesterol; HDL-C: High-density lipoprotein ; TG: Triglyceride; BUN: Blood Urea Nitrogen;

Table 7. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on blood glucose, lipid profile and hepatic and renal damage markers in serum in control and

Our work on interaction of OPI with diabetic component clearly shows that OPI can act as both predisposing and aggravating factors for diabetes. The inference becomes an important consideration to be made as the modern world is facing an escalating situation of alarming increase in the incidence of diabetes. Our study employed a low dose of monocrotophos,

Control Mono STZ STZ+Mono

382.71 ± 14.0ab

50.45 ± 1.6a

37.84 ± 1.4

125.44 ± 9.2a

71.18 ± 10.1a

0.81 ± 0.1

80.03 ± 2.4

199.78 ± 14.2de

597.94 ± 12.5cde

50.42 ± 1.2a

35.48 ± 1.3

193.52 ± 19.4bcd

78.05 ± 5.2b

0.84 ± 0.04

120.77 ± 9.2abc

341.55 ± 8.5abc

cardiovascular index in control and diabetic rats (Begum and Rajini, 2011a).

101.58 ± 1.4

38.00 ± 2.1

31.09 ± 1.2

66.74 ± 3.5

33.08 ± 5.6

0.63 ± 0.06

66.19 ± 2.0

126.57 ± 0.2

Data analyzed by Tukey's HSD test; Mean SEM (n=4)

Blood glucose (mg/dl)

> TC (mg/dl)

HDL-C (mg/dl)

TG (mg/dl)

BUN (mg/dl)

SC (mg/dl)

Serum ALT (U/L)

Serum AST (U/L)

SC: Serum creatinine

diabetic rat.

which *per se* did not interfere with lipid profile in rats, yet causing augmentation of alteration in lipid profile in diabetic rats. However, other studies have clearly demonstrated that several OPI cause alterations in lipid profile, particularly hypertriglyceridemia (Ryhänen et al, 1984; Ibrahim & El-Gamal, 2003; Rezg et al., 2010). Dyslipidemia or lipid abnormalities play an important role in the progression of diabetes (Goldberg, 2001) and these are characterized by lipid derangements including hypertriglyceridemia, low highdensity cholesterol (HDL-C), and a high concentration of small dense low-density lipoprotein (LDL) particles. Further, a state of elevated hypertriglyceridemia is commonly associated with insulin resistance and represents a valuable clinical marker of the metabolic syndrome (Grundy et al., 1999). Propensity of OPI to induce hypertriglyceridemia coupled with their permissive effects of gluconeogenesis in liver creates a serious threat to glucose homeostasis.

Fig. 9. Effect of repeated oral doses of monocrotophos at 1/20 LD50 (0.9 mg/kg b.w) on oxidative balance in kidney of control and diabetic rats (Begum and Rajini, 2011b).

#### **4. Conclusion**

Given the status of OPI as environmental pollutant with residues being detected in biosphere around, which are now being shown to make it into human body, it is almost certain that OPI will interact with etiological factors of diabetes at toxicologically significant levels. Interaction of living system with OPI may have severe two-way impact on glycemic control. As documented facilitators of hepatic glucose output via glycogenolysis and gluconeogenesis, OPI are most likely to elicit hyperglycaemic responses in humans during exposure. Further, OPI may also affect the responsiveness to human system to insulin via

Organophosphorus Insecticides and Glucose Homeostasis 81

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#### **5. Acknowledgments**

The authors we wish to thank the Director, CFTRI for extending support for this research. Indian Council of Medical Research (New Delhi) is gratefully acknowledged for funding the research programme described herein. The first author (AKRJ) thanks the Council of Industrial and Scientific Research (New Delhi) for award of Research Fellowship.

## **6. References**


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**4** 

*Japan* 

**The Toxicity of Fenitrothion and Permethrin** 

*Fenitrothion*: An organophosphorus insecticide, fenitrothion (*O*,*O*-dimethyl *O*-4-nitro-*m*-tolyl phosphorothioate; CAS No. 122-14-5), which is a yellow-brown liquid with an unpleasant odor at room temperature, was introduced in 1959 by both Sumitomo Chemical Company and Bayer Leverkusen, and later by American Cyanamid Company (Hayes, 1982; Hayes and

Organophosphorus insecticides began with the massive development of agriculture and agribusiness after World War II. At that time, parathion, one of the famous organophosphorus insecticides, was used in large quantities for preventing rice-stem borer worldwide. However, because of the high acute toxicity, parathion was thought to be an extremely hazardous substance. In man, an oral dose of 3-5 mg/kg is usually fatal. The following case report additionally came across the high toxicity and persistence in humans (Clifford and Nies, 1989). A 25-year-old worker in a pesticide-formulating plant was contaminated after accidentally spilling a 76% parathion solution on his groin and scrotal areas. Although he showered and changed clothes immediately, the resulting nausea and diarrhea made him consult a doctor two days later. The worker placed the parathionsaturated uniform in a plastic bag to be burned. But the contaminated clothing was laundered, and then was used in succession by a second worker, who wore it until he had complaints similar to the first worker. The coveralls were again laundered and used by still a third intoxicated worker. Totally, three workers suffered from toxic reaction to parathion. This case shows the toxic nature of parathion and its persistence on clothing even after successive laundering. Moreover, Etzel et al. (1987) reported that 49 persons in Sierra Leone were acutely poisoned by parathion in May and June 1986, 14 of whom later died. The casecontrol study of the employed 21 cases and 22 household controls was undertaken to explore which factors were associated with the development of the symptoms such as excess salivation, excess tearing, increased urination, diarrhea, convulsions, and loss of consciousness. Each case and control were questioned about foods and beverages that had been consumed during the 4 hours before becoming ill (for cases) or on the day of a case's illness (for controls). The odds ratio of cases (12.7; 95% confidence interval (CI), 2.4-83.8) for taking bread was significantly increased, suggesting that cases were more likely than controls to have eaten bread within the 4 hours before becoming ill. In addition, when stratified by age, the odds ratio was far higher in children under 18 years (odds ratio, 21.7; 95% CI, 2.4-264.6) than adults (odds ratio, 2.3; 95% CI, 0.02-195.9). This may be due to the higher consumption of parathion based on body weight or higher susceptibility to the

**1. Introduction** 

Laws, 1990; Worthing and Walker, 1987).

Dong Wang, Hisao Naito and Tamie Nakajima

*Nagoya University Graduate School of Medicine* 


## **The Toxicity of Fenitrothion and Permethrin**

Dong Wang, Hisao Naito and Tamie Nakajima

*Nagoya University Graduate School of Medicine Japan* 

#### **1. Introduction**

84 Insecticides – Pest Engineering

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RA90.

*Fenitrothion*: An organophosphorus insecticide, fenitrothion (*O*,*O*-dimethyl *O*-4-nitro-*m*-tolyl phosphorothioate; CAS No. 122-14-5), which is a yellow-brown liquid with an unpleasant odor at room temperature, was introduced in 1959 by both Sumitomo Chemical Company and Bayer Leverkusen, and later by American Cyanamid Company (Hayes, 1982; Hayes and Laws, 1990; Worthing and Walker, 1987).

Organophosphorus insecticides began with the massive development of agriculture and agribusiness after World War II. At that time, parathion, one of the famous organophosphorus insecticides, was used in large quantities for preventing rice-stem borer worldwide. However, because of the high acute toxicity, parathion was thought to be an extremely hazardous substance. In man, an oral dose of 3-5 mg/kg is usually fatal. The following case report additionally came across the high toxicity and persistence in humans (Clifford and Nies, 1989). A 25-year-old worker in a pesticide-formulating plant was contaminated after accidentally spilling a 76% parathion solution on his groin and scrotal areas. Although he showered and changed clothes immediately, the resulting nausea and diarrhea made him consult a doctor two days later. The worker placed the parathionsaturated uniform in a plastic bag to be burned. But the contaminated clothing was laundered, and then was used in succession by a second worker, who wore it until he had complaints similar to the first worker. The coveralls were again laundered and used by still a third intoxicated worker. Totally, three workers suffered from toxic reaction to parathion. This case shows the toxic nature of parathion and its persistence on clothing even after successive laundering. Moreover, Etzel et al. (1987) reported that 49 persons in Sierra Leone were acutely poisoned by parathion in May and June 1986, 14 of whom later died. The casecontrol study of the employed 21 cases and 22 household controls was undertaken to explore which factors were associated with the development of the symptoms such as excess salivation, excess tearing, increased urination, diarrhea, convulsions, and loss of consciousness. Each case and control were questioned about foods and beverages that had been consumed during the 4 hours before becoming ill (for cases) or on the day of a case's illness (for controls). The odds ratio of cases (12.7; 95% confidence interval (CI), 2.4-83.8) for taking bread was significantly increased, suggesting that cases were more likely than controls to have eaten bread within the 4 hours before becoming ill. In addition, when stratified by age, the odds ratio was far higher in children under 18 years (odds ratio, 21.7; 95% CI, 2.4-264.6) than adults (odds ratio, 2.3; 95% CI, 0.02-195.9). This may be due to the higher consumption of parathion based on body weight or higher susceptibility to the

The Toxicity of Fenitrothion and Permethrin 87

dimethylphosphate and 3-methyl-4-nitrophenol (MNP) by paraoxonase 1 (PON1). MNP and methylphosphate are also produced by glutathione-S-aryltransferase (GST) and PON1. In another pathway, fenitrothion is directly metabolized to MNP and dimethylthiophosphate by PON1 or MNP and methylthiophosphate by GST and PON1 (Figure 1). Interestingly, its major metabolic route differs between mammals and birds as mentioned later. Most of the metabolites are excreted in urine within 24 hours in humans (Nosal and Hladka, 1968), and within 2-4 days in the rat, guinea-pig, mouse, and dog (Miyamoto et al., 1963; Miyamoto, 1964). Species and sexes differences are observed in the composition of the metabolites. MNP, which is also contained in diesel exhaust emissions, has potential adverse effects on the reproductive systems in mammals and birds. Fenitrothion at doses of 0.18 and 0.36 mg/kg per day was administered to 12 human volunteers for 4 days (Meaklim et al., 2003). Pharmacokinetic parameters could only be determined at the high dosage, because the blood levels of fenitrothion at the low dosage were below the detectable level. Fenitrothion concentrations showed a wide range of interindividual variability, with peak blood levels achieved 1-4 hours after dosing, and the half-life ranged from 0.8 to 4.5 hours. Serum concentrations of fenitrothion were measured in 15 patients after acute fenitrothion intoxication, who admitted to the hospital 0.5-12 hours after the ingestion of 5-50 g fenitrothion (Koyama et al., 2006). The serum fenitrothion concentrations ranged from undetectable (< 0.01 µg/ml) to 9.73 µg/ml. Serum fenitrothion concentrations were less than 7 µg/ml in the patients with mild intoxication, while in the severe cases, the levels were more than 7 µg/ml. The elimination half-lives in the mild cases were 9.9 ± 7.7 hours (mean ± SD), and the serum fenitrothion concentrations declined below the detectable level in 48 hours. The elimination half-lives relating to two severe cases were 5.3 and 6.7 hours in the alpha phase (under direct hemoperfusion), and 35 and 52 hours in the beta phase, respectively. The serum fenitrothion

*Permethrin*: Regarding its metabolism, permethrin is converted to 2,2-dichlorovinyl-2,2 dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzyl alcohol (3PBAlc) by carboxylesterase. The latter metabolite is followed by oxidation to form 3-phenoxybenzaldehyde, and finally 3-phenoxybenzoic acid (3PBA) (Figure 2). The metabolites are reported to be endocrinedisrupting agents, but most studies mention permethrin toxicity is derived from itself (Yuan et al., 2010). In general, *trans* isomer is more rapidly metabolized than *cis* isomer, which is related to the lower susceptibility of *cis* isomer to enzymatic hydrolysis of the ester linkage (Soderlund and Casida, 1977; Zhang et al., 2008). Besides hydrolytic pathway by carboxylesterase, oxidative metabolic pathway of both *cis*-and *trans*-permethrin in rat and human hepatic microsomes was recently reported (Scollon et al., 2009). The toxicokinetics of permethrin (with a *cis*:*trans* ratio of 25:75) was investigated after single oral doses to rats (Anadon et al., 1991). The plasma level of permethrin was maximal within 4 hours after dosing, and then was slowly eliminated from plasma with a half-life of 12.4 hours. The bioavailability of permethrin was found to be 60.7%. The maximum permethrin concentrations in the central and peripheral nervous system were higher than plasma concentrations, and declined with half-life similar to those of plasma. Clearance of *trans*- and *cis*-permethrin from the blood was also investigated in a man who drank an emulsifiable concentrate formulation of permethrin (consisting of 43.5% *cis* and 56.5% *trans*) (Gotoh et al., 1998). The serum concentrations of *cis*- and *trans*-permethrin peaked 3-4 hours after ingestion and then declined, with *trans*-permethrin cleared from the blood more quickly than *cis*-permethrin. Levels of the *trans* isomer were below the detectability threshold within 25 hours after exposure, whereas *cis* isomer was still detectable 10 days after exposure. The present study indicated that the differential persistence of *cis* and

concentrations fell below the detectable level in 300 hours.

insecticide in the former than the latter. Parathion was detected from residue floor on the truck that had brought the wheat flour from the milling factory to the general store where the baker purchased it, suggesting that the flour had been contaminated during transport. The authors estimated that 10-15 ml of parathion may have spilled onto a 22.5 kg bag of flour in the truck. Besides these, many cases of parathion intoxication have been reported to date (Aardema et al., 2008; Eyer et al., 2003; Hoffmann and Papendorf, 2006; Laynez et al., 1997).

In light of this background, fenitrothion was developed in place of parathion for its highly selective toxicity to insects over humans and animals. While the structure of fenitrothion is similar to that of parathion, its residual effects and acute toxicity are lower than parathion (Miyamoto, 1969). An oral LD50 of parathion is approximately 6 mg/kg for rats, against 330 mg/kg for fenitrothion, which is more rapidly broken down and does not persist in areas where they are used. Fenitrothion is effective against a wide range of pests on rice, cereals, fruits, vegetables, stored grains, cotton and forests, and also in public health programs for control of flies, mosquitoes and cockroaches. Fenitrothion is produced at the rate of 15,000 to 20,000 tons per year worldwide, and is available in emulsifiable concentrates, ultra-lowvolume concentrates, powders, granules, dustable powders, oil-based sprays and in combination with other pesticides.

*Permethrin*: Permethrin (3-phenoxybenzyl (1*RS*,3*RS*;1*RS*,3*SR*)-3-(2,2-dichlorovinyl)-2,2 dimethylcyclopropanecarboxylate; CAS No. 52645-53-1) was first synthesized in 1973 and marketed in 1977 as a photostable pyrethroid (Elliott et al., 1973). Approximately 600 tons per year is at present used worldwide not only in agriculture but also in forestry, household settings, and public health programs.

Pyrethroids represented a major advancement as a high insecticide potential, but showed relatively low potential for mammals. Their development was especially timely with the identification of problems with DDT use. Pyrethroids consist first of identifying components of pyrethrum, which were extracted from East African chrysanthemum flowers, long known to have insecticidal properties. Pyrethrum rapidly knocks down flying insects, but has low mammalian toxicity and negligible persistence, which are good for the environment but yield poor efficacy when applied in the field. In the 1960s, 1st-generation pyrethroids, including bioallethrin, tetramethrin, resmethrin and bioresmethrin, were developed. They are more active than natural pyrethrum, but are unstable in sunlight. Then, permethrin, cypermethrin and deltamethrin were discovered as a 2nd generation of more persistent compounds. They are substantially more resistant to degradation by light and air, thus making them suitable for use in agriculture.

Permethrin is highly effective for protection of stored grains, cotton and other crops, and the control of body lice and household noxious insects. Technical products, which are a brown or yellowish-brown liquid, are a mixture of *cis* and *trans* isomers in the ratio of 40:60 or 25:75, and are available in emulsifiable concentrates, ultra-low-volume concentrates, wettable powders, and dustable powders.

## **2. Absorption, metabolism and excretion in laboratory animals and humans**

In mammals, fenitrothion and permethrin are absorbed via gastrointestinal or respiratory tract and skin, and are rapidly metabolized and excreted.

*Fenitrothion*: After uptake into the body, fenitrothion is metabolized by hepatic cytochrome P450 (CYP) to form fenitrooxon, which is thought to have a higher potential acute neurotoxicity than the parent compound. Fenitrooxon is further metabolized to

insecticide in the former than the latter. Parathion was detected from residue floor on the truck that had brought the wheat flour from the milling factory to the general store where the baker purchased it, suggesting that the flour had been contaminated during transport. The authors estimated that 10-15 ml of parathion may have spilled onto a 22.5 kg bag of flour in the truck. Besides these, many cases of parathion intoxication have been reported to date (Aardema et al., 2008; Eyer et al., 2003; Hoffmann and Papendorf, 2006; Laynez et al., 1997). In light of this background, fenitrothion was developed in place of parathion for its highly selective toxicity to insects over humans and animals. While the structure of fenitrothion is similar to that of parathion, its residual effects and acute toxicity are lower than parathion (Miyamoto, 1969). An oral LD50 of parathion is approximately 6 mg/kg for rats, against 330 mg/kg for fenitrothion, which is more rapidly broken down and does not persist in areas where they are used. Fenitrothion is effective against a wide range of pests on rice, cereals, fruits, vegetables, stored grains, cotton and forests, and also in public health programs for control of flies, mosquitoes and cockroaches. Fenitrothion is produced at the rate of 15,000 to 20,000 tons per year worldwide, and is available in emulsifiable concentrates, ultra-lowvolume concentrates, powders, granules, dustable powders, oil-based sprays and in

*Permethrin*: Permethrin (3-phenoxybenzyl (1*RS*,3*RS*;1*RS*,3*SR*)-3-(2,2-dichlorovinyl)-2,2 dimethylcyclopropanecarboxylate; CAS No. 52645-53-1) was first synthesized in 1973 and marketed in 1977 as a photostable pyrethroid (Elliott et al., 1973). Approximately 600 tons per year is at present used worldwide not only in agriculture but also in forestry, household

Pyrethroids represented a major advancement as a high insecticide potential, but showed relatively low potential for mammals. Their development was especially timely with the identification of problems with DDT use. Pyrethroids consist first of identifying components of pyrethrum, which were extracted from East African chrysanthemum flowers, long known to have insecticidal properties. Pyrethrum rapidly knocks down flying insects, but has low mammalian toxicity and negligible persistence, which are good for the environment but yield poor efficacy when applied in the field. In the 1960s, 1st-generation pyrethroids, including bioallethrin, tetramethrin, resmethrin and bioresmethrin, were developed. They are more active than natural pyrethrum, but are unstable in sunlight. Then, permethrin, cypermethrin and deltamethrin were discovered as a 2nd generation of more persistent compounds. They are substantially more resistant to degradation by light and air, thus

Permethrin is highly effective for protection of stored grains, cotton and other crops, and the control of body lice and household noxious insects. Technical products, which are a brown or yellowish-brown liquid, are a mixture of *cis* and *trans* isomers in the ratio of 40:60 or 25:75, and are available in emulsifiable concentrates, ultra-low-volume concentrates,

**2. Absorption, metabolism and excretion in laboratory animals and humans**  In mammals, fenitrothion and permethrin are absorbed via gastrointestinal or respiratory

*Fenitrothion*: After uptake into the body, fenitrothion is metabolized by hepatic cytochrome P450 (CYP) to form fenitrooxon, which is thought to have a higher potential acute neurotoxicity than the parent compound. Fenitrooxon is further metabolized to

combination with other pesticides.

settings, and public health programs.

making them suitable for use in agriculture.

wettable powders, and dustable powders.

tract and skin, and are rapidly metabolized and excreted.

dimethylphosphate and 3-methyl-4-nitrophenol (MNP) by paraoxonase 1 (PON1). MNP and methylphosphate are also produced by glutathione-S-aryltransferase (GST) and PON1. In another pathway, fenitrothion is directly metabolized to MNP and dimethylthiophosphate by PON1 or MNP and methylthiophosphate by GST and PON1 (Figure 1). Interestingly, its major metabolic route differs between mammals and birds as mentioned later. Most of the metabolites are excreted in urine within 24 hours in humans (Nosal and Hladka, 1968), and within 2-4 days in the rat, guinea-pig, mouse, and dog (Miyamoto et al., 1963; Miyamoto, 1964). Species and sexes differences are observed in the composition of the metabolites. MNP, which is also contained in diesel exhaust emissions, has potential adverse effects on the reproductive systems in mammals and birds. Fenitrothion at doses of 0.18 and 0.36 mg/kg per day was administered to 12 human volunteers for 4 days (Meaklim et al., 2003). Pharmacokinetic parameters could only be determined at the high dosage, because the blood levels of fenitrothion at the low dosage were below the detectable level. Fenitrothion concentrations showed a wide range of interindividual variability, with peak blood levels achieved 1-4 hours after dosing, and the half-life ranged from 0.8 to 4.5 hours. Serum concentrations of fenitrothion were measured in 15 patients after acute fenitrothion intoxication, who admitted to the hospital 0.5-12 hours after the ingestion of 5-50 g fenitrothion (Koyama et al., 2006). The serum fenitrothion concentrations ranged from undetectable (< 0.01 µg/ml) to 9.73 µg/ml. Serum fenitrothion concentrations were less than 7 µg/ml in the patients with mild intoxication, while in the severe cases, the levels were more than 7 µg/ml. The elimination half-lives in the mild cases were 9.9 ± 7.7 hours (mean ± SD), and the serum fenitrothion concentrations declined below the detectable level in 48 hours. The elimination half-lives relating to two severe cases were 5.3 and 6.7 hours in the alpha phase (under direct hemoperfusion), and 35 and 52 hours in the beta phase, respectively. The serum fenitrothion concentrations fell below the detectable level in 300 hours.

*Permethrin*: Regarding its metabolism, permethrin is converted to 2,2-dichlorovinyl-2,2 dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzyl alcohol (3PBAlc) by carboxylesterase. The latter metabolite is followed by oxidation to form 3-phenoxybenzaldehyde, and finally 3-phenoxybenzoic acid (3PBA) (Figure 2). The metabolites are reported to be endocrinedisrupting agents, but most studies mention permethrin toxicity is derived from itself (Yuan et al., 2010). In general, *trans* isomer is more rapidly metabolized than *cis* isomer, which is related to the lower susceptibility of *cis* isomer to enzymatic hydrolysis of the ester linkage (Soderlund and Casida, 1977; Zhang et al., 2008). Besides hydrolytic pathway by carboxylesterase, oxidative metabolic pathway of both *cis*-and *trans*-permethrin in rat and human hepatic microsomes was recently reported (Scollon et al., 2009). The toxicokinetics of permethrin (with a *cis*:*trans* ratio of 25:75) was investigated after single oral doses to rats (Anadon et al., 1991). The plasma level of permethrin was maximal within 4 hours after dosing, and then was slowly eliminated from plasma with a half-life of 12.4 hours. The bioavailability of permethrin was found to be 60.7%. The maximum permethrin concentrations in the central and peripheral nervous system were higher than plasma concentrations, and declined with half-life similar to those of plasma. Clearance of *trans*- and *cis*-permethrin from the blood was also investigated in a man who drank an emulsifiable concentrate formulation of permethrin (consisting of 43.5% *cis* and 56.5% *trans*) (Gotoh et al., 1998). The serum concentrations of *cis*- and *trans*-permethrin peaked 3-4 hours after ingestion and then declined, with *trans*-permethrin cleared from the blood more quickly than *cis*-permethrin. Levels of the *trans* isomer were below the detectability threshold within 25 hours after exposure, whereas *cis* isomer was still detectable 10 days after exposure. The present study indicated that the differential persistence of *cis* and

The Toxicity of Fenitrothion and Permethrin 89

CH3

C

O

Permethrin (*cis* and *trans* isomers)

+

2,2-dichlorovinyl-

C CH

Cl Cl

1-carboxylic acid

2,2-dimethylcyclopropane-

CH3

C CH

CH3

C

OH

O

CH3

Cl Cl

carboxylesterase

O CH2 O

3-phenoxybenzyl alcohol (3PBAlc)

HO CH2 O

3-phenoxybenzylaldehyde

O CH O

O C O

OH

3-phenoxybenzoic acid (3PBA)

oxidation

oxidation

Fig. 2. Metabolic pathway of permethrin in mammals. This figure was adopted from WHO (1990) with slight modifications. Carboxylesterase plays an important role in the hydrolytic biotransformation of permethrin. The expression of carboxylesterase is ubiquitous with high levels in various tissues. Amon various animal tissues, the highest hydrolase activity is typically found in the liver and other tissues, such as testis, kidney and plasma. Unlike

mouse, rat, rabbit, horse and cat, human plasma contains no carboxylesterase.

*trans* isomers in human is consistent with a difference in the metabolic rate of *cis*- and *trans*permethrin in animal studies (Anadon et al., 1991).

Fig. 1. Metabolic pathway of fenitrothion in mammals or birds. This figure was adopted from WHO (1992) with slight modifications. In mammals, dimethylthiophosphate, dimethylphosphate and MNP were the major urinary metabolites of fenitrothion and fenitrooxon. In birds, MNP was the major urinary metabolite of fenitrothion and fenitrooxon. GST activity was found to be lower than in mammals.

*trans* isomers in human is consistent with a difference in the metabolic rate of *cis*- and *trans*-

dimethylthiophosphate dimethylphosphate

Fenitrothion Fenitrooxon

PON1

GST GST

CYP

CH3

CH3

P N O O2

P O NO2

PON1

O

HO

OP

O

CH3O

O

CH3O

CH3O

CH3O

CH3O

Fig. 1. Metabolic pathway of fenitrothion in mammals or birds. This figure was adopted from WHO (1992) with slight modifications. In mammals, dimethylthiophosphate, dimethylphosphate and MNP were the major urinary metabolites of fenitrothion and fenitrooxon. In birds, MNP was the major urinary metabolite of fenitrothion and

3-methyl-4-nitrophenol (MNP)

HO NO2

PON1 PON1

CH3

fenitrooxon. GST activity was found to be lower than in mammals.

permethrin in animal studies (Anadon et al., 1991).

S

CH3O

CH3O

S

CH3O

CH3O

S

HO

CH3O

PON1

P O NO2

CH3

P O NO2

CH3

OP

3-phenoxybenzoic acid (3PBA)

Fig. 2. Metabolic pathway of permethrin in mammals. This figure was adopted from WHO (1990) with slight modifications. Carboxylesterase plays an important role in the hydrolytic biotransformation of permethrin. The expression of carboxylesterase is ubiquitous with high levels in various tissues. Amon various animal tissues, the highest hydrolase activity is typically found in the liver and other tissues, such as testis, kidney and plasma. Unlike mouse, rat, rabbit, horse and cat, human plasma contains no carboxylesterase.

The Toxicity of Fenitrothion and Permethrin 91

performance, organ weights, histopathology, and sperm analytical parameters were not influenced. In the offspring, no effects on anogenital distance, retention of areolae/nipples, onset of puberty, organ weights, histopathological findings, and sperm parameters were observed. In conclusion, fenitrothion had no effects on the reproductive or endocrine systems of the parental animals and their offspring, even at a toxic dose suppressing brain cholinesterase activity in parental animals. The concentration of 60 ppm (3.81 mg/kg per day) is 750 times higher than the acceptable daily intake (ADI) of fenitrothion (0.005 mg/kg body weight). Therefore, any potential risk of exposure may be negligible, and fenitrothion at in-use levels in the environment may be unlikely to cause disruption of human endocrine

*Human*: WHO (1990) classified technical grade fenitrothion as "moderately hazardous" (Class II). ADI of fenitrothion was established as 0.005 mg/kg body weight by the Joint FAO/WHO Expert Committee on Pesticide Residues in 2000. Nosal and Hladka (1968) reported that administration of fenitrothion at a single oral dose of 0.042-0.33 mg/kg body weight and repeated administration of 0.04-0.08 mg/kg body weight to human volunteers did not cause inhibition in plasma and erythrocyte ChE, and the urinary MNP was completely excreted within 24 hours. Chronic symptoms of exposure to fenitrothion in humans include general malaise, fatigue, headache, loss of memory and ability to concentrate, anorexia,

Permethrin acts on the axons in the peripheral and central nervous systems, causing prolonged opening of sodium channels. The acute toxicity of permethrin in mammalians is relatively low, though the LD50 value varies considerably according to the vehicle used and

*Animal*: NOAEL is assigned at 5 mg/kg body weight per day for permethrin with an isomer ratio of *cis*:*trans* 40:60 from the viewpoint of the effects on liver weight in 2-year and 26 week studies in rats, and a 3-month study in dogs. NOAEL is not available for respective *cis* and *trans* isomers (WHO, 1990). The rat appeared to be the most sensitive species with an oral LD50 of 400 mg/kg body weight for *cis*:*trans* 40:60 permethrin administered in corn oil, against 650 mg/kg body weight in mice. The neurotoxicity of intravenous- or orallyadministered *cis*-permethrin is over 10-fold greater than that of *trans* isomer. Neonatal rats are more sensitive than adult rats to the acute toxic effects of permethrin, which are thought

In their acute neurotoxicity study, Freeman (1993a) performed a functional observational battery (FOB) approximately 12 hours following administration of 10, 150 or 300 mg/kg of technical grade permethrin (mixture of *cis* and *trans*) in corn oil to male and female Sprague-Dawley rats. At doses of 150 and 300 mg/kg, permethrin caused salivation, tremor, splayed hindlimbs, abnormal posture, staggered gait, decreased grip strength, exaggerated reaction to sound, exaggerated hindlimb flexion, convulsions, and mortality. No treatment-related effects were observed at the lowest dose of 10 mg/kg. In a behavioral neurotoxicity study (McDaniel and Moser, 1993), technical grade permethrin was administered by gavage in corn oil to Long-Evans hooded rats, at doses of 25, 75 and 150 mg/kg, and the FOB were evaluated at 2 and 4 hours following treatment. Results of the present study are consistent with the acute regulatory study (Freeman, 1993a) including tremor, chromodacryorrhea, decreased grip strength and an exaggerated startle response. However, the absence of salivation, splayed hindlimbs and convulsions and the presence of aggressive sparring in

nausea, thirst, loss of weight, cramps, muscular weakness and tremors.

the *cis*:*trans* isomeric ratio (FAO, 1999; U.S. EPA, 2007).

to be related to differences in permethrin metabolism.

systems.

**Permethrin** 

## **3. Toxicity for experimental animals and humans**

## **Fenitrothion**

Since it is a cholinesterase (ChE) inhibitor, exposure to fenitrothion causes ChE activity depression in plasma, red blood cells, brain, and liver tissues. The acute toxicity of fenitrothion is considered to be low in mammals, because of the high metabolic rate (Hayes, 1982; Spencer, 1981).

*Animal*: The no-observed-adverse-effect levels (NOAEL), based on brain-ChE activity, were 10 mg/kg diet in both short- and long-term studies on rats, in long-term studies on mice, and were 50 mg/kg diet in short-term studies on dogs, respectively (WHO, 1992). Fenitrothion was given to female rats by gavage every other day from gestational day 6-15 at doses 3, 15, 30 and 45 mg/kg (Berlińska and Sitarek 1997). The maternal death rates were 39% and 88% at doses of 30 and 45 mg/kg, respectively. At 30 mg/kg, fenitrothion caused a significant decrease in maternal body weight gain, food consumption, hemoglobin and hematocrit values, and absolute weights of liver and kidney, but an increase in relative weights of adrenal and ovary. At 15 mg/kg, fenitrothion significantly decreased maternal relative liver weight. Although fenitrothion at doses of 3-30 mg/kg did not induced teratogenic effects, at 30 mg/kg it showed embryotoxicity, such as a significant increase in the frequency of early resorption per litter, postimplantation loss, and fetuses and litters with dilation of the cerebral ventricles. Furthermore, fenitrothion produced delayed ossification of sternum and cranium, and decreased fetal body weight and length. The frequency of fetuses and litters with dilation of the cerebral ventricles was increased at a dose of 15 mg/kg. Thus, Berlińska and Sitarek concluded that the NOAEL for developmental toxicity in rats was 3 mg/kg per day, and the lowest-observed-adverse-effect level was 15 mg/kg per day.

Recent studies showed the endocrine-disrupting effect of fenitrothion. Berger and Sultatos (1997) demonstrated that fenitrothion treatment caused a dose-dependent decrease in 2 hydroxyestradiol and 4-hydroxyestradiol production in mouse hepatic microsomes even at a dosage as low as 7 mg/kg, and an increase in 16 alpha-hydroxyestrone and estriol production. In another study, 7-week-old castrated Sprague-Dawley rats were subcutaneously treated with testosterone propionate (50 μg/day in 0.2 ml corn oil) and orally with corn oil vehicle or fenitrothion (15 or 30 mg/kg per day) once a day for 7 days (Tamura et al. 2001). Both fenitrothion doses caused significant decreases in the weights of ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscles. In contrast, blood acetylcholinesterase activity was only inhibited at the higher dose (30 mg/kg). Tamura et al. also demonstrated in an *in vitro* experiment that fenitrothion blocked dihydrotestosterone-dependent androgen receptor (AR) activity in a concentrationdependent and competitive manner in HepG2 human hepatoma liver cells, which were transiently transfected with human AR and an AR-dependent luciferase reporter gene, suggesting that fenitrothion may be a competitive androgen receptor antagonist.

On the other hand, Okahashi et al. (2005) suggested that lower-dose fenitrothion did not cause disruption of endocrine systems in animals. They administered fenitrothion to Crj:CD(SD)IGS parental rats at concentrations of 10, 20, and 60 (3.81 mg/kg per day) ppm in the diet for 10 weeks prior to mating, and throughout mating, gestation and lactation. Their offspring were exposed from weaning until maturation at the age of 10 weeks. In the parental animals, brain cholinesterase activity was remarkably reduced in males exposed to 60 ppm fenitrothion and in females exposed to 20 and 60 ppm fenitrothion. Reproductive performance, organ weights, histopathology, and sperm analytical parameters were not influenced. In the offspring, no effects on anogenital distance, retention of areolae/nipples, onset of puberty, organ weights, histopathological findings, and sperm parameters were observed. In conclusion, fenitrothion had no effects on the reproductive or endocrine systems of the parental animals and their offspring, even at a toxic dose suppressing brain cholinesterase activity in parental animals. The concentration of 60 ppm (3.81 mg/kg per day) is 750 times higher than the acceptable daily intake (ADI) of fenitrothion (0.005 mg/kg body weight). Therefore, any potential risk of exposure may be negligible, and fenitrothion at in-use levels in the environment may be unlikely to cause disruption of human endocrine systems.

*Human*: WHO (1990) classified technical grade fenitrothion as "moderately hazardous" (Class II). ADI of fenitrothion was established as 0.005 mg/kg body weight by the Joint FAO/WHO Expert Committee on Pesticide Residues in 2000. Nosal and Hladka (1968) reported that administration of fenitrothion at a single oral dose of 0.042-0.33 mg/kg body weight and repeated administration of 0.04-0.08 mg/kg body weight to human volunteers did not cause inhibition in plasma and erythrocyte ChE, and the urinary MNP was completely excreted within 24 hours. Chronic symptoms of exposure to fenitrothion in humans include general malaise, fatigue, headache, loss of memory and ability to concentrate, anorexia, nausea, thirst, loss of weight, cramps, muscular weakness and tremors.

#### **Permethrin**

90 Insecticides – Pest Engineering

Since it is a cholinesterase (ChE) inhibitor, exposure to fenitrothion causes ChE activity depression in plasma, red blood cells, brain, and liver tissues. The acute toxicity of fenitrothion is considered to be low in mammals, because of the high metabolic rate (Hayes,

*Animal*: The no-observed-adverse-effect levels (NOAEL), based on brain-ChE activity, were 10 mg/kg diet in both short- and long-term studies on rats, in long-term studies on mice, and were 50 mg/kg diet in short-term studies on dogs, respectively (WHO, 1992). Fenitrothion was given to female rats by gavage every other day from gestational day 6-15 at doses 3, 15, 30 and 45 mg/kg (Berlińska and Sitarek 1997). The maternal death rates were 39% and 88% at doses of 30 and 45 mg/kg, respectively. At 30 mg/kg, fenitrothion caused a significant decrease in maternal body weight gain, food consumption, hemoglobin and hematocrit values, and absolute weights of liver and kidney, but an increase in relative weights of adrenal and ovary. At 15 mg/kg, fenitrothion significantly decreased maternal relative liver weight. Although fenitrothion at doses of 3-30 mg/kg did not induced teratogenic effects, at 30 mg/kg it showed embryotoxicity, such as a significant increase in the frequency of early resorption per litter, postimplantation loss, and fetuses and litters with dilation of the cerebral ventricles. Furthermore, fenitrothion produced delayed ossification of sternum and cranium, and decreased fetal body weight and length. The frequency of fetuses and litters with dilation of the cerebral ventricles was increased at a dose of 15 mg/kg. Thus, Berlińska and Sitarek concluded that the NOAEL for developmental toxicity in rats was 3 mg/kg per day, and the lowest-observed-adverse-effect

Recent studies showed the endocrine-disrupting effect of fenitrothion. Berger and Sultatos (1997) demonstrated that fenitrothion treatment caused a dose-dependent decrease in 2 hydroxyestradiol and 4-hydroxyestradiol production in mouse hepatic microsomes even at a dosage as low as 7 mg/kg, and an increase in 16 alpha-hydroxyestrone and estriol production. In another study, 7-week-old castrated Sprague-Dawley rats were subcutaneously treated with testosterone propionate (50 μg/day in 0.2 ml corn oil) and orally with corn oil vehicle or fenitrothion (15 or 30 mg/kg per day) once a day for 7 days (Tamura et al. 2001). Both fenitrothion doses caused significant decreases in the weights of ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscles. In contrast, blood acetylcholinesterase activity was only inhibited at the higher dose (30 mg/kg). Tamura et al. also demonstrated in an *in vitro* experiment that fenitrothion blocked dihydrotestosterone-dependent androgen receptor (AR) activity in a concentrationdependent and competitive manner in HepG2 human hepatoma liver cells, which were transiently transfected with human AR and an AR-dependent luciferase reporter gene,

suggesting that fenitrothion may be a competitive androgen receptor antagonist.

On the other hand, Okahashi et al. (2005) suggested that lower-dose fenitrothion did not cause disruption of endocrine systems in animals. They administered fenitrothion to Crj:CD(SD)IGS parental rats at concentrations of 10, 20, and 60 (3.81 mg/kg per day) ppm in the diet for 10 weeks prior to mating, and throughout mating, gestation and lactation. Their offspring were exposed from weaning until maturation at the age of 10 weeks. In the parental animals, brain cholinesterase activity was remarkably reduced in males exposed to 60 ppm fenitrothion and in females exposed to 20 and 60 ppm fenitrothion. Reproductive

**3. Toxicity for experimental animals and humans** 

**Fenitrothion** 

1982; Spencer, 1981).

level was 15 mg/kg per day.

Permethrin acts on the axons in the peripheral and central nervous systems, causing prolonged opening of sodium channels. The acute toxicity of permethrin in mammalians is relatively low, though the LD50 value varies considerably according to the vehicle used and the *cis*:*trans* isomeric ratio (FAO, 1999; U.S. EPA, 2007).

*Animal*: NOAEL is assigned at 5 mg/kg body weight per day for permethrin with an isomer ratio of *cis*:*trans* 40:60 from the viewpoint of the effects on liver weight in 2-year and 26 week studies in rats, and a 3-month study in dogs. NOAEL is not available for respective *cis* and *trans* isomers (WHO, 1990). The rat appeared to be the most sensitive species with an oral LD50 of 400 mg/kg body weight for *cis*:*trans* 40:60 permethrin administered in corn oil, against 650 mg/kg body weight in mice. The neurotoxicity of intravenous- or orallyadministered *cis*-permethrin is over 10-fold greater than that of *trans* isomer. Neonatal rats are more sensitive than adult rats to the acute toxic effects of permethrin, which are thought to be related to differences in permethrin metabolism.

In their acute neurotoxicity study, Freeman (1993a) performed a functional observational battery (FOB) approximately 12 hours following administration of 10, 150 or 300 mg/kg of technical grade permethrin (mixture of *cis* and *trans*) in corn oil to male and female Sprague-Dawley rats. At doses of 150 and 300 mg/kg, permethrin caused salivation, tremor, splayed hindlimbs, abnormal posture, staggered gait, decreased grip strength, exaggerated reaction to sound, exaggerated hindlimb flexion, convulsions, and mortality. No treatment-related effects were observed at the lowest dose of 10 mg/kg. In a behavioral neurotoxicity study (McDaniel and Moser, 1993), technical grade permethrin was administered by gavage in corn oil to Long-Evans hooded rats, at doses of 25, 75 and 150 mg/kg, and the FOB were evaluated at 2 and 4 hours following treatment. Results of the present study are consistent with the acute regulatory study (Freeman, 1993a) including tremor, chromodacryorrhea, decreased grip strength and an exaggerated startle response. However, the absence of salivation, splayed hindlimbs and convulsions and the presence of aggressive sparring in

The Toxicity of Fenitrothion and Permethrin 93

The presence of micro-flora may accelerate degradation. Miyamoto et al. (1966) studied the degradation of fenitrothion by *B. subtilis*. The major metabolite was aminofenitrothion, and other minor metabolites detected were dimethyl thiophosphoric acid and desmethyl fenitrothion. In the bacteria, aminofenitrothion is further degraded to desmethyl aminofenitrothion, but the rate is slower than the parent compound. No reduction of desmethyl fenitrothion to desmethyl aminofenitrothion was detected, and dimethyl phosphoric acid was not formed from aminofenitrothion (Figure 3). Thus, the degradation of fenitrothine in *B. subtilis*, may be quite different from the metabolic route of experimental animals and humans. In the absence of sunlight or microbial contamination, fenitrothion is stable in water. In soil, biodegradation is the primary route, though photolysis may also play a role. Airborne concentrations of fenitrothion and its levels in water may decrease rapidly by photolysis and hydrolysis. The concentrations of fenitrothion that are likely to be found in the environment do not have any effects on microorganisms in soil or water. In laboratory studies, fenitrothion is highly toxic for aquatic invertebrates in both freshwater and seawater, while fish are less sensitive to fenitrothion than invertebrates, and the most sensitive life stage is early larva. Fenitrothion is highly toxic to bees (LD50, 0.03-0.04 µg/bee) when bees are exposed to direct treatment or to dried residues on foliage (U.S. EPA, 1987). Furthermore, fenitrothion was found to be highly toxic to upland game birds, but not so toxic to waterfowl. Indeed, the acute oral LD50 values were determined to be 23.6 and 1190 mg/kg body weight for bobwhite quail and mallards, respectively. Even in reproduction studies, NOEL was 10 mg/kg body weight for the quail and 100 mg/kg body weight for the mallard, respectively. There are quantitative differences in the composition of metabolites of fenitrothion between mammalian and avian species. For example, in rats, mice and dogs, demethylated products at the *P*-*O*-methyl linkage by GST accounted for 30 to 60% of the total urinary metabolites (Hollingworth et al., 1967; Miyamoto et al., 1976), whereas in the birds only 10 to 15%. This may be due to lower GST activity in avian species compared with that in mammalians. Mihara et al. (1979) also revealed that oxidative activities of the *m*methyl group of fenitrothion and fenitrooxon in livers from hen, quail, pheasant and duck were higher than those of mammalian liver, while *O*-demethylate activity for fenitrothion or fenitrooxon was lower in these birds. In birds, MNP is the major metabolite of fenitrothion by hydrolysis, though a pathway exists with oxidation of the *m*-methyl group of fenitrothion or fenitrooxon. The metabolite MNP is then conjugated with uridine diphosphate glucuronic acid or 3'-phosphoadenosine-5'-phosphosulfate by catalytic action of uridine diphosphate glucuronosyltranferase (UGT) and sulfotransferase (SULT), respectively (Mackenzie et al., 1997). Hepatic UGT and SULT activities investigated in vitro for MNP in Japanese quail, mice and rats revealed lower UGT activity for MNP in quail than rats and mice, but no significant difference in SULT activity (Lee et al., 2007). In addition, the SULT activity was only one-tenth of the UGT activity, suggesting that the latter enzyme plays an important role in MNP elimination in vivo. Li et al. (2008) reported that the birds treated with 100 mg/kg of MNP induced acute toxicological responses such as dyspnea and tremor, and finally death. MNP may cause acute toxicity and death, possibly by a rapid decrease in blood pressure followed by ischemic shock, because the potential vasodilatory action of MNP had been reported in rats (Mori et al., 2003; Taneda et al., 2004). However, none of the rats died after treatment with 100 mg/kg of MNP (Li et al., 2007), suggesting that the sensitivity to MNP is higher in quail than in rats. For these reasons, fenitrothion causes

higher toxicity in birds than in mammals.

the latter study (McDaniel and Moser, 1993) were inconsistent with the findings of the regulatory acute neurotoxicity study (Freeman, 1993a).

In a subchronic neurotoxicity study (Freeman, 1993b), technical grade permethrin was administered through the diet to male and female Sprague-Dawley rats, at concentrations of 250, 1500 and 2500 ppm (18, 101 and 170 mg/kg per day, respectively). At the 1500 and 2500 ppm dietary levels, permethrin produced tremor, splayed hindlimbs, abnormal posture, a staggered gait, and decreased grip strength. No such effects were observed at the lowest dose of 250 ppm.

Effects of permethrin on endocrine or reproductive function are investigated, but the report is very limited. Castrated rats (5-week-old) were pretreated with testosterone propionate and orally given permethrin (mixture of *cis* and *trans*, 24.8% and 71.8%) at doses of 10, 50 and 100 mg/kg per day for 10 days. A mixture of *cis*- and *trans*-permethrin showed anti-androgen-like effects on male rats such as significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights (Kim et al., 2005). *cis*-Permethrin at 0, 35 and 70 mg/kg was orally administered to IRC mice for 6 weeks, and male reproductive toxicity was investigated. This chemical dose-dependently decreased testicular and plasma testosterone levels, along with a dose-dependent increase in circulating LH and declines in epididymal sperm count and sperm motility (Zhang et al., 2007). Testicular residue concentrations of *cis*-permethrin from the individual animals were also strongly inversely correlated with testicular testosterone levels. The exposure-related reductions in mRNA and protein expression levels of peripheral benzodiazepine receptor, steroidogenic acute regulatory protein and cytochrome P450 sidechain cleavage, which are involved in testosterone synthesis in testis, were observed, as well as structural changes in Leydig cell mitochondria, suggesting that the mitochondrial damage caused by permethrin exposure may result in a reduction of testosterone synthesizing elements and thereby decrease testosterone levels. In a follow-up study in mice, *cis*-permethrin induces reproductive toxicity whereas at the same dose *trans*-permethrin does not because of a faster metabolic rate than *cis* isomer (Zhang et al., 2008). Zhang et al. also reported that *cis*permethrin caused structural abnormalities in the seminiferous tubules. However, it must be noted that these studies to date have used dose levels much higher than encountered by nonoccupationally exposed humans.

*Human*: Permethrin is a moderately to practically non-toxic pesticide in EPA toxicity class II or III, depending on the formulation. Formulations in the case of possible eye and skin irritation are grouped into class II. Permethrin belongs to the type I group of pyrethroids because it lacks a cyano group, and typically causes tremor (T-syndrome), incoordination, hyperactivity, prostration, and paralysis. An ADI of 0.05 mg/kg body weight for *cis*:*trans* 40:60 or 25:75 permethrin was established in 1987. Rishikesh et al. (1978) evaluated staff involved with bagging, mixing, or spraying a 5% preparation of permethrin (cis/trans ratio, 25:75) in Nigeria by a questionnaire and urinalysis. Regardless of the protective equipment worn by the sprayers, only 2 mg of permethrin was absorbed after exposure to 6 kg of permethrin, which was excreted in 24 hours.

#### **4. Toxicity for ecosystem**

#### **Fenitrothion**

In the environment, fenitrothion is degraded by photolysis and hydrolysis. In the presence of ultraviolet radiation or sunlight, the half-life of fenitrothion in water is less than 24 hours.

the latter study (McDaniel and Moser, 1993) were inconsistent with the findings of the

In a subchronic neurotoxicity study (Freeman, 1993b), technical grade permethrin was administered through the diet to male and female Sprague-Dawley rats, at concentrations of 250, 1500 and 2500 ppm (18, 101 and 170 mg/kg per day, respectively). At the 1500 and 2500 ppm dietary levels, permethrin produced tremor, splayed hindlimbs, abnormal posture, a staggered gait, and decreased grip strength. No such effects were observed at the lowest

Effects of permethrin on endocrine or reproductive function are investigated, but the report is very limited. Castrated rats (5-week-old) were pretreated with testosterone propionate and orally given permethrin (mixture of *cis* and *trans*, 24.8% and 71.8%) at doses of 10, 50 and 100 mg/kg per day for 10 days. A mixture of *cis*- and *trans*-permethrin showed anti-androgen-like effects on male rats such as significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights (Kim et al., 2005). *cis*-Permethrin at 0, 35 and 70 mg/kg was orally administered to IRC mice for 6 weeks, and male reproductive toxicity was investigated. This chemical dose-dependently decreased testicular and plasma testosterone levels, along with a dose-dependent increase in circulating LH and declines in epididymal sperm count and sperm motility (Zhang et al., 2007). Testicular residue concentrations of *cis*-permethrin from the individual animals were also strongly inversely correlated with testicular testosterone levels. The exposure-related reductions in mRNA and protein expression levels of peripheral benzodiazepine receptor, steroidogenic acute regulatory protein and cytochrome P450 sidechain cleavage, which are involved in testosterone synthesis in testis, were observed, as well as structural changes in Leydig cell mitochondria, suggesting that the mitochondrial damage caused by permethrin exposure may result in a reduction of testosterone synthesizing elements and thereby decrease testosterone levels. In a follow-up study in mice, *cis*-permethrin induces reproductive toxicity whereas at the same dose *trans*-permethrin does not because of a faster metabolic rate than *cis* isomer (Zhang et al., 2008). Zhang et al. also reported that *cis*permethrin caused structural abnormalities in the seminiferous tubules. However, it must be noted that these studies to date have used dose levels much higher than encountered by non-

*Human*: Permethrin is a moderately to practically non-toxic pesticide in EPA toxicity class II or III, depending on the formulation. Formulations in the case of possible eye and skin irritation are grouped into class II. Permethrin belongs to the type I group of pyrethroids because it lacks a cyano group, and typically causes tremor (T-syndrome), incoordination, hyperactivity, prostration, and paralysis. An ADI of 0.05 mg/kg body weight for *cis*:*trans* 40:60 or 25:75 permethrin was established in 1987. Rishikesh et al. (1978) evaluated staff involved with bagging, mixing, or spraying a 5% preparation of permethrin (cis/trans ratio, 25:75) in Nigeria by a questionnaire and urinalysis. Regardless of the protective equipment worn by the sprayers, only 2 mg of permethrin was absorbed after exposure to 6 kg of

In the environment, fenitrothion is degraded by photolysis and hydrolysis. In the presence of ultraviolet radiation or sunlight, the half-life of fenitrothion in water is less than 24 hours.

regulatory acute neurotoxicity study (Freeman, 1993a).

dose of 250 ppm.

occupationally exposed humans.

**4. Toxicity for ecosystem** 

**Fenitrothion** 

permethrin, which was excreted in 24 hours.

The presence of micro-flora may accelerate degradation. Miyamoto et al. (1966) studied the degradation of fenitrothion by *B. subtilis*. The major metabolite was aminofenitrothion, and other minor metabolites detected were dimethyl thiophosphoric acid and desmethyl fenitrothion. In the bacteria, aminofenitrothion is further degraded to desmethyl aminofenitrothion, but the rate is slower than the parent compound. No reduction of desmethyl fenitrothion to desmethyl aminofenitrothion was detected, and dimethyl phosphoric acid was not formed from aminofenitrothion (Figure 3). Thus, the degradation of fenitrothine in *B. subtilis*, may be quite different from the metabolic route of experimental animals and humans. In the absence of sunlight or microbial contamination, fenitrothion is stable in water. In soil, biodegradation is the primary route, though photolysis may also play a role. Airborne concentrations of fenitrothion and its levels in water may decrease rapidly by photolysis and hydrolysis. The concentrations of fenitrothion that are likely to be found in the environment do not have any effects on microorganisms in soil or water. In laboratory studies, fenitrothion is highly toxic for aquatic invertebrates in both freshwater and seawater, while fish are less sensitive to fenitrothion than invertebrates, and the most sensitive life stage is early larva. Fenitrothion is highly toxic to bees (LD50, 0.03-0.04 µg/bee) when bees are exposed to direct treatment or to dried residues on foliage (U.S. EPA, 1987). Furthermore, fenitrothion was found to be highly toxic to upland game birds, but not so toxic to waterfowl. Indeed, the acute oral LD50 values were determined to be 23.6 and 1190 mg/kg body weight for bobwhite quail and mallards, respectively. Even in reproduction studies, NOEL was 10 mg/kg body weight for the quail and 100 mg/kg body weight for the mallard, respectively. There are quantitative differences in the composition of metabolites of fenitrothion between mammalian and avian species. For example, in rats, mice and dogs, demethylated products at the *P*-*O*-methyl linkage by GST accounted for 30 to 60% of the total urinary metabolites (Hollingworth et al., 1967; Miyamoto et al., 1976), whereas in the birds only 10 to 15%. This may be due to lower GST activity in avian species compared with that in mammalians. Mihara et al. (1979) also revealed that oxidative activities of the *m*methyl group of fenitrothion and fenitrooxon in livers from hen, quail, pheasant and duck were higher than those of mammalian liver, while *O*-demethylate activity for fenitrothion or fenitrooxon was lower in these birds. In birds, MNP is the major metabolite of fenitrothion by hydrolysis, though a pathway exists with oxidation of the *m*-methyl group of fenitrothion or fenitrooxon. The metabolite MNP is then conjugated with uridine diphosphate glucuronic acid or 3'-phosphoadenosine-5'-phosphosulfate by catalytic action of uridine diphosphate glucuronosyltranferase (UGT) and sulfotransferase (SULT), respectively (Mackenzie et al., 1997). Hepatic UGT and SULT activities investigated in vitro for MNP in Japanese quail, mice and rats revealed lower UGT activity for MNP in quail than rats and mice, but no significant difference in SULT activity (Lee et al., 2007). In addition, the SULT activity was only one-tenth of the UGT activity, suggesting that the latter enzyme plays an important role in MNP elimination in vivo. Li et al. (2008) reported that the birds treated with 100 mg/kg of MNP induced acute toxicological responses such as dyspnea and tremor, and finally death. MNP may cause acute toxicity and death, possibly by a rapid decrease in blood pressure followed by ischemic shock, because the potential vasodilatory action of MNP had been reported in rats (Mori et al., 2003; Taneda et al., 2004). However, none of the rats died after treatment with 100 mg/kg of MNP (Li et al., 2007), suggesting that the sensitivity to MNP is higher in quail than in rats. For these reasons, fenitrothion causes higher toxicity in birds than in mammals.

The Toxicity of Fenitrothion and Permethrin 95

mammals. However, the extreme susceptibility to permethrin may be ascribed to its high sensitivity to sodium channels rather than low carboxylesterase activity. Permethrin is also highly toxic to honey bees (LD50, 0.11 µg/bee), yet exhibits very low toxicity to birds when given orally or fed in the diet. LD50 is >3000 mg/kg body weight for a single oral dosage and >5000 mg/kg diet for dietary exposure, respectively. One of the reasons for the different toxicity of permethrin among species is negatively correlated its toxic action to their body temperature, thus generally showing more acute effects on cold-blooded animals (insects,

Organophosphorus insecticides are being increasingly used in combination with pyrethroid insecticides. Fenitrothion is used in combination with other pesticides to enhance ChE inhibition by nature. However, fenitrothion inhibits not only ChE but also other esterase activity such as carboxylesterase. Trottier et al. (1980) reported that the oral administration of fenitrothion to male CD rats at a dose of 0, 2.5, 5, 10, or 20 mg/kg per day for 30 consecutive days significantly decreased liver carboxylesterase activity (by 50-80%) on days 8-30 at doses more than 2.5 mg/kg per day but had returned to control values by day 45 (15 days after termination of treatment) at all doses except 20 mg/kg per day, at which a decrease of 25% was still observed. At this dose, the values had returned to normal by day 87 (57 days after termination of treatment). A significant decrease in renal carboxylesterase activity (by 20-70%) was also observed on days 8-30 at doses over 5 mg/kg per day. Recovery of the activity was rapid, and the values were comparable to those of controls by

As described at the metabolism of permethrin, carboxylesterase plays an important role in detoxication of permethrin. Ortiz et al. (1995) examined the interactions between a commercial formulation of methyl parathion and a commercially formulated product of permethrin in male rats. When rats were treated with the mixture, 380 mg/kg of methyl parathion reduced the LD50 of permethrin by only 9.0%, whereas when rats received methyl parathion at 464 mg/kg, the LD50 of permethrin was reduced by 37% (P < 0.001). Results indicated that methyl parathion modified the acute toxicity of permethrin. Another study examined the effect of organophosphorus insecticide dichlorvos on excretion levels of urinary *cis*-permethrin-derived 3PBA in rats (Hirosawa et al., 2011). After *cis*-permethrin injection (20 mg/kg) via the tail vein of rats pretreated intraperitoneally with dichlorvos (low dose, 0.3 mg/kg; high dose, 1.5 mg/kg), the amounts of urinary 3PBA excretion over 48 hours were decreased to 81.1% and 70.3% of dichlorvos non-treated rats in the low- and high-dose dichlorvos groups, respectively. The plasma concentration of *cis*-permethrinderived 3PBAlc in high-dose dichlorvos group was significantly lower than that in the dichlorvos non-treated group one hour after *cis*-permethrin injection. In contrast, no differences were observed in the excretion levels of urinary 3PBA after injection of 3PBAlc between the dichlorvos non-treated group and the high-dose dichlorvos group. These results suggested that dichlorvos may have inhibited the metabolism of the co-exposed *cis*permethrin and thereby decreased the amount of urinary 3PBA excretion. In our recent study, we evaluated male reproductive toxicity after co-exposure to diazinon (3 mg/kg) and *cis*-permethrin (35 mg/kg) in mice. Exposure to diazinon alone and the mixture with *cis*permethrin inhibited plasma and liver carboxylesterase activities. In the co-exposed mice,

fish, etc) than warm-blooded animals (mammals and birds).

**5. Interactive toxicity of insecticide mixture** 

day 38 (8 days after the end of treatment).

Fig. 3. Pathway of degradation of fenitrothion by *B. subtilis* in environment. This figure was adopted from Miyamoto et al. (1966) with slight modifications. Aminofenitrothion was the major metabolite of fenitrothion in *B. subtilis.*

#### **Permethrin**

Permethrin is photodegraded by sunlight in water and on soil surfaces. Under aerobic conditions in soil, permethrin degrades with a half-life of 28 days. Permethrin deposited on plants degrades with a half-life of approximately 10 days. Thus, in the environment, permethrin is hydrolyzed, and the resultant acid and alcohol are conjugated. However, permethrin itself evidences very little movement within the environment, because it binds very strongly to soil particles and is nearly insoluble in water and not expected to leach or contaminate groundwater. Permethrin has been shown to be highly toxic for aquatic arthropods and fish, because they have lower levels of carboxylesterase activity than

CH3

CH3

P O NH2

desmethyl aminofenitrothion

P O NH2

Aminofenitrothion

O

O

HO

CH3O

CH3O

CH3O

Fig. 3. Pathway of degradation of fenitrothion by *B. subtilis* in environment. This figure was adopted from Miyamoto et al. (1966) with slight modifications. Aminofenitrothion was the

Permethrin is photodegraded by sunlight in water and on soil surfaces. Under aerobic conditions in soil, permethrin degrades with a half-life of 28 days. Permethrin deposited on plants degrades with a half-life of approximately 10 days. Thus, in the environment, permethrin is hydrolyzed, and the resultant acid and alcohol are conjugated. However, permethrin itself evidences very little movement within the environment, because it binds very strongly to soil particles and is nearly insoluble in water and not expected to leach or contaminate groundwater. Permethrin has been shown to be highly toxic for aquatic arthropods and fish, because they have lower levels of carboxylesterase activity than

major metabolite of fenitrothion in *B. subtilis.*

desmethyl fenitrothion

Fenitrothion

dimethyl thiophosphoric acid

CH3O

S

S

HO

CH3O

CH3O

CH3O

CH3O

P O NO2

P O NO2

OP

S

CH3

CH3

**Permethrin** 

mammals. However, the extreme susceptibility to permethrin may be ascribed to its high sensitivity to sodium channels rather than low carboxylesterase activity. Permethrin is also highly toxic to honey bees (LD50, 0.11 µg/bee), yet exhibits very low toxicity to birds when given orally or fed in the diet. LD50 is >3000 mg/kg body weight for a single oral dosage and >5000 mg/kg diet for dietary exposure, respectively. One of the reasons for the different toxicity of permethrin among species is negatively correlated its toxic action to their body temperature, thus generally showing more acute effects on cold-blooded animals (insects, fish, etc) than warm-blooded animals (mammals and birds).

## **5. Interactive toxicity of insecticide mixture**

Organophosphorus insecticides are being increasingly used in combination with pyrethroid insecticides. Fenitrothion is used in combination with other pesticides to enhance ChE inhibition by nature. However, fenitrothion inhibits not only ChE but also other esterase activity such as carboxylesterase. Trottier et al. (1980) reported that the oral administration of fenitrothion to male CD rats at a dose of 0, 2.5, 5, 10, or 20 mg/kg per day for 30 consecutive days significantly decreased liver carboxylesterase activity (by 50-80%) on days 8-30 at doses more than 2.5 mg/kg per day but had returned to control values by day 45 (15 days after termination of treatment) at all doses except 20 mg/kg per day, at which a decrease of 25% was still observed. At this dose, the values had returned to normal by day 87 (57 days after termination of treatment). A significant decrease in renal carboxylesterase activity (by 20-70%) was also observed on days 8-30 at doses over 5 mg/kg per day. Recovery of the activity was rapid, and the values were comparable to those of controls by day 38 (8 days after the end of treatment).

As described at the metabolism of permethrin, carboxylesterase plays an important role in detoxication of permethrin. Ortiz et al. (1995) examined the interactions between a commercial formulation of methyl parathion and a commercially formulated product of permethrin in male rats. When rats were treated with the mixture, 380 mg/kg of methyl parathion reduced the LD50 of permethrin by only 9.0%, whereas when rats received methyl parathion at 464 mg/kg, the LD50 of permethrin was reduced by 37% (P < 0.001). Results indicated that methyl parathion modified the acute toxicity of permethrin. Another study examined the effect of organophosphorus insecticide dichlorvos on excretion levels of urinary *cis*-permethrin-derived 3PBA in rats (Hirosawa et al., 2011). After *cis*-permethrin injection (20 mg/kg) via the tail vein of rats pretreated intraperitoneally with dichlorvos (low dose, 0.3 mg/kg; high dose, 1.5 mg/kg), the amounts of urinary 3PBA excretion over 48 hours were decreased to 81.1% and 70.3% of dichlorvos non-treated rats in the low- and high-dose dichlorvos groups, respectively. The plasma concentration of *cis*-permethrinderived 3PBAlc in high-dose dichlorvos group was significantly lower than that in the dichlorvos non-treated group one hour after *cis*-permethrin injection. In contrast, no differences were observed in the excretion levels of urinary 3PBA after injection of 3PBAlc between the dichlorvos non-treated group and the high-dose dichlorvos group. These results suggested that dichlorvos may have inhibited the metabolism of the co-exposed *cis*permethrin and thereby decreased the amount of urinary 3PBA excretion. In our recent study, we evaluated male reproductive toxicity after co-exposure to diazinon (3 mg/kg) and *cis*-permethrin (35 mg/kg) in mice. Exposure to diazinon alone and the mixture with *cis*permethrin inhibited plasma and liver carboxylesterase activities. In the co-exposed mice,

The Toxicity of Fenitrothion and Permethrin 97

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Mori Y, Kamata K, Toda N, et al: Isolation of nitrophenols from diesel exhaust particles (DEP) as vasodilatation compounds. *Biol Pharm Bull* 26:394-395, 2003. Nosál M, Hladká A: Determination of the exposure to fenitrothion (*O*,*O*-dimethyl-O-3-

cresol by the urine of the persons tested. *Int Arch Arbeitsmed* 25:28-38, 1968. Okahashi N, Sano M, Miyata K, et al: Lack of evidence for endocrine disrupting effects in

compounds. Part I. Metabolic fate of 32P-labelled Sumithion and methylparathion

Activation and degradation of Sumithion and methylparathion in vivo. Agric biol

methyl-4-nitrophenyl-thiophosphate) on the basis of the excretion of p-nitro-m-

rats exposed to fenitrothion in utero and from weaning to maturation. *Toxicology*

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diphosphate glucuronosyltransferase and sulfotransferase in liver microsomes of mice, rats, and Japanese quail (Coturnix japonica). *Environ Toxicol Chem* 26:1873-

toxicity in female Japanese quail (Coturnix japonica). *Biol Pharm Bull* 31:2158-2161,

recommended nomenclature update based on evolutionary divergence.

the effects of two pyrethroids, permethrin and cypermethrin. *Neurotoxicol Teratol*

acute intoxication patients. *Chudoku Kenkyu* 19:41-47, 2006.

*Intensive Care Med* 32:464-468, 2006.

in rats. *J Reprod Dev* 51:201-210, 2005.

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2008.

the urinary *cis*-permethrin metabolite decreased compared to that in mice exposed to *cis*permethrin alone. The co-exposure significantly decreased plasma testosterone levels and increased the number of degenerated germ cells within the seminiferous tubule, whereas exposure to each chemical did not. We concluded that diazinon inhibited the plasma and liver carboxylesterase activities and the metabolic rate of co-exposed *cis*-permethrin, which resulted in accentuating the reproductive toxicity of *cis*-permethrin (Wang et al., unpublished data submitted to the Journal).

Recently, since we could not find any study on the interaction between fenitrothion and permethrin, the toxicity of permethrin may be enhanced by fenitrothion via depression in carboxylesterase activity. Since fenitrothion and permethrin are used in the same place, if not purposefully in mixtures, the two insecticides could conceivably be combined. Until now, the risk assessments of combined toxicity to mammals are still insufficient and further detailed studies are warranted.

## **6. References**


the urinary *cis*-permethrin metabolite decreased compared to that in mice exposed to *cis*permethrin alone. The co-exposure significantly decreased plasma testosterone levels and increased the number of degenerated germ cells within the seminiferous tubule, whereas exposure to each chemical did not. We concluded that diazinon inhibited the plasma and liver carboxylesterase activities and the metabolic rate of co-exposed *cis*-permethrin, which resulted in accentuating the reproductive toxicity of *cis*-permethrin (Wang et al.,

Recently, since we could not find any study on the interaction between fenitrothion and permethrin, the toxicity of permethrin may be enhanced by fenitrothion via depression in carboxylesterase activity. Since fenitrothion and permethrin are used in the same place, if not purposefully in mixtures, the two insecticides could conceivably be combined. Until now, the risk assessments of combined toxicity to mammals are still insufficient and further

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Anadon A, Martinez-Larranaga MR, Diaz MJ, et al: Toxicokinetics of permethrin in the rat.

Berger CW Jr, Sultatos LG: The effects of the phosphorothioate insecticide fenitrothion on

Berlińska B, Sitarek K: Disturbances of prenatal development in rats exposed to fenitrothion.

Clifford NJ, Nies AS: Organophosphate poisoning from wearing a laundered uniform previously contaminated with parathion. *JAMA* 262:3035-3036, 1989. Elliott M, Farnham AW, Janes NF, et al: A photostable pyrethroid. *Nature* 246:169-170, 1973 Etzel RA, Forthal DN, Hill RH Jr, et al: Fatal parathion poisoning in Sierra Leone. *Bull World* 

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Hirosawa N, Ueyama J, Kondo T, et al: Effect of DDVP on urinary excretion levels of pyrethroid metabolite 3-phenoxybenzoic acid in rats. *Toxicol Lett* 203:28-32, 2011. Hoilingworth RM, Metcalf RL, Fukuto TR: The selectivity of sumithion compared with

methylparathion: Metabolism in the white mouse. *J Agric Food Chem* 15:242-249,

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1967.

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detailed studies are warranted.

**6. References** 


**5** 

**DDT and Its Metabolites in Mexico** 

*Laboratorio de Toxicología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí,* 

*San Luis Potosí, SLP,* 

*México* 

Iván Nelinho Pérez Maldonado, Jorge Alejandro Alegría-Torres,

Sandra Teresa Orta-Garcia and Lucia Guadalupe Pruneda Álvarez

DDT (dichlorodiphenyltrichloroethane) was first synthesized in 1874, and its insecticidal properties were discovered in 1939 by Paul Hermann Müller (Stapleton 1998). The U.S. military began using DDT extensively for mosquito control in 1944, particularly in the Pacific, where much of the action of World War II took place in highly malarious areas (Stapleton 1998). In 1955, the World Health Organization (WHO) started a global malaria control program with DDT; by 1958, 75 countries had joined and, at the peak of the campaign, 69,500 tons of pesticides mainly DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl)ethane] were applied to 100 million dwellings each year (Wijeyaratne, 1993). For the control of malaria, houses were sprayed twice a year with DDT wettable powder to kill resting mature Anopheles mosquito. Later, the Stockholm Convention on Persistent Organic Pollutants, which came into force on 17 May 2004, outlawed the use of 12 chemicals including DDT (UNEP, 2004). However, one exemption clause allows malaria-endemic nations to use DDT, strictly for disease vector control. The United Nations Environment Program estimates that about 25 countries will use DDT under exemptions from the DDT pesticide ban (POPs, 2009). Thus, in regard to presence of DDT around the world can be divided into three scenarios: Sites where DDT is still in use; sites where the presence is due to DDT sprayed several years ago, and sites where the presence of DDT is the result of a long-range transport of the insecticide to areas where it was never used like the Antarctic. In Mesoamerica (Mexico, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, Nicaragua and Panama) DDT was used until the year 2000, Mexico and Nicaragua being the last nations that applied the insecticide in agriculture and for the control of malaria. Table 1 lists the period and the total amount of DDT used in each Mesoamerican country by the malaria control programs. The amount used (approximately 85,000 tons between 1946 and 1999) together with the high environmental persistence of DDT and its metabolites, provide the necessary conditions for DDT to become a contaminant of concern for this region of the world (ISAT, 2002). Taking into account the environmental persistence and the toxicity of DDT, a program for the control of malaria without using insecticides in Mesoamerica was developed between 2004 and 2007, with assistance from the Pan American Health Organization [PAHO; (Chanon et al., 2003; PAHO, 2008)]. The phase-out of DDT in Costa

**1. Introduction** 

Octavio Gaspar-Ramírez, Francisco Javier Pérez Vázquez,


## **DDT and Its Metabolites in Mexico**

Iván Nelinho Pérez Maldonado, Jorge Alejandro Alegría-Torres, Octavio Gaspar-Ramírez, Francisco Javier Pérez Vázquez, Sandra Teresa Orta-Garcia and Lucia Guadalupe Pruneda Álvarez *Laboratorio de Toxicología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP,* 

*México* 

## **1. Introduction**

98 Insecticides – Pest Engineering

Ortiz D, Yáñez L, Gómez H, et al: Acute toxicological effects in rats treated with a mixture of

Rishikesh N, Clarke JL, Martin HL, et al: Evaluation of Decamethrins and Permethrin

Soderlund DM, Casida JE: Effects of pyrethroid structure on rates of hydrolysis and

Spencer EY: Guide to the Chemicals Used in Crop Protection. 7th edition. Publication 1093.

Tamura H, Maness SC, Reischmann K, et al: Androgen receptor antagonism by the organophosphate insecticide fenitrothion. *Toxicol Sci* 60:56-62, 2001. Taneda S, Kamata K, Hayashi H, et al: Investigation of vasodilatory substances in diesel

Trottier B, Fraser AR, Planet G, et al: Subacute toxicity of technical fenitrothion in male rats.

U.S. Environmental Protection Agency: Guidance for the Reregistration of Pesticide

U.S. Environmental Protection Agency: Reregistration Eligibility Decision (RED) for

Yuan C, Wang C, Gao SQ, et al: Effects of permethrin, cypermethrin and 3-phenoxybenzoic

Zhang SY, Ito Y, Yamanoshita O, et al: Permethrin may disrupt testosterone biosynthesis via

Zhang SY, Ueyama J, Ito Y, et al: Permethrin may induce adult male mouse reproductive toxicity due to *cis* isomer not *trans* isomer. *Toxicology* 248:136-141, 2008.

acid on rat sperm motility in vitro evaluated with computer-assisted sperm

mitochondrial membrane damage of Leydig cells in adult male mouse.

Registration Div., Washington, DC. 132 pp. July, 1987.

Published by The British Crop Protection Council, 1987.

analysis. *Toxicol In Vitro* 24:382-386, 2010.

*Endocrinology* 148:3941-3949, 2007.

World Health Organization, Division of Vector Biological Control; 1978. Scollon EJ, Starr JM, Godin SJ, et al: In vitro metabolism of pyrethroid pesticides by rat and

*Ecotoxicol Environ Saf* 32:154-158, 1995.

*Research Branch. Agriculture Canada.* 1981.

*Health Sci* 50:133-141, 2004.

*Toxicology* 17:29-38, 1980.

37:221-228, 2009.

1977.

commercially formulated products containing methyl parathion and permethrin.

Against *Anopheles gambiae* and *Anopheles funestus* in a Village Trial in Nigeria.

human hepatic microsomes and cytochrome P450 isoforms. *Drug Metab Dispos* 

oxidation by mouse liver microsomal enzymes. *Pestic Biochem Physiol* 7:391-401,

exhaust particles (DEP): isolation and identification of nitrophenol derivatives. *J* 

Products Containing Fenitrothion. US EPA, Office of Pesticide Programs,

Permethrin. US EPA, Office of Prevention, Pesticides and Toxic Substance, Office of Pesticide Programs, U.S. Government Printing Office: Washington, DC, 2007. WHO: Environmental Health Criteria 133: Fenitrothion. World Health Organization, International Programme on Chemical Safety, Geneva, Switzerland, 1992. WHO: Environmental Health Criteria 94: Permethrin. World Health Organization, International Programme on Chemical Safety, Geneva, Switzerland, 1990. WHO: The WHO recommended classification of pesticides by hazard and Guidelines to classification 1990-1991, Geneva, World Health Organization (WHO/PCS/90.1), 1990. Worthing CR, Walker SB: The Pesticide Manual: A World Compendium. 8th edition. DDT (dichlorodiphenyltrichloroethane) was first synthesized in 1874, and its insecticidal properties were discovered in 1939 by Paul Hermann Müller (Stapleton 1998). The U.S. military began using DDT extensively for mosquito control in 1944, particularly in the Pacific, where much of the action of World War II took place in highly malarious areas (Stapleton 1998). In 1955, the World Health Organization (WHO) started a global malaria control program with DDT; by 1958, 75 countries had joined and, at the peak of the campaign, 69,500 tons of pesticides mainly DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl)ethane] were applied to 100 million dwellings each year (Wijeyaratne, 1993). For the control of malaria, houses were sprayed twice a year with DDT wettable powder to kill resting mature Anopheles mosquito. Later, the Stockholm Convention on Persistent Organic Pollutants, which came into force on 17 May 2004, outlawed the use of 12 chemicals including DDT (UNEP, 2004). However, one exemption clause allows malaria-endemic nations to use DDT, strictly for disease vector control. The United Nations Environment Program estimates that about 25 countries will use DDT under exemptions from the DDT pesticide ban (POPs, 2009). Thus, in regard to presence of DDT around the world can be divided into three scenarios: Sites where DDT is still in use; sites where the presence is due to DDT sprayed several years ago, and sites where the presence of DDT is the result of a long-range transport of the insecticide to areas where it was never used like the Antarctic. In Mesoamerica (Mexico, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, Nicaragua and Panama) DDT was used until the year 2000, Mexico and Nicaragua being the last nations that applied the insecticide in agriculture and for the control of malaria. Table 1 lists the period and the total amount of DDT used in each Mesoamerican country by the malaria control programs. The amount used (approximately 85,000 tons between 1946 and 1999) together with the high environmental persistence of DDT and its metabolites, provide the necessary conditions for DDT to become a contaminant of concern for this region of the world (ISAT, 2002). Taking into account the environmental persistence and the toxicity of DDT, a program for the control of malaria without using insecticides in Mesoamerica was developed between 2004 and 2007, with assistance from the Pan American Health Organization [PAHO; (Chanon et al., 2003; PAHO, 2008)]. The phase-out of DDT in Costa

DDT and Its Metabolites in Mexico 101

the 1980s and 1990s, and the use of the pesticide was gradually reduced. In 1997, the Intergovernmental Forum on Chemical Safety agreed there was sufficient evidence to take

**Country Period of use Total tons** 

México 1957-2000 69,545.00

Nicaragua 1959-1991 2,172.00

Costa Rica 1957-1985 1,387.00

Guatemala 1958-1979 4,790.00

Honduras 1950-1978 2,640.00

El Salvador 1946-1973 4,271.00

Panamá 1967-1971 189.00

The physicochemical properties of DDTs (Table 2) show the extent of their volatility and the high KOW/KOA value shows that they are more likely to partition into environmental sectors which exhibit greater organic phases (biota, soil, and sediment). The concentration of DDTs in the water samples may be limited due to characteristically low water solubility. In other hand, the exposure pathways are the processes by which DDT may be transported from the pollution source to living organisms. In the malaria areas, the source of DDT was the household-spraying of the insecticide. Since the beginning of the control program of malaria, DDT was sprayed on the ceilings and walls, both indoors and outdoors. Therefore, after spraying, indoor dust (or indoor soil in some cases), and the external surface soil in those areas next to the dwellings, were the media first to become contaminated with DDT. From these points, the insecticide could be transported from one medium to another by

Table 1. History of DDT use in Mesoamerica countries (ISAT, 2002).

**3. Environmental pathways of exposure to DDT** 

different processes.

international action to restrict and reduce the use of DDT.

Rica, El Salvador, Guatemala, Honduras, Mexico, Nicaragua and Panama was part of a regional proposal supported by the Global Environment Facility (GEF) and the United Nations Environmental Program with the participation of the North American Commission for Environmental Cooperation (CEC).

## **2. DDT in Mexico**

In 1944, and for the first time, houses were sprayed with DDT in Temixco, Morelos, Mexico (Stapleton 1998). The spray was applied to the walls and ceilings of residences. Studies done two months after the spraying, showed that there was a 99% reduction in the incidence of Anopheles (Stapleton 1998). In 1947–48, the spraying of DDT began in other Mexican regions, such as Veracruz, Mexico City and Baja California (Stapleton 1998). By 1948, the first clear evidence of malaria control appeared in the areas first sprayed with DDT; the overall parasite rate in the state of Morelos was found to be 10%, and the rate in the sprayed towns was found to be 1% (Stapleton 1998). In 1936 it was estimated that half of the Mexican population lived in endemic regions and was subject to a malaria mortality rate of 0.5%, or about 36,000 deaths per year (Stapleton 1998).During the 1930s and 1940s, malaria became the third cause of death in the country. However, the antimalaria campaign was not generalized until 1956 (CCE 1998). The success of DDT was outstanding, malaria cases decreased from 41,000 in 1955 to 4,000 in 1960 (Fernández de Castro 1998); in 1970 the campaign was relaxed and the cases increased to 57,000 (Fernández de Castro 1998). However, this was also the time in which DDT production peaked in Mexico, with more than 80 thousand tonnes produced annually (CCE 1998). In recent years, the incidences of malaria have declined significantly, to less than 5,000 cases. Since 1982 there have been no deaths from this disease. As, showed, Malaria is a longstanding public health problem that has inhibited development in large areas of the country. Approximately 60% of the Mexican territory, representing an area inhabited by close to 45 million people, provides an environment suitable for malaria transmission. This includes the Pacific coast, the Gulf of Mexico slopes, the Yucatan peninsula and interior basins of the high plateau. (CCE 1998). In actuality, Mexico operates a malaria control program that has substantially reduced the incidence of this disease. In 1995, Mexico initiated an integrated pest management approach for malaria to reduce the heavy dependence on pesticides. Much of the success of Mexico's malaria control program (there have been no recorded deaths from malaria since 1982) is due to improvements in sanitation, increased disease surveillance, and integrated pest management schemes that focus pesticide applications on critical habitats and stages in the mosquito's life cycle (Government of Mexico 1998). Since 1998, DDT was substituted with pyrethroids in the malaria control program. In other hand, In the area of agriculture, as much as 1,000 tonnes per year were used (CCE 1998).Application rates in the north of Mexico, were among the highest in the world (CCE 1998). However, the growing concern about DDT persistence has had a significant impact on agricultural practices in Mexico. During the early 1970s the US Food and Drug Administration (USFDA) began rejecting the importation of commodities due to high residue levels, especially of DDT (CCE 1998). Therefore, some agricultural areas changed to newer pesticides in order to comply with the USFDA regulations. By 1990, DDT was limited to campaigns addressing public sanitation (CCE 1998). In recognition of DDT's environmental and human health effects, Mexico shifted the emphasis of its anti-malarial campaigns away from DDT beginning in

Rica, El Salvador, Guatemala, Honduras, Mexico, Nicaragua and Panama was part of a regional proposal supported by the Global Environment Facility (GEF) and the United Nations Environmental Program with the participation of the North American Commission

In 1944, and for the first time, houses were sprayed with DDT in Temixco, Morelos, Mexico (Stapleton 1998). The spray was applied to the walls and ceilings of residences. Studies done two months after the spraying, showed that there was a 99% reduction in the incidence of Anopheles (Stapleton 1998). In 1947–48, the spraying of DDT began in other Mexican regions, such as Veracruz, Mexico City and Baja California (Stapleton 1998). By 1948, the first clear evidence of malaria control appeared in the areas first sprayed with DDT; the overall parasite rate in the state of Morelos was found to be 10%, and the rate in the sprayed towns was found to be 1% (Stapleton 1998). In 1936 it was estimated that half of the Mexican population lived in endemic regions and was subject to a malaria mortality rate of 0.5%, or about 36,000 deaths per year (Stapleton 1998).During the 1930s and 1940s, malaria became the third cause of death in the country. However, the antimalaria campaign was not generalized until 1956 (CCE 1998). The success of DDT was outstanding, malaria cases decreased from 41,000 in 1955 to 4,000 in 1960 (Fernández de Castro 1998); in 1970 the campaign was relaxed and the cases increased to 57,000 (Fernández de Castro 1998). However, this was also the time in which DDT production peaked in Mexico, with more than 80 thousand tonnes produced annually (CCE 1998). In recent years, the incidences of malaria have declined significantly, to less than 5,000 cases. Since 1982 there have been no deaths from this disease. As, showed, Malaria is a longstanding public health problem that has inhibited development in large areas of the country. Approximately 60% of the Mexican territory, representing an area inhabited by close to 45 million people, provides an environment suitable for malaria transmission. This includes the Pacific coast, the Gulf of Mexico slopes, the Yucatan peninsula and interior basins of the high plateau. (CCE 1998). In actuality, Mexico operates a malaria control program that has substantially reduced the incidence of this disease. In 1995, Mexico initiated an integrated pest management approach for malaria to reduce the heavy dependence on pesticides. Much of the success of Mexico's malaria control program (there have been no recorded deaths from malaria since 1982) is due to improvements in sanitation, increased disease surveillance, and integrated pest management schemes that focus pesticide applications on critical habitats and stages in the mosquito's life cycle (Government of Mexico 1998). Since 1998, DDT was substituted with pyrethroids in the malaria control program. In other hand, In the area of agriculture, as much as 1,000 tonnes per year were used (CCE 1998).Application rates in the north of Mexico, were among the highest in the world (CCE 1998). However, the growing concern about DDT persistence has had a significant impact on agricultural practices in Mexico. During the early 1970s the US Food and Drug Administration (USFDA) began rejecting the importation of commodities due to high residue levels, especially of DDT (CCE 1998). Therefore, some agricultural areas changed to newer pesticides in order to comply with the USFDA regulations. By 1990, DDT was limited to campaigns addressing public sanitation (CCE 1998). In recognition of DDT's environmental and human health effects, Mexico shifted the emphasis of its anti-malarial campaigns away from DDT beginning in

for Environmental Cooperation (CEC).

**2. DDT in Mexico** 

the 1980s and 1990s, and the use of the pesticide was gradually reduced. In 1997, the Intergovernmental Forum on Chemical Safety agreed there was sufficient evidence to take international action to restrict and reduce the use of DDT.


Table 1. History of DDT use in Mesoamerica countries (ISAT, 2002).

## **3. Environmental pathways of exposure to DDT**

The physicochemical properties of DDTs (Table 2) show the extent of their volatility and the high KOW/KOA value shows that they are more likely to partition into environmental sectors which exhibit greater organic phases (biota, soil, and sediment). The concentration of DDTs in the water samples may be limited due to characteristically low water solubility. In other hand, the exposure pathways are the processes by which DDT may be transported from the pollution source to living organisms. In the malaria areas, the source of DDT was the household-spraying of the insecticide. Since the beginning of the control program of malaria, DDT was sprayed on the ceilings and walls, both indoors and outdoors. Therefore, after spraying, indoor dust (or indoor soil in some cases), and the external surface soil in those areas next to the dwellings, were the media first to become contaminated with DDT. From these points, the insecticide could be transported from one medium to another by different processes.

DDT and Its Metabolites in Mexico 103

located in tropical areas, DDTs were quantified in a relatively small stream. The levels of total DDT found in the tropical area was 280 pg/L (Carvalho et al. 2009). In other hand, levels of total DDT found in the tropical area in United states of America was 10300 pg/L

As stated above, sediments act as the primary reservoir for excess quantities of DDT. Therefore, it is very important to analyze the concentrations in this medium. In Table 6 it is shown that DDT concentrations in Mexican samples are lower than those detected in other countries, where DDT was used either for the control of malaria or for agricultural practices. Whether this difference can be explained by an increased degradation or by a DDT mass reduction caused by water currents carrying suspended DDTs out of the contaminated area, are issues that deserve further research. However, we cannot exclude another explanation. The Mexican studies, results of which are shown in Table 6, were not designed to assess the amount of DDT in sediments due to spraying. In fact, a sediment sample collected in a river near an area where the insecticide was used intensively for vector control, had DDT concentrations of up to 70.0 mg/kg (Gonzalez-Mille et al. 2010).

**(mg/Kg) Region Reference** 

al. 2011

2011

2011

al. 2010

2008

Chiapas 0.95 Southeastern Martínez-Salinas et

Chiapas 8.20 Southeastern Yañez et al. 2002

Oaxaca 0.90 Southeastern Yañez et al. 2002

Tabasco 0.04 Southeastern Torres-Dosal et al.

Chihuahua 0.45 North Díaz-Barriga et al.

Veracruz 0.01 Southeastern Espinosa-Reyes et

Puebla and Mexico 0.07 Central Waliszewski et al.

Table 3. Total DDT levels in outdoor surface soil (mg/Kg) in different Mexican Regions.

(California 1999)

Discvutir disminución.

**Location Total DDT** 

**Sediments** 


Table 2. Physicochemical properties of DDT and its metabolites at 250 C. (Sahsuvar et al. 2003; Shen and Wania 2005).

#### **Soil and Dust**

Several studies have identified indoor house dust as an important pathway of toxicant exposure. Often levels of pollutants found in house dust, including compounds banned long ago such as DDT, are significant sources of exposure for the general population, especially children (Butte and Heinzow 2002; Hwang et al. 2008; Rudel et al. 2003). Moreover, analyses of compounds in house dust are a measure of indoor contamination, but may also provide valuable information on the assessment of human indoor exposure (Butte and Heinzow 2002). Also, outdoor soil is considered an important exposure pathway for the general population and children to compounds banned long ago (Herrera-Portugal et al. 2005). However, it is important to note that longer residence times and elevated contaminant concentrations in the indoor environment may increase the chance of exposure to these contaminants by 1,000-fold compared to outdoor exposure (Hwang et al. 2008).

Tables 3 and 4 show DDT levels in outdoor and indoor surface soils, respectively. Taking into account the guideline for DDT in residential soil: 0.7 mg/kg from Canada (Environment Canada, 2007) different scenarios have been observed in Mexico. Regarding outdoor levels, in general lower levels were found in household outdoor samples (Table 3). With exception of levels found in Chiapas and Oaxaca, that have levels lower than Canadian guide (Table 3). In other hand, high levels are recorded in indoor levels in different regions of Mexico, generally higher than Canadian guideline (Table 4). Also, we can note that the higher levels of DDT in those environment media were found in indoor dust samples, generally with levels above the Canadian guideline (Table 5). Moreover, the data in Tables 3, 4 and 5 indicate high levels of total DDT in soil and dust in all regions studied in Mexico when compared with studies around the world.

#### **Water**

DDT, DDD and DDE (DDTs) are only slightly soluble in water, with solubilities of 3.4 ppb, 160 ppb and 120 ppb, respectively (ATSDR 2010). In this regard, sedimentation is the most important factor for the disappearance of DDT from water. However, it has also been suggested that contaminated sediments are a main source of DDT inputs to the water column (Zeng 1999). In order to study the degree of pollution in water bodies located in tropical areas, DDTs were quantified in a relatively small stream. The levels of total DDT found in the tropical area was 280 pg/L (Carvalho et al. 2009). In other hand, levels of total DDT found in the tropical area in United states of America was 10300 pg/L (California 1999)

## **Sediments**

102 Insecticides – Pest Engineering

p´p-DDT 354.5 0.00048 0.00042 1.1 6.39 9.73

p´p-DDE 319.0 0.00340 0.00079 4.2 6.93 9.70

p´p-DDD 321.0 0.00120 0.00230 0.5 6.33 10.03

Several studies have identified indoor house dust as an important pathway of toxicant exposure. Often levels of pollutants found in house dust, including compounds banned long ago such as DDT, are significant sources of exposure for the general population, especially children (Butte and Heinzow 2002; Hwang et al. 2008; Rudel et al. 2003). Moreover, analyses of compounds in house dust are a measure of indoor contamination, but may also provide valuable information on the assessment of human indoor exposure (Butte and Heinzow 2002). Also, outdoor soil is considered an important exposure pathway for the general population and children to compounds banned long ago (Herrera-Portugal et al. 2005). However, it is important to note that longer residence times and elevated contaminant concentrations in the indoor environment may increase the chance of exposure to these

Tables 3 and 4 show DDT levels in outdoor and indoor surface soils, respectively. Taking into account the guideline for DDT in residential soil: 0.7 mg/kg from Canada (Environment Canada, 2007) different scenarios have been observed in Mexico. Regarding outdoor levels, in general lower levels were found in household outdoor samples (Table 3). With exception of levels found in Chiapas and Oaxaca, that have levels lower than Canadian guide (Table 3). In other hand, high levels are recorded in indoor levels in different regions of Mexico, generally higher than Canadian guideline (Table 4). Also, we can note that the higher levels of DDT in those environment media were found in indoor dust samples, generally with levels above the Canadian guideline (Table 5). Moreover, the data in Tables 3, 4 and 5 indicate high levels of total DDT in soil and dust in all regions studied in Mexico when

DDT, DDD and DDE (DDTs) are only slightly soluble in water, with solubilities of 3.4 ppb, 160 ppb and 120 ppb, respectively (ATSDR 2010). In this regard, sedimentation is the most important factor for the disappearance of DDT from water. However, it has also been suggested that contaminated sediments are a main source of DDT inputs to the water column (Zeng 1999). In order to study the degree of pollution in water bodies

Table 2. Physicochemical properties of DDT and its metabolites at 250 C. (Sahsuvar et al.

contaminants by 1,000-fold compared to outdoor exposure (Hwang et al. 2008).

Aqueous solubility (mol/m3)

Henry´s law constant (Pa m3/mol)

Log

Kow\* Log Koa

Vapor pressure (Pa)

Compound

2003; Shen and Wania 2005).

compared with studies around the world.

**Soil and Dust** 

**Water** 

Molecular weight

> As stated above, sediments act as the primary reservoir for excess quantities of DDT. Therefore, it is very important to analyze the concentrations in this medium. In Table 6 it is shown that DDT concentrations in Mexican samples are lower than those detected in other countries, where DDT was used either for the control of malaria or for agricultural practices. Whether this difference can be explained by an increased degradation or by a DDT mass reduction caused by water currents carrying suspended DDTs out of the contaminated area, are issues that deserve further research. However, we cannot exclude another explanation. The Mexican studies, results of which are shown in Table 6, were not designed to assess the amount of DDT in sediments due to spraying. In fact, a sediment sample collected in a river near an area where the insecticide was used intensively for vector control, had DDT concentrations of up to 70.0 mg/kg (Gonzalez-Mille et al. 2010). Discvutir disminución.


Table 3. Total DDT levels in outdoor surface soil (mg/Kg) in different Mexican Regions.

DDT and Its Metabolites in Mexico 105

**Location Total DDT (µg/Kg) Region Reference** 

(bay) 1.5 Southeastern Noreña-Barroso et

Mexico (bay) 0.6 Southeastern Noreña-Barroso et

Mexico (river) 74.0 Southeastern Gonzalez-Mille et al.

Mexico (lagoon) 4.9 Southeastern Botello et al. 2000

China (Bay) 7.8 Liu et al. 2011

China (river) 3.8 Tan et al. 2009

Korea (bay) 3.4 Khim et al. 2001

Japan (bay) 1.2 Kim et al. 2007

Because DDTs have a Henry's Law constant value of 10–4/10–5 atm m3 mol, they are considered moderate volatile compounds [5]. Therefore, these compounds can be transported by air, either in the gaseous phase or adsorbed to atmospheric particles [5].Photodegradation of DDT occurs slowly; thus, residues of these pesticides are ubiquitous in the atmosphere, although at lower concentrations. Information on the atmospheric levels of OCs in Mexico is scarce. Previous studies in southern Mexico found that DDT and toxaphene concentrations in air were 1-2 orders of magnitude above levels in the Laurentian Great Lakes and arctic regions (24-26). Atmospheric levels in southern Mexico were generally higher than those in central Mexico (27), Costa Rica (28, 29) and Cuba (30), and

Recently, two important studies regarding DDT and its metabolites in air has been developed. Passive air samplers (PAS) were deployed at four sampling sites at the southern Mexico in 2002-2004 and eleven sampling sites across Mexico during 2005-2006 (referencia). The total DDT levels ranged from 239 to 2360 pg/m3 in 2002-2004 (referencia) and from 15 to 1975 pg/m3 in 2005-2006 (refernecia). Table ? shows the Total DDT air levels in the sampling sites in both studies. Total DDT tended to be higher in the south (poner siglas) and

Table 6. Total DDT levels in sediment (mg/Kg) in different Mexican Regions.

al. 1998

al. 2007

2010

Estado, Mexico

**Air** 

comparable to those in Belize (31).


Table 4. Total DDT levels in indoor soil (mg/Kg) in different Mexican Regions.

#### **Food and Biota**

Due to their lipophilic attributes and high persistence, the DDTs may bioaccumulate significantly in animal species (Fisher 1995). Furthermore, biomagnification has been observed; for example, DDT concentration increased with each successive trophic level in a food chain (Fisher 1995).Taking into account these properties, food ingestion can be considered a pathway of exposure. In Mexico, studies have been done in different food items, such as fish, hen's egg, butter and cow's milk and muscle. In Table 7, total DDT levels in different food items are presented. Considering fish, the concentrations of DDT, in organisms collected in Mexico, are above normal values. As is shown in Table 8, where DDTs levels in Fish are depicted for different countries. We can note that the food item with high levels of DDT are food rich in fat as butter and cow´s milk (Table 7).


Table 5. Total DDT levels in dust (mg/Kg) in different Mexican Regions.

Chiapas 6.8 Southeastern Martínez-Salinas

Chiapas 7.1 Southeastern Yañez et al. 2002

Oaxaca 0.15 Southeastern Yañez et al. 2002

Chihuahua 0.95 North Díaz-Barriga et al.

Due to their lipophilic attributes and high persistence, the DDTs may bioaccumulate significantly in animal species (Fisher 1995). Furthermore, biomagnification has been observed; for example, DDT concentration increased with each successive trophic level in a food chain (Fisher 1995).Taking into account these properties, food ingestion can be considered a pathway of exposure. In Mexico, studies have been done in different food items, such as fish, hen's egg, butter and cow's milk and muscle. In Table 7, total DDT levels in different food items are presented. Considering fish, the concentrations of DDT, in organisms collected in Mexico, are above normal values. As is shown in Table 8, where DDTs levels in Fish are depicted for different countries. We can note that the food item with

Table 4. Total DDT levels in indoor soil (mg/Kg) in different Mexican Regions.

high levels of DDT are food rich in fat as butter and cow´s milk (Table 7).

Table 5. Total DDT levels in dust (mg/Kg) in different Mexican Regions.

**Location Total DDT** 

**(mg/Kg) Region Reference** 

**(mg/Kg) Region Reference** 

Chiapas 6.9 Southeastern Martínez-Salinas

Chihuahua 1.0 North Díaz-Barriga et al.

et al. 2011

2011

et al. 2011

2011

**Mexican state Total DDT** 

**Food and Biota** 


Table 6. Total DDT levels in sediment (mg/Kg) in different Mexican Regions.

#### **Air**

Because DDTs have a Henry's Law constant value of 10–4/10–5 atm m3 mol, they are considered moderate volatile compounds [5]. Therefore, these compounds can be transported by air, either in the gaseous phase or adsorbed to atmospheric particles [5].Photodegradation of DDT occurs slowly; thus, residues of these pesticides are ubiquitous in the atmosphere, although at lower concentrations. Information on the atmospheric levels of OCs in Mexico is scarce. Previous studies in southern Mexico found that DDT and toxaphene concentrations in air were 1-2 orders of magnitude above levels in the Laurentian Great Lakes and arctic regions (24-26). Atmospheric levels in southern Mexico were generally higher than those in central Mexico (27), Costa Rica (28, 29) and Cuba (30), and comparable to those in Belize (31).

Recently, two important studies regarding DDT and its metabolites in air has been developed. Passive air samplers (PAS) were deployed at four sampling sites at the southern Mexico in 2002-2004 and eleven sampling sites across Mexico during 2005-2006 (referencia). The total DDT levels ranged from 239 to 2360 pg/m3 in 2002-2004 (referencia) and from 15 to 1975 pg/m3 in 2005-2006 (refernecia). Table ? shows the Total DDT air levels in the sampling sites in both studies. Total DDT tended to be higher in the south (poner siglas) and

DDT and Its Metabolites in Mexico 107

Veracruz, Mexico 25.0 Southeastern Gonzalez-Mille et al.

Chiapas, Mexico 4.7 Southeastern Pérez-Maldonado et

Hidalgo, Mexico 7.3 Central Fernandez-Bringas et

Costa Rica 0.6 Pérez-Maldonado et

Nicaragua 3.9 Pérez-Maldonado et

El Salvador 3.8 Pérez-Maldonado et

Guatemala < LOD Pérez-Maldonado et

Baja California 338 North Wong et al. 2009

Chihuahua 34 North Wong et al. 2009

Yucatan 1975 Southeastern Wong et al. 2009

Colima 750 North Wong et al. 2009

Veracruz 129 Southeastern Wong et al. 2009

**(pg/m3) Region Reference** 

**lipid) Region Reference** 

2010

al. 2010

al. 2008

al. 2010

Pérez-Maldonado et al. 2010

al. 2010

al. 2010

al. 2010

**Location Total DDT (ng/g** 

Honduras < LOD (below

**Community Total DDT** 

detection limit)

Table 8. Total DDT levels in fish (mg/Kg) in different Mexican Regions.

some central sites (sigles). While, the other central and northern sites had lower total DDT levels (poner siglas). It is important to note that the higher levels were found in tropical sites where DDT was used for health campagnes or for agriculture as MT, CEL and VC (Table ?).


Table 7. Total DDT levels in food items (mg/Kg) in different Mexican Regions.

some central sites (sigles). While, the other central and northern sites had lower total DDT levels (poner siglas). It is important to note that the higher levels were found in tropical sites where DDT was used for health campagnes or for agriculture as MT, CEL and VC (Table ?).

Mexico country Butter 88.0 Waliszewski et

Veracruz Cow´s milk 39.0 Southeastern Pardio et al. 2003

Campeche Oysters 5.9 Southeastern Carvalho et al.

Campeche Oysters 1.5 Southeastern Gold-Bouchot et

Tabasco Oysters 6.2 Southeastern Botello et al. 1994

Campeche Shrimps 0.25 Southeastern Gold-Bouchot et

Campeche Mussels 1.44 Southeastern Gold-Bouchot et

Baja California Mussels 9.16 North Gutierrez-

Table 7. Total DDT levels in food items (mg/Kg) in different Mexican Regions.

**(mg/Kg) Region Reference** 

al. 2003

2009

al. 1995

al. 1995

al. 1995

Galindo et al. 1988

**Location Food Total DDT** 


Table 8. Total DDT levels in fish (mg/Kg) in different Mexican Regions.


DDT and Its Metabolites in Mexico 109

In other hand, a biomarker of exposure is a xenobiotic substance or its metabolite (s), that is measured within a compartment of an organism. The preferred biomarkers of exposure are generally the substance itself or substance-specific metabolites in readily obtainable body fluid(s) or excreta. DDT and its metabolites DDD,DDE, DDA, and MeSO2-DDE (3 methylsulphonyl-DDE), can be measured in adipose tissue, blood serum, urine, feces,

Psychological and medical studies have underlined the benefits of nursing which raises immunological defenses and provides a healthier development of the baby. Parallel findings have increased concern about the excretion of drugs and environmental contaminants contained in breast milk, since it is considered the main route for eliminating deposited organochlorine pesticides from a mother's body (Jensen and Slorach 1991; Sonawane 1995;

Because of their lipophilic nature and high persistence, DDT and its metabolites accumulate in lipophilic human body parts, particularly in lipid-rich tissues such as adipose tissue and subsequently translocated and excreted through milk fat. A major concern is that milk is the

Concentrations of DDTs (DDT, DDD and DDE) in human milk have been shown to be higher in communities exposed to this insecticide, than in non-exposed populations (Table 10). For example, levels from a cotton area where DDT was used for agricultural purposes (Coahuila) were higher or similar to those obtained in samples collected in a malarious area (Veracruz, Yucatan) where DDT was extensively used (Table 10). And both were higher than the concentrations quantified in urban areas (Mexico DF), where DDT has never been

The World Health Organization's Acceptable Daily Intake (ADI) for DDTs is 20 mg/kg/day (Lu 1995). In this regard, considering a body mass of 5 kg, a milk intake of 0.85 kg/day, a proportion of fat in milk of 0.035, and a DDT concentration in milk of 10.4 mg/kg (total DDT concentration in samples collected during 1996 1997 in a suburban malarious area; Albert et al. 1980), the estimated daily intake is three times higher than the ADI. Furthermore, if the maximum range concentration found in some studies (36.5 mg/kg; Albert et al. 1980) is taken into account, the ADI is surpassed 11 times. However, when calculated the ADI with DDT concentration in milk of 2.4 mg/kg (Waliszewski et al. 2009), the ADI calculated is lower than 20 mg/kg/day. It is important to remark that the chronological levels obtained by Waliszewski et al. (1996, 1999, 2001, 2002, 2009) have a decreasing tendency. That result

In this document, we presented data regarding DDT and its metabolites levels in children

Children appear to be particularly suitable for a monitoring program, as they are not directly exposed to occupational pollution; thus, children normally reflect present trends of environmental exposure more accurately than do adults (Link et al., 2005). Moreover, it is well established that children are potentially at a higher risk than adults for adverse health effects from exposure to many environmental chemicals (Guzelian et al., 1992; Bearer, 1995; Carlson, 1998; Galson et al., 1998; Aprea et al., 2000; Needham and Sexton, 2000; Adgate and

first (and in some areas the only) food for the newborn child.

coincides with the restriction and prohibition for DDT use in Mexico.

semen, or breast milk .

Cupul-Uicab et al. 2008).

**Breast Milk** 

used.

**Serum** 

(Table 11) and adults (Table 12)

Sexton, 2001; Brent et al., 2004; IPCS, 2006).


Table 9. Total DDT levels in air (pg/m3) in different Mexican Regions.

## **4. Human exposure to DDT**

Biomonitoring studies are a useful instrument in formulating environmental health policies. For example, in Mexico during the last decade, studies in children led to the reduction or elimination of different chemicals such as lead, lindane and inclusive DDT. Furthermore, the biomonitoring of susceptible populations is a valuable method for the identification of critical contaminants, as has been shown in the United States with the National Health and Nutritional Examination Survey (NHANES III; Needham et al., 2005). Information about human exposure to chemicals is very limited; besides, in relation to children, the information is even more scarce. In this regard, in this text, we shall present data for breast milk, blood serum and adipose tisssue. Results have been done in adults and children.

In other hand, a biomarker of exposure is a xenobiotic substance or its metabolite (s), that is measured within a compartment of an organism. The preferred biomarkers of exposure are generally the substance itself or substance-specific metabolites in readily obtainable body fluid(s) or excreta. DDT and its metabolites DDD,DDE, DDA, and MeSO2-DDE (3 methylsulphonyl-DDE), can be measured in adipose tissue, blood serum, urine, feces, semen, or breast milk .

## **Breast Milk**

108 Insecticides – Pest Engineering

Morelos 500 Central Wong et al. 2009

Sinaloa 76 North Wong et al. 2009

Mexico DF 55 Central Wong et al. 2009

Nuevo Leon 15 North Wong et al. 2009

San Luis Potosi 21 Central Wong et al. 2009

Veracruz 50 Southeastern Wong et al. 2009

Tabasco 239 Southeastern Alegría et al. 2008

Chiapas 2360 Southeastern Alegría et al. 2008

Chiapas 547 Southeastern Alegría et al. 2008

Veracruz 1200 Southeastern Alegría et al. 2008

Biomonitoring studies are a useful instrument in formulating environmental health policies. For example, in Mexico during the last decade, studies in children led to the reduction or elimination of different chemicals such as lead, lindane and inclusive DDT. Furthermore, the biomonitoring of susceptible populations is a valuable method for the identification of critical contaminants, as has been shown in the United States with the National Health and Nutritional Examination Survey (NHANES III; Needham et al., 2005). Information about human exposure to chemicals is very limited; besides, in relation to children, the information is even more scarce. In this regard, in this text, we shall present data for breast milk, blood serum and adipose tisssue. Results have been done in adults and children.

Table 9. Total DDT levels in air (pg/m3) in different Mexican Regions.

**4. Human exposure to DDT** 

**(pg/m3) Region Reference** 

**Community Total DDT** 

Psychological and medical studies have underlined the benefits of nursing which raises immunological defenses and provides a healthier development of the baby. Parallel findings have increased concern about the excretion of drugs and environmental contaminants contained in breast milk, since it is considered the main route for eliminating deposited organochlorine pesticides from a mother's body (Jensen and Slorach 1991; Sonawane 1995; Cupul-Uicab et al. 2008).

Because of their lipophilic nature and high persistence, DDT and its metabolites accumulate in lipophilic human body parts, particularly in lipid-rich tissues such as adipose tissue and subsequently translocated and excreted through milk fat. A major concern is that milk is the first (and in some areas the only) food for the newborn child.

Concentrations of DDTs (DDT, DDD and DDE) in human milk have been shown to be higher in communities exposed to this insecticide, than in non-exposed populations (Table 10). For example, levels from a cotton area where DDT was used for agricultural purposes (Coahuila) were higher or similar to those obtained in samples collected in a malarious area (Veracruz, Yucatan) where DDT was extensively used (Table 10). And both were higher than the concentrations quantified in urban areas (Mexico DF), where DDT has never been used.

The World Health Organization's Acceptable Daily Intake (ADI) for DDTs is 20 mg/kg/day (Lu 1995). In this regard, considering a body mass of 5 kg, a milk intake of 0.85 kg/day, a proportion of fat in milk of 0.035, and a DDT concentration in milk of 10.4 mg/kg (total DDT concentration in samples collected during 1996 1997 in a suburban malarious area; Albert et al. 1980), the estimated daily intake is three times higher than the ADI. Furthermore, if the maximum range concentration found in some studies (36.5 mg/kg; Albert et al. 1980) is taken into account, the ADI is surpassed 11 times. However, when calculated the ADI with DDT concentration in milk of 2.4 mg/kg (Waliszewski et al. 2009), the ADI calculated is lower than 20 mg/kg/day. It is important to remark that the chronological levels obtained by Waliszewski et al. (1996, 1999, 2001, 2002, 2009) have a decreasing tendency. That result coincides with the restriction and prohibition for DDT use in Mexico.

#### **Serum**

In this document, we presented data regarding DDT and its metabolites levels in children (Table 11) and adults (Table 12)

Children appear to be particularly suitable for a monitoring program, as they are not directly exposed to occupational pollution; thus, children normally reflect present trends of environmental exposure more accurately than do adults (Link et al., 2005). Moreover, it is well established that children are potentially at a higher risk than adults for adverse health effects from exposure to many environmental chemicals (Guzelian et al., 1992; Bearer, 1995; Carlson, 1998; Galson et al., 1998; Aprea et al., 2000; Needham and Sexton, 2000; Adgate and Sexton, 2001; Brent et al., 2004; IPCS, 2006).

DDT and Its Metabolites in Mexico 111

p,p′-DDE conncentrations in adipose tissue. The index of chronic exposure was constructed according to worker position and based on the historical duration and intensity of DDT

When the concentrations of DDTs in adipose tissue were expressed by age group, two groups were identified as the most exposed. Those groups were children and elderly people [49]. The levels in elderly people can be explained by the accumulation of DDT in a chronic exposure scenario,whereas the concentration in children may be the result of an exposure to multiple pathways (soil, household dust, air, water, food, etc.). It is interesting that the group less exposed to DDT was the 0–2 years, a group that may be exposed to DDT through

Chiapas 22280.0 Southeastern Trejo-Acevedo et al.

Oaxaca 7500.00 Southeastern Perez-Maldonado et al.

Quintana Roo 11300.0 Southeastern Perez-Maldonado et al.

Chihuahua 35000.00 North Díaz-Barriga et al. 2011

Queretaro 2170.0 Central Trejo-Acevedo et al.

Durango 2270.0 North Trejo-Acevedo et al.

San Luis Potosí 1990.0 Central Trejo-Acevedo et al.

Guanajuato 940.0 Central Trejo-Acevedo et al.

Veracruz 1910.0 Southeastern Trejo-Acevedo et al.

Michoacan 550.0 Central Trejo-Acevedo et al.

Zacatecas 700.0 Central Trejo-Acevedo et al.

Table 11. Total DDT levels in serum (ng/g lipid) of children living in different Mexican

**lipid) Region Reference** 

2009

2006

2006

2009

2009

2009

2009

2009

2009

2009

application [48].

lactation [49].

Regions.

**Location Total DDT (ng/g** 


Table 10. Total DDT levels in human milk (ng/g lipid) in different Mexican Regions.

#### **Adipose tissue**

Adipose tissue biopsy has been used in epidemiological studies to assess chronic exposure to DDT. This is a logical choice because the DDTs are accumulated in adipose tissue due to its lipid solubility. The half-life of DDT in human adipose tissue is approximately seven years (Woodruff et al. 1994).

As in serum, DDTs in adipose tissue are a good biomarker of exposure for communities exposed to DDT. When compared to an urban non-exposed community [46], the levels of DDTs (especially those of DDE), were higher in the exposed population (Table 9). In the same table it can be observed that the concentrations of DDT in adipose tissue from workers of the malaria program were higher than the levels found in people living in an agricultural area or in malarious areas. In the workers, a linear model that included an index of chronic exposure, the use of protective gear,and recent weight loss explained 55% of the variation of

**Location Total DDT Region Reference** 

Coahuila 10400.0 North Albert et al. 1980

Mexico DF 900.0 Central Torres-Arreola et al.

Veracruz 7815.0 Southeastern Pardio et al. 1998

Veracruz 6280.0 Southeastern Waliszewski et al.

Veracruz 4700.0 Southeastern Waliszewski et al.

Veracruz 4700.0 Southeastern Waliszewski et al.

Veracruz 3740.0 Southeastern Waliszewski et al.

Morelos 4320.0 Central Lara et al. 2000

Yucatan 3065.0 Southeastern Rodas-.Ortíz et al.

Veracruz 2335.0 Southeastern Waliszewski et al.

Adipose tissue biopsy has been used in epidemiological studies to assess chronic exposure to DDT. This is a logical choice because the DDTs are accumulated in adipose tissue due to its lipid solubility. The half-life of DDT in human adipose tissue is approximately seven

As in serum, DDTs in adipose tissue are a good biomarker of exposure for communities exposed to DDT. When compared to an urban non-exposed community [46], the levels of DDTs (especially those of DDE), were higher in the exposed population (Table 9). In the same table it can be observed that the concentrations of DDT in adipose tissue from workers of the malaria program were higher than the levels found in people living in an agricultural area or in malarious areas. In the workers, a linear model that included an index of chronic exposure, the use of protective gear,and recent weight loss explained 55% of the variation of

Table 10. Total DDT levels in human milk (ng/g lipid) in different Mexican Regions.

**Adipose tissue** 

years (Woodruff et al. 1994).

1999

1996

1999

2001

2002

2008

2009

p,p′-DDE conncentrations in adipose tissue. The index of chronic exposure was constructed according to worker position and based on the historical duration and intensity of DDT application [48].

When the concentrations of DDTs in adipose tissue were expressed by age group, two groups were identified as the most exposed. Those groups were children and elderly people [49]. The levels in elderly people can be explained by the accumulation of DDT in a chronic exposure scenario,whereas the concentration in children may be the result of an exposure to multiple pathways (soil, household dust, air, water, food, etc.). It is interesting that the group less exposed to DDT was the 0–2 years, a group that may be exposed to DDT through lactation [49].


Table 11. Total DDT levels in serum (ng/g lipid) of children living in different Mexican Regions.

DDT and Its Metabolites in Mexico 113

**Location Total DDT Region Reference** 

Coahuila 18400.0 Central Albert et al. 1980

Mexico DF 6100.0 Central Albert et al. 1980

Puebla 2700.0 Central Albert et al. 1980

Veracruz 10000.0 Southeastern Waliszewski et al.

Veracruz 61000.0 Southeastern Rivero-Rodriguez et

Veracruz 5700.0 Southeastern Waliszewski et al.

Veracruz 2600.0 Southeastern Waliszewski et al.

Puebla 800.0 Central Waliszewski et al.

Veracruz 1900.0 Southeastern Waliszewski et al.

Veracruz 1400.0 Southeastern Herrero-Mercado et

Veracruz 900.0 Southeastern Herrero-Mercado et

Although it has been suggested that the estrogenic activity of DDE may be a contributing factor for development of breast cancer in women, levels of these compounds are not consistently elevated in breast cancer patients. It was initially reported that levels of *p,p*- DDE were elevated in breast cancer patients (serum or tissue) versus controls [52].More recent studies and analysis of organochlorine levels in breast cancer patients versus controls

Table 13. Total DDT levels in Adipose Tissue (ng/g lipid) of adults living in different

Mexican Regions.

**5.1 Cancer** 

1996

al. 1997

2001

2010

2010

2011

al. 2010

al. 2011

A monitoring program of DDTs in adipose tissue is needed in order to assess the body burden, now that in Mexico this insecticide has been eliminated from the malaria program. However, due to ethical constraints, it is not always possible to obtain adipose tissue samples form healthy individuals. Therefore, alternative matrices are needed; for example, a good correlation between adipose tissue concentration and levels in human milk [50] or human serum [51] has been reported .When the geometric DDE levels in lipid bases are used for the estimation of the adipose tissue/serum DDE ratio, a value near unity is obtained [51].

## **5. Health effects**

DDT and its metabolites have been associated with neurological effects (Dorner and Plagemann 2002; Fenster et al. 2007; Torres-Sánchez et al. 2007; Rocha-Amador et al. 2009), asthma (Sunyer et al. 2006), immunodeficiency (Dewailly et al. 2000; Vine et al. 2000; Vine et al. 2001; Belles-Isles et al. 2002; Bilrha et al. 2003; Cooper et al. 2004; Dallaireet al. 2004), apoptosis (Pérez-Maldonado et al. 2004) and DNA damage in immune cells in children (Yáñez et al. 2004; Herrera-Portugal et al. 2005b).


Table 12. Total DDT levels in serum (ng/g lipid) of adults living in different Mexican Regions.

A monitoring program of DDTs in adipose tissue is needed in order to assess the body burden, now that in Mexico this insecticide has been eliminated from the malaria program. However, due to ethical constraints, it is not always possible to obtain adipose tissue samples form healthy individuals. Therefore, alternative matrices are needed; for example, a good correlation between adipose tissue concentration and levels in human milk [50] or human serum [51] has been reported .When the geometric DDE levels in lipid bases are used for the estimation of the adipose tissue/serum DDE ratio, a value near unity is

DDT and its metabolites have been associated with neurological effects (Dorner and Plagemann 2002; Fenster et al. 2007; Torres-Sánchez et al. 2007; Rocha-Amador et al. 2009), asthma (Sunyer et al. 2006), immunodeficiency (Dewailly et al. 2000; Vine et al. 2000; Vine et al. 2001; Belles-Isles et al. 2002; Bilrha et al. 2003; Cooper et al. 2004; Dallaireet al. 2004), apoptosis (Pérez-Maldonado et al. 2004) and DNA damage in immune cells in children

Chiapas 12750.0 Southeastern Yañez et al. 2002

Oaxaca 8050.0 Southeastern Yañez et al. 2002

Tabasco 8700.0 Southeastern Torres-Dosal et al.

Mexico, DF 20.0 Central Lopez-Carrillo et al.

Veracruz 4500.00 Southeastern Waliszewski et al.

Morelos 20.0 Central Lopez-Carrillo et al.

San Luis Potosí 1715.0 Central Yañez et al. 2004

Table 12. Total DDT levels in serum (ng/g lipid) of adults living in different Mexican

**lipid) Region Reference** 

2011

1997

2000

2001

obtained [51].

Regions.

**5. Health effects** 

(Yáñez et al. 2004; Herrera-Portugal et al. 2005b).

**Location Total DDT (ng/g** 


Table 13. Total DDT levels in Adipose Tissue (ng/g lipid) of adults living in different Mexican Regions.

#### **5.1 Cancer**

Although it has been suggested that the estrogenic activity of DDE may be a contributing factor for development of breast cancer in women, levels of these compounds are not consistently elevated in breast cancer patients. It was initially reported that levels of *p,p*- DDE were elevated in breast cancer patients (serum or tissue) versus controls [52].More recent studies and analysis of organochlorine levels in breast cancer patients versus controls

DDT and Its Metabolites in Mexico 115

well as levels of LH, FSH, prolactine, and testosterone. Participants ranged in age from 22 to 69 years, and had been employed in the sanitation campaign from 4 to 37 years. Ninetyseven percent of the participants were sprayers of DDT at some time in their occupational history, and 15% are current sprayers. Average levels of DDT and metabolites expressed as μg/g of extractable lipids were: total DDT, 60.1; *p,p*-DDE, 37.41; *p,p*-DDT, 21.52; *p,p*- DDD, 1.07 and *op*-DDT 0.11. Results show a positive association of LH and FSH with DDT metabolites. An increase of 10 *m*g/g of *p,p*-DDE was associated with an increase of 1.95 UI/L in LH (p=0.01); and 1.10 UI/L per each 10 *m*g/g of *p,p*-DDT (p=0.02). FSH increased 1.09 UI/L per each 10 *m*g/g of *p,p*- DDT (p=0.03). There was a negative association of DDE with testosterone, DDT in Mexico 383 especially for those participants under 55 years of age. These associations suggest direct toxicity to the testicles, especially the Leydig cells, as

Some studies have reported genotoxic effects in humans heavily exposed to DDT [8]. Therefore, this area has been studied in Mexico. Studies were done in workers from the control program of malaria, and in women living in malarious areas. Herrera et al. [63] evaluated chromosomal translocations in a sample of the above-mentioned workers.Nineteen male sprayers (median age 46 years; range: 22–64),working in campaigns to control malaria vectors in the state of Guerrero, Mexico were included in this study.DDT data obtained in a previous study from eleven individuals, 5 women and 6 men, living in Mexico City, and occupationally unexposed to DDT were used as reference group. Chromosomal aberrations in lymphocytes were analyzed with a chromosome painting technique, a highsensitivity technique for detecting complex chromosomal translocations. *o,p*- DDT was the only isomer significantly associated with the frequency of chromosomal translocations (p=0.003). Individuals presenting the higher levels of *o,p*-DDT in serum (>0.79 *m*g/g fat; n=4) had a mean frequency of chromosomal translocations (5.1*¥*1000 metaphases), two times higher than that observed in workers occupationally exposed to 0.5 Gy of radiation.A positive relationship between the duration of exposure to DDT, measured as years working for the vector control program, and chromosomal translocations was observed (Fig.2). These results suggest an increased risk for diseases with a genetic

Yañez et al. [64] evaluated the association of blood DDT levels and DNA damage using the single cell gel electrophoresis assay. A group of 53 postpartum women were selected from two different areas in San Luis Potosí to assure different exposure levels, one with antecedents of malaria and DDT spraying and the other without malaria. Mean and range levels of DDT, DDE and DDD in whole blood were 5.57 ppm (0.02–20.69), 6.24 ppm (0.04– 39.16) and 1.16 ppm (0.01–5.63), respectively. The significant correlation of DNA damage, measured as DNA migration with the logarithm of DDT,DDE and DDD was 0.60, 0.62 and 0.43, respectively. Fig. 3 shows the shape of the association of DNA migration with DDE concentration in whole blood, as obtained by the regression: DNA migration= 71.58+7.62(logDDE), this association was not modified by age, smoking habits, nutrition or occupation. This observational finding in the epidemiological study with postpartum women was reevaluated in an in vitro study.Human blood cells were exposed to three doses of DDT, DDD and DDE.DNA damage was assessed by two different techniques: single-cell electrophoresis and flow cytometry. Results obtained by either technique showed that DNA damage was induced by the three organochlorides and a dose-response was observed with

observed with antineoplasic drugs.

**5.3 Genotoxicity** 

component, such as cancer.

show that these contaminants are not elevated in the latter group [53–56]. The study of occupationally exposed workers has not found clear increased risks for other cancers [57]. Two case-control studies of breast cancer have been carried out in Mexico City, with conflicting results. The first study, conducted by Lopez-Carrillo et al. [58] in Mexico City, compared 141 cases of breast cancer with 141 age-matched controls. All subjects were identified at three referral hospitals between March 1994 and April 1996. The arithmetic mean of serum DDE in lipid basis was 562 ppb±676 for the cases and 505 ppb±567 for the controls.The age-adjusted odds ratios for breast cancer regarding the serum level of DDE were 0.69 (95% confidence interval, 0.38–1.24) and 0.97 (CI, 0.55–1.70) for the contrasts between tertile 1 (lowest level) and tertiles 2 and 3, respectively. These estimates were unaffected by adjustment for body mass, accumulated time of breast-feeding and menopause, and other breast cancer risk factors. These results do not lend support to the hypothesis that DDT is causally related to breast cancer. The second study conducted by Romieu et al. [59] compared 120 cases and 126 controls, selected from six hospitals in Mexico City, from 1989 to 1995. Serum DDE levels in lipid basis were higher among cases (mean=3840 ppb±5980) than among controls (mean=2510 ppb±1970 ).After adjusting for age, age at menarche,duration of lactation, Quetelet index, and serum DDT levels, serum DDE levels were positively r elated to t he risk for b reast cancer ( adjusted O RQ1–Q2=1.24), 382 F. Díaz-Barriga et al. (CI, 0.50–3.06; ORQ1 –Q3=2.31, 95 percent, CI, 0.92–5.86; ORQ1 –Q4=3.81, CI, 1.14–12.80). The increased risk associated with higher serum DDE levels was more apparent among postmenopausal women (ORQ1 –Q4=5.26, 95%, CI, 0.80–34.30). Serum DDT level was not related to the risk for breast cancer. In addition to the differences in the comparison of cases and controls, the difference in the serum DDE levels among the women studied is remarkable. Participants from both studies came from similar hospitals, and there were no apparent differences between case and control selection that could explain this divergence. Differences in laboratory procedures is the most feasible explanation.

#### **5.2 Endocrine disruption**

DDT is known to have adverse effects on wildlife via endocrine disruption.Clear effects include thinning of the eggshell, feminization, reproduction impairment and development effects [60]. In Mexico two studies in humans have reported findings in this area. Gladen and Rogan [61] found that DDE might affect women's ability to lactate in a study conducted in an agricultural town in northern Mexico. Two hundred and twenty-nine women were followed from childbirth until weaning or until the child reached 18 months of age.DDE was measured in breast milk samples taken at birth, and women were followed to see how long they lactated.Median duration was 7.5 months in the lowest DDE group and 3 months in the highest. The effect was confined to those who had lactated previously – but not for first pregnancies – and it persisted after statistical adjustment for other factors. Rodriguez et al. [62] conducted a study aimed at determining the capability of long-term exposure to DDT of altering the normal endocrine function of the hypothalamus-hypophysis-gonads axis in humans. This included 70 workers dedicated to control malaria in the State of Guerrero,Mexico. The main activities of these workers were the application of pesticides,detection of malaria cases and promotion of preventive measures for control vectors. The average time of exposure to technical grade DDT was 25 years (range: 4–35), their last exposure being 5 months before sampling. An interview gathered information on the occupational history, reproductive performance, life styles and other relevant factors. Blood and urine samples were collected to measure serum levels of DDT and metabolites as

show that these contaminants are not elevated in the latter group [53–56]. The study of occupationally exposed workers has not found clear increased risks for other cancers [57]. Two case-control studies of breast cancer have been carried out in Mexico City, with conflicting results. The first study, conducted by Lopez-Carrillo et al. [58] in Mexico City, compared 141 cases of breast cancer with 141 age-matched controls. All subjects were identified at three referral hospitals between March 1994 and April 1996. The arithmetic mean of serum DDE in lipid basis was 562 ppb±676 for the cases and 505 ppb±567 for the controls.The age-adjusted odds ratios for breast cancer regarding the serum level of DDE were 0.69 (95% confidence interval, 0.38–1.24) and 0.97 (CI, 0.55–1.70) for the contrasts between tertile 1 (lowest level) and tertiles 2 and 3, respectively. These estimates were unaffected by adjustment for body mass, accumulated time of breast-feeding and menopause, and other breast cancer risk factors. These results do not lend support to the hypothesis that DDT is causally related to breast cancer. The second study conducted by Romieu et al. [59] compared 120 cases and 126 controls, selected from six hospitals in Mexico City, from 1989 to 1995. Serum DDE levels in lipid basis were higher among cases (mean=3840 ppb±5980) than among controls (mean=2510 ppb±1970 ).After adjusting for age, age at menarche,duration of lactation, Quetelet index, and serum DDT levels, serum DDE levels were positively r elated to t he risk for b reast cancer ( adjusted O RQ1–Q2=1.24), 382 F. Díaz-Barriga et al. (CI, 0.50–3.06; ORQ1 –Q3=2.31, 95 percent, CI, 0.92–5.86; ORQ1 –Q4=3.81, CI, 1.14–12.80). The increased risk associated with higher serum DDE levels was more apparent among postmenopausal women (ORQ1 –Q4=5.26, 95%, CI, 0.80–34.30). Serum DDT level was not related to the risk for breast cancer. In addition to the differences in the comparison of cases and controls, the difference in the serum DDE levels among the women studied is remarkable. Participants from both studies came from similar hospitals, and there were no apparent differences between case and control selection that could explain this divergence.

Differences in laboratory procedures is the most feasible explanation.

DDT is known to have adverse effects on wildlife via endocrine disruption.Clear effects include thinning of the eggshell, feminization, reproduction impairment and development effects [60]. In Mexico two studies in humans have reported findings in this area. Gladen and Rogan [61] found that DDE might affect women's ability to lactate in a study conducted in an agricultural town in northern Mexico. Two hundred and twenty-nine women were followed from childbirth until weaning or until the child reached 18 months of age.DDE was measured in breast milk samples taken at birth, and women were followed to see how long they lactated.Median duration was 7.5 months in the lowest DDE group and 3 months in the highest. The effect was confined to those who had lactated previously – but not for first pregnancies – and it persisted after statistical adjustment for other factors. Rodriguez et al. [62] conducted a study aimed at determining the capability of long-term exposure to DDT of altering the normal endocrine function of the hypothalamus-hypophysis-gonads axis in humans. This included 70 workers dedicated to control malaria in the State of Guerrero,Mexico. The main activities of these workers were the application of pesticides,detection of malaria cases and promotion of preventive measures for control vectors. The average time of exposure to technical grade DDT was 25 years (range: 4–35), their last exposure being 5 months before sampling. An interview gathered information on the occupational history, reproductive performance, life styles and other relevant factors. Blood and urine samples were collected to measure serum levels of DDT and metabolites as

**5.2 Endocrine disruption** 

well as levels of LH, FSH, prolactine, and testosterone. Participants ranged in age from 22 to 69 years, and had been employed in the sanitation campaign from 4 to 37 years. Ninetyseven percent of the participants were sprayers of DDT at some time in their occupational history, and 15% are current sprayers. Average levels of DDT and metabolites expressed as μg/g of extractable lipids were: total DDT, 60.1; *p,p*-DDE, 37.41; *p,p*-DDT, 21.52; *p,p*- DDD, 1.07 and *op*-DDT 0.11. Results show a positive association of LH and FSH with DDT metabolites. An increase of 10 *m*g/g of *p,p*-DDE was associated with an increase of 1.95 UI/L in LH (p=0.01); and 1.10 UI/L per each 10 *m*g/g of *p,p*-DDT (p=0.02). FSH increased 1.09 UI/L per each 10 *m*g/g of *p,p*- DDT (p=0.03). There was a negative association of DDE with testosterone, DDT in Mexico 383 especially for those participants under 55 years of age. These associations suggest direct toxicity to the testicles, especially the Leydig cells, as observed with antineoplasic drugs.

#### **5.3 Genotoxicity**

Some studies have reported genotoxic effects in humans heavily exposed to DDT [8]. Therefore, this area has been studied in Mexico. Studies were done in workers from the control program of malaria, and in women living in malarious areas. Herrera et al. [63] evaluated chromosomal translocations in a sample of the above-mentioned workers.Nineteen male sprayers (median age 46 years; range: 22–64),working in campaigns to control malaria vectors in the state of Guerrero, Mexico were included in this study.DDT data obtained in a previous study from eleven individuals, 5 women and 6 men, living in Mexico City, and occupationally unexposed to DDT were used as reference group. Chromosomal aberrations in lymphocytes were analyzed with a chromosome painting technique, a highsensitivity technique for detecting complex chromosomal translocations. *o,p*- DDT was the only isomer significantly associated with the frequency of chromosomal translocations (p=0.003). Individuals presenting the higher levels of *o,p*-DDT in serum (>0.79 *m*g/g fat; n=4) had a mean frequency of chromosomal translocations (5.1*¥*1000 metaphases), two times higher than that observed in workers occupationally exposed to 0.5 Gy of radiation.A positive relationship between the duration of exposure to DDT, measured as years working for the vector control program, and chromosomal translocations was observed (Fig.2). These results suggest an increased risk for diseases with a genetic component, such as cancer.

Yañez et al. [64] evaluated the association of blood DDT levels and DNA damage using the single cell gel electrophoresis assay. A group of 53 postpartum women were selected from two different areas in San Luis Potosí to assure different exposure levels, one with antecedents of malaria and DDT spraying and the other without malaria. Mean and range levels of DDT, DDE and DDD in whole blood were 5.57 ppm (0.02–20.69), 6.24 ppm (0.04– 39.16) and 1.16 ppm (0.01–5.63), respectively. The significant correlation of DNA damage, measured as DNA migration with the logarithm of DDT,DDE and DDD was 0.60, 0.62 and 0.43, respectively. Fig. 3 shows the shape of the association of DNA migration with DDE concentration in whole blood, as obtained by the regression: DNA migration= 71.58+7.62(logDDE), this association was not modified by age, smoking habits, nutrition or occupation. This observational finding in the epidemiological study with postpartum women was reevaluated in an in vitro study.Human blood cells were exposed to three doses of DDT, DDD and DDE.DNA damage was assessed by two different techniques: single-cell electrophoresis and flow cytometry. Results obtained by either technique showed that DNA damage was induced by the three organochlorides and a dose-response was observed with

**6** 

*Croatia* 

**Presence of Dichlorodiphenyltrichloroethane** 

Pesticide is any substance or mixture of substances used for preventing, destroying or controlling any pest. According to the Food and Agriculture Organization (FAO) pest is defined as an organism that: (1) is a vector of human and animal disease; (2) can interfere with or cause harm to any step in food, wood and agricultural goods production, transport and storage; (3) can lower the quality of animal food or drugs. Extended definition of pesticide includes all substances: (1) used as plant growth regulator, defoliant and desiccant or agent for prevention of premature fruit fall; (2) applied to crops in order to protect them from deterioration during storage and transport (Food and Agriculture Organization [FAO], 2005). Every pesticide consists of active ingredient whose main function is to cause harm on the pest and inert carrier substance to improve storage, handling, application, efficiency and

For easier dealing with large numbers of pesticides, there are several classifications which divide pesticides in some classes. According to the chemical structure pesticides can be classified as: pyrethroids, carbamates, thiocarbamates, dithiocarbamates, organophosphates, organochlorides, phenoxy and benzoic acid herbicides, triazines, triazoles, ureas etc. (Kamir, 2000). Another classification is done considering the target organism, so pesticides can be classified as: insecticides, fungicides, herbicides, virucides, avicides, molluscicides, nematocides, rodenticides etc. (WHO, 1990). It is important to notice that pesticide's mode of action is not restricted just to the target organism; it also has negative effects on other organisms. That is why the World Health Organization (WHO) recommends the classification of pesticides by hazard. The base of this classification is determining the lethal dose (LD50) for 50% of rat population after acute oral and dermal pesticide exposure. Depending of the LD50 value, as presented in Table 1, pesticides are classified as extremely,

It can be assumed that fighting against the pests started when human civilization was introduced to agriculture which ensured enough food for further development. The first

safety of the pesticide (World Health Organization [WHO], 1990).

highly, moderately or slightly hazardous (WHO, 2009a).

**1. Introduction** 

**(DDT) in Croatia and Evaluation** 

Goran Gajski1, Marko Gerić1, Sanda Ravlić2, Željka Capuder3 and Vera Garaj-Vrhovac1 *1Institute for Medical Research and Occupational Health 2Ruđer Bošković Institute, Center for Marine Research 3Dr. Andrija Štampar Institute of Public Health* 

**of Its Genotoxicity** 

DDT. The data suggest a DDT-induced DNA fragmentation, and this outcome was also observed with DDE and DDD.

## **6. References**


## **Presence of Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity**

Goran Gajski1, Marko Gerić1, Sanda Ravlić2, Željka Capuder3 and Vera Garaj-Vrhovac1 *1Institute for Medical Research and Occupational Health 2Ruđer Bošković Institute, Center for Marine Research 3Dr. Andrija Štampar Institute of Public Health Croatia* 

#### **1. Introduction**

116 Insecticides – Pest Engineering

DDT. The data suggest a DDT-induced DNA fragmentation, and this outcome was also

[2] North American Commission for Environmental Cooperation (1998) North American

[3] Fernández de Castro J (1988) Panorama histórico y epidemiológico del paludismo en

[4] Implementation Plan to Control Malaria with Alternatives Methods to DDT in Mexico

[5] ISAT, 2002. Diagnóstico situacional del uso de DDT y el control de la malaria. Informe

[6]ATSDR (2010) Toxicological Profile for DDT,DDE, and DDD.Agency for Toxic Substances

<http://www.cec.org/files/PDF/POLLUTANTS/InfRegDDTb\_ES\_EN.pdf>

[11] Woodruff T,Wolff MS, Davis DL,Hayward D (1994) Environ Res 65:132

Regional Commitments and Action Plans.Montreal, Canada, p 67

Regional Action Plan on DDT. In: The Sound Management of Chemicals Initiative.

regional para México y Centroamérica. Instituto Salud Ambiente y Trabajo, México.

observed with DDE and DDD.

[1] Stapleton DH (1998) Parassitologia 40 :149

(accessed 07.07.09.).

México. Secretaría de Salud,México

and Disease Registry,Atlanta,Georgia [7] Zeng EY,Yu CC, Tran K (1999) Environ Sci Technol 33:39 [8] Fisher SW (1995) Rev Environ Contam Toxicol 142:87 [9] Albert LA (1996) Rev Environ Contam Toxicol 147:1 [10] Lu FC (1995) Reg Toxicol Pharmacol 21 :352

1999–2001.Government of Mexico, January 1999

**6. References** 

Pesticide is any substance or mixture of substances used for preventing, destroying or controlling any pest. According to the Food and Agriculture Organization (FAO) pest is defined as an organism that: (1) is a vector of human and animal disease; (2) can interfere with or cause harm to any step in food, wood and agricultural goods production, transport and storage; (3) can lower the quality of animal food or drugs. Extended definition of pesticide includes all substances: (1) used as plant growth regulator, defoliant and desiccant or agent for prevention of premature fruit fall; (2) applied to crops in order to protect them from deterioration during storage and transport (Food and Agriculture Organization [FAO], 2005). Every pesticide consists of active ingredient whose main function is to cause harm on the pest and inert carrier substance to improve storage, handling, application, efficiency and safety of the pesticide (World Health Organization [WHO], 1990).

For easier dealing with large numbers of pesticides, there are several classifications which divide pesticides in some classes. According to the chemical structure pesticides can be classified as: pyrethroids, carbamates, thiocarbamates, dithiocarbamates, organophosphates, organochlorides, phenoxy and benzoic acid herbicides, triazines, triazoles, ureas etc. (Kamir, 2000). Another classification is done considering the target organism, so pesticides can be classified as: insecticides, fungicides, herbicides, virucides, avicides, molluscicides, nematocides, rodenticides etc. (WHO, 1990). It is important to notice that pesticide's mode of action is not restricted just to the target organism; it also has negative effects on other organisms. That is why the World Health Organization (WHO) recommends the classification of pesticides by hazard. The base of this classification is determining the lethal dose (LD50) for 50% of rat population after acute oral and dermal pesticide exposure. Depending of the LD50 value, as presented in Table 1, pesticides are classified as extremely, highly, moderately or slightly hazardous (WHO, 2009a).

It can be assumed that fighting against the pests started when human civilization was introduced to agriculture which ensured enough food for further development. The first

Presence of

Chemical

Chemical structure

Molecular mass

Physical

Melting

Solubility

Solubility in organic solvents

Colour Colourless crystals,

Slightly soluble in ethanol, very soluble in ethyl ether and acetone

2002), a room temperature, b at 25°C, \*NA – not available.

testicular cancer etc.; Aubé et al., 2008; McGlynn et al., 2008).

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 119

(Brooks, 1986; Gold & Brunk, 1982, 1983; Morgan & Roan, 1974; Peterson & Robinson, 1964). As mentioned in the book *Silent spring*, DDT (especially metabolite DDE) has negative effects on bird's reproductive system by reducing Ca2+ transport which results with

formula C14H9Cl5 C14H8Cl4 C14H10Cl4

state Solida Cristalline solida Solida

point 109 °C 89 °C 109 – 110 °C

in water 0.025 mg/Lb 0.12 mg/Lb 0.09 mg/Lb

Table 2. Chemicals properties of *p,p'*-DDT, *p,p'*-DDE, and *p,p'*-DDD (adapted from ATSDR,

Not only birds are affected by this persistent pollutant, animals with higher amounts of fatty tissue have high levels of DDT and its metabolites (e.g. polar bears, orcas, belugas etc. (Crinnion, 2009; Galassi et al., 2008; Glynn et al., 2011; Okonkwo et al., 2008)). Although the acute DDT poisoning is rare, there are reports of negative effects on human health when exposed to low concentrations of DDT and its metabolites for longer periods. As shown in animals, the major damage was done on reproductive system where DDT can interfere with reproductive hormones. Also, there have been some reports regarding neurotoxicity, hepatotoxicity, imunotoxicity and genotoxicity (ATSDR, 2002). According to International Agency for Research on Cancer (IARC) DDT is classified as possible carcinogen to humans (IARC, 2009) and the increase in frequency of some cancers was detected (e.g. breast,

As mentioned above, DDT introduced revolution in control of vector-borne diseases. Today, there are reports that some *Anopheles* species are resistant to DDT (Morgan et al., 2010) which implies that new methods of fighting vectors should be introduced. According to WHO one can observe the decrease in both number of countries using pesticides to control

Boiling point Decomposes 336 °C 350 °C

*p,p'*-DDT *p,p'*-DDE *p,p'*-DDD

354.49 318.03 320.05

Lipids and most organic solvents

white powder White Colourless crystals, white

powder

NA\*

eggshell thinning thus increasing lethality (Carson, 2002).

known historical use of pesticide was observed in Mesopotamia 2500 BC. People in ancient Sumer used sulphur to treat crops thus killing pests (Miller, 2004). Around 500 BC ancient Greek historian Herodotus described the use of castor-oil plant as a mosquito repellent. People in Egypt who lived in swampy areas lit the castor-oil laps which had an unpleasant smell. Also they fixed fishing nets around their beds before going to sleep. This information suggests that ancient Egyptians used similar techniques to deal with mosquitos as today's insecticide-treated nets (Charlwood, 2003; WHO, 2007). After long period of natural pesticides use (e.g. arsenic, mercury, nicotine sulphate, pyrethrum, etc.), in 20th century the revolution of synthesized pesticides started.



DDT (1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane) was one of the most used pesticides in the mid 20th century. This organochloride was synthesized in 1874 by Othmar Zeidler and its pesticides properties were discovered in 1939 by Paul Hermann Müller who was awarded Nobel Prize in Physiology or Medicine for this discovery (Agency for Toxic Substances and Disease Service [ATSDR], 2002; Turusov et al., 2002). Commercially used DDT is a mixture of several isomeric forms: *p,p'-*DDT (85%), *o,p'*-DDT (15%), *o,o'*-DDT (trace amounts) and its breakdown products DDE (1,1-dichloro-2,2-bis(*p*-chlorophenyl)ethylene) and DDD (1,1-dichloro-2,2-bis(*p*-chlorophenyl)ethane). The basic chemical properties of *p,p'*- DDT, *p,p'*-DDE and *p,p'*-DDD are shown in Table 2. According to WHO, DDT is moderately hazardous pesticide with LD50 of 113 mg/kg (WHO, 2009a).

In the period of almost 35 years, 2 million tons of DDT was used to control malaria and typhus, thus contaminating water, soil and air. When DDT and its metabolites enter the environment they have the potential to stay adsorbed in the sediment for more than 100 years. The best example of DDT's persistency is that it has been found in the areas 1000 km off the spraying spot and even in the Arctic animals and ice where it has never been used (ATSDR, 2002). One of the most popular acts that contributed global DDT ban in 1973, was Rachel Carson's book *Silent Spring* written in 1962 where she emphasized the problem of the DDT use (Carson, 2002). Today DDT is still legally produced (China, India, and North Korea) and used in the countries of the third world (e.g. India, Ethiopia, South Africa etc.) for malaria control (WHO, 2009b).

There are four possible ways for DDT to enter organisms: (1) ingestion, (2) inhalation, (3) dermal exposure, and (4) placental transport. Once in the body DDT is metabolized mainly in the liver and partly in the kidneys to its most familiar metabolites DDE and DDD. Detailed metabolic pathways are shown in Figure 1. Conjugated forms of 2,2-bis(4 chlorophernyl)-acetic acid (DDA) are then excreted through urine and faeces. The most dangerous property of DDT is that it possesses high bioaccumulation and biomagnification potential. As the trophic level rises, the concentration of DDT and/or its metabolites increases. All three compounds are reported to be harmful to either human or animals

known historical use of pesticide was observed in Mesopotamia 2500 BC. People in ancient Sumer used sulphur to treat crops thus killing pests (Miller, 2004). Around 500 BC ancient Greek historian Herodotus described the use of castor-oil plant as a mosquito repellent. People in Egypt who lived in swampy areas lit the castor-oil laps which had an unpleasant smell. Also they fixed fishing nets around their beds before going to sleep. This information suggests that ancient Egyptians used similar techniques to deal with mosquitos as today's insecticide-treated nets (Charlwood, 2003; WHO, 2007). After long period of natural pesticides use (e.g. arsenic, mercury, nicotine sulphate, pyrethrum, etc.), in 20th century the

I a Extremely hazardous 5 or less 20 or less 10 or less 40 or less I b Highly hazardous 5 – 50 20 – 200 10 – 100 40 – 400 II Moderately hazardous 50 – 500 200 – 2000 100 – 1000 400 – 4000 III Slightly hazardous over 500 over 2000 over 1000 over 4000

DDT (1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane) was one of the most used pesticides in the mid 20th century. This organochloride was synthesized in 1874 by Othmar Zeidler and its pesticides properties were discovered in 1939 by Paul Hermann Müller who was awarded Nobel Prize in Physiology or Medicine for this discovery (Agency for Toxic Substances and Disease Service [ATSDR], 2002; Turusov et al., 2002). Commercially used DDT is a mixture of several isomeric forms: *p,p'-*DDT (85%), *o,p'*-DDT (15%), *o,o'*-DDT (trace amounts) and its breakdown products DDE (1,1-dichloro-2,2-bis(*p*-chlorophenyl)ethylene) and DDD (1,1-dichloro-2,2-bis(*p*-chlorophenyl)ethane). The basic chemical properties of *p,p'*- DDT, *p,p'*-DDE and *p,p'*-DDD are shown in Table 2. According to WHO, DDT is moderately

In the period of almost 35 years, 2 million tons of DDT was used to control malaria and typhus, thus contaminating water, soil and air. When DDT and its metabolites enter the environment they have the potential to stay adsorbed in the sediment for more than 100 years. The best example of DDT's persistency is that it has been found in the areas 1000 km off the spraying spot and even in the Arctic animals and ice where it has never been used (ATSDR, 2002). One of the most popular acts that contributed global DDT ban in 1973, was Rachel Carson's book *Silent Spring* written in 1962 where she emphasized the problem of the DDT use (Carson, 2002). Today DDT is still legally produced (China, India, and North Korea) and used in the countries of the third world (e.g. India, Ethiopia, South Africa etc.)

There are four possible ways for DDT to enter organisms: (1) ingestion, (2) inhalation, (3) dermal exposure, and (4) placental transport. Once in the body DDT is metabolized mainly in the liver and partly in the kidneys to its most familiar metabolites DDE and DDD. Detailed metabolic pathways are shown in Figure 1. Conjugated forms of 2,2-bis(4 chlorophernyl)-acetic acid (DDA) are then excreted through urine and faeces. The most dangerous property of DDT is that it possesses high bioaccumulation and biomagnification potential. As the trophic level rises, the concentration of DDT and/or its metabolites increases. All three compounds are reported to be harmful to either human or animals

Table 1. The WHO pesticide classification based on LD50 (adapted from WHO, 2005).

Oral Dermal Solids Liquids Solids Liquids

revolution of synthesized pesticides started.

Class LD50 for rats (mg/kg of body mass)

hazardous pesticide with LD50 of 113 mg/kg (WHO, 2009a).

for malaria control (WHO, 2009b).

(Brooks, 1986; Gold & Brunk, 1982, 1983; Morgan & Roan, 1974; Peterson & Robinson, 1964). As mentioned in the book *Silent spring*, DDT (especially metabolite DDE) has negative effects on bird's reproductive system by reducing Ca2+ transport which results with eggshell thinning thus increasing lethality (Carson, 2002).


Table 2. Chemicals properties of *p,p'*-DDT, *p,p'*-DDE, and *p,p'*-DDD (adapted from ATSDR, 2002), a room temperature, b at 25°C, \*NA – not available.

Not only birds are affected by this persistent pollutant, animals with higher amounts of fatty tissue have high levels of DDT and its metabolites (e.g. polar bears, orcas, belugas etc. (Crinnion, 2009; Galassi et al., 2008; Glynn et al., 2011; Okonkwo et al., 2008)). Although the acute DDT poisoning is rare, there are reports of negative effects on human health when exposed to low concentrations of DDT and its metabolites for longer periods. As shown in animals, the major damage was done on reproductive system where DDT can interfere with reproductive hormones. Also, there have been some reports regarding neurotoxicity, hepatotoxicity, imunotoxicity and genotoxicity (ATSDR, 2002). According to International Agency for Research on Cancer (IARC) DDT is classified as possible carcinogen to humans (IARC, 2009) and the increase in frequency of some cancers was detected (e.g. breast, testicular cancer etc.; Aubé et al., 2008; McGlynn et al., 2008).

As mentioned above, DDT introduced revolution in control of vector-borne diseases. Today, there are reports that some *Anopheles* species are resistant to DDT (Morgan et al., 2010) which implies that new methods of fighting vectors should be introduced. According to WHO one can observe the decrease in both number of countries using pesticides to control

Presence of

(Dugački, 2005).

**2. Presence of DDT in Croatia 2.1 Production and use in Croatia** 

(Table 4; National Implementation Plan [NIP], 2004).

1959. consumption DDT 3122

1963. production DDT 2327

Yugoslavia in some years (adapted from Hamel, 2003).

Year and purpose Type of pesticide Amount (t) Note

DDT + Lindane 687

DDT + Lindane 1854

1958. production DDT 2150 1600 t sprayer concentrate 1959. plan DDT 3000 550 t emulsifiable concentrate

Table 3. The planned and produced quantities of DDT, according to the available data for

4.6-6.6 % DDT + 0.3-0.7 lindane content of active substance in preparation

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 121

Croatia, as a Mediterranean country, has been affected by malaria since the ancient times, especially in the coastal area. Only areas along the Velebit and Biokovo and the districts Hvar and Supetar have been spared from the disease. Most exposed to malaria have been the cities Nin, Benkovac, Obrovac, Skradin, Knin, Drniš, Šibenik, Imotski, Vrgorac and Metković area, the valley of the Neretva River, where malaria was called *Morbus naronianus* - Neretva disease. In 1820 French pharmacists Pierre Joseph Pelletier & Jean Biename Caventou isolated the alkaloid quinine, while doctor Lujo Adam from the island Lošinj was probably the first in the world who injected quinine sulphate into the veins of people sick from malaria. In 1902 doctor Rudolf Battara conducted the first controlled study of drug quinine prophylaxis on the overall population in Nin. The success of the study encouraged implementation of this prophylaxis in other places in Dalmatia in the upcoming years

DDT was first used in Croatia in 1941, when spraying was carried out against the lice of people as part of educational campaigns in the School of Public Health. The first mass use of DDT was carried out on the front of Srijem in 1945 by spraying soldiers as prevention of typhoid fever with the preparation "neocid", which was donated by the International Red Cross (Bakić, 2011). Companies Chromos from Zagreb and Zorka from Šabac, with their preparations Pantakan and Pepein, were the first producers of DDT in Croatia and the former Yugoslavia. Since 1946 DDT was produced in the form of dust, and since 1949 in the form of concentrated emulsions (Table 3). Production of pesticides in Croatia was small, but large quantities of pesticides were imported. Data on quantities of imported pesticides does not exist, except for the year 1957, when active substances for plant protection products in quantities of 1435 tons were imported. The reason for the continuing production decline of DDT was the lack of foreign exchange quotas for the purchase of organic substances necessary for the production of pesticides. Production and use of DDT in Croatia was until 1972, when its use was banned in agriculture, while in forestry DDT was still used until 1984

vectors (from 72 countries in 2006 to 61 countries in 2007) and in total amount of used DDT for vector control (from 5.1 million kg in 2006 to 3.7 million kg in 2007). Also WHO stimulates the import of new technologies to improve efficiency of insecticide-treated nets and long-lasting insecticidal nets (WHO, 2007). The importance of introducing new technologies for non harmful and efficient global pest fighting can be seen in estimation that only in United States, environmental and social costs of pesticide use are 9645 million dollars per year (Pimentel, 2005).

Fig. 1. Models of DDT metabolic pathways (adapted from Gold & Brunk, 1982 and Peterson & Robinson, 1964).

## **2. Presence of DDT in Croatia**

120 Insecticides – Pest Engineering

vectors (from 72 countries in 2006 to 61 countries in 2007) and in total amount of used DDT for vector control (from 5.1 million kg in 2006 to 3.7 million kg in 2007). Also WHO stimulates the import of new technologies to improve efficiency of insecticide-treated nets and long-lasting insecticidal nets (WHO, 2007). The importance of introducing new technologies for non harmful and efficient global pest fighting can be seen in estimation that only in United States, environmental and social costs of pesticide use are 9645 million

Fig. 1. Models of DDT metabolic pathways (adapted from Gold & Brunk, 1982 and Peterson

dollars per year (Pimentel, 2005).

& Robinson, 1964).

## **2.1 Production and use in Croatia**

Croatia, as a Mediterranean country, has been affected by malaria since the ancient times, especially in the coastal area. Only areas along the Velebit and Biokovo and the districts Hvar and Supetar have been spared from the disease. Most exposed to malaria have been the cities Nin, Benkovac, Obrovac, Skradin, Knin, Drniš, Šibenik, Imotski, Vrgorac and Metković area, the valley of the Neretva River, where malaria was called *Morbus naronianus* - Neretva disease. In 1820 French pharmacists Pierre Joseph Pelletier & Jean Biename Caventou isolated the alkaloid quinine, while doctor Lujo Adam from the island Lošinj was probably the first in the world who injected quinine sulphate into the veins of people sick from malaria. In 1902 doctor Rudolf Battara conducted the first controlled study of drug quinine prophylaxis on the overall population in Nin. The success of the study encouraged implementation of this prophylaxis in other places in Dalmatia in the upcoming years (Dugački, 2005).

DDT was first used in Croatia in 1941, when spraying was carried out against the lice of people as part of educational campaigns in the School of Public Health. The first mass use of DDT was carried out on the front of Srijem in 1945 by spraying soldiers as prevention of typhoid fever with the preparation "neocid", which was donated by the International Red Cross (Bakić, 2011). Companies Chromos from Zagreb and Zorka from Šabac, with their preparations Pantakan and Pepein, were the first producers of DDT in Croatia and the former Yugoslavia. Since 1946 DDT was produced in the form of dust, and since 1949 in the form of concentrated emulsions (Table 3). Production of pesticides in Croatia was small, but large quantities of pesticides were imported. Data on quantities of imported pesticides does not exist, except for the year 1957, when active substances for plant protection products in quantities of 1435 tons were imported. The reason for the continuing production decline of DDT was the lack of foreign exchange quotas for the purchase of organic substances necessary for the production of pesticides. Production and use of DDT in Croatia was until 1972, when its use was banned in agriculture, while in forestry DDT was still used until 1984 (Table 4; National Implementation Plan [NIP], 2004).


Table 3. The planned and produced quantities of DDT, according to the available data for Yugoslavia in some years (adapted from Hamel, 2003).

Presence of

transferred to vials for the quantitative analysis.

(annually) in intercalibration study proficiency test.

Milk (liquid) and milk products (milk fat)

Fish and fish products

(wet weight)

**2.2.2 Gas chromatograph analysis** 

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 123

method EN 1528 1-4 (1996). Approximately 10-20 g of food were homogenized and extracted with hexane, the extract was evaporated in a rotary evaporator and in nitrogen stream to ensure dryness. Contents of fat were determined weighing a dry sample. Milk fat portions were extracted from each individual sample of milk, cheese, cream and yoghurt according to the method by Sannino et al. (1996). Butter does not normally require extraction procedures. An automated gel permeation chromatographic (GPC) procedure was used to determine DDT residues in fatty foods. About three grams of the fat or less was dissolved into methylene chloride and cleaned up by GPC with a Biobeads SX3 column (OI Analytical, College Station, TX, USA) and a methylene chloride as eluant. About 10 g of fish tissue was weighed, homogenized with anhydrous sodium sulphate and extracted with pesticide grade hexane (Merck, UNEP/IAEA, 1982). The extract was condensed in a rotary flask vaporator to a specific aliquot (5 ml). The aliquot was then subjected to acid treatment by adding concentrated sulphuric acid (Merck). All samples were cleaned up with florisil, commercially available cartridges, 3-6 mL, 500-1000 mg (Kodba & Voncina, 2007). Extracts were concentrated using evaporator in nitrogen stream. An aliquot of each extract was

The samples were analyzed by gas chromatograph (GC) Shimadzu (Models GC 2010 and GC 17A series, Tokyo, Japan) equipped with autosampler and electron capture detector (ECD) on two fused silica capillary columns of different polarity. Nitrogen was used as the carrier gas with a flow rate of about 1.4 mL/min and as the makeup gas. The injection was set at splitless mode. The injection port and detector temperature were 250 and 300 0C, respectively. The compounds *p,p'*-DDE, *p,p'*-DDD, *o,p'*-DDT and *p,p'*-DDT were identified by comparing peak retention times between samples and known standards. The standard samples of the 18 pesticides were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany, and AccuStandard Inc., New Haven, CT, USA) with the purities of 97-99%. Calibration standard curves were created and DDT residues were quantitatively determined by comparison of the retention times and peak areas of the sample chromatogram with those of standard solutions run under the same operating conditions. Peak confirmation was done by running the samples and the standard on another column and comparing. The concentrations of DDT residues in each sample were reported as ng/L or μg/kg. Limit of quantification for every matrix is presented in Table 5. The laboratory has participated

Limit of quantification

Water μg/L 0.0005 0.001 0.001 0.001 Soil (dry mater) μg/g 0.002 0.003 0.004 0.003

Food (fat) μg/g 0.001 0.002 0.004 0.002

Table 5. Limit of quantification for *p,p'*-DDE, *p,p'*-DDD, *o,p'*-DDT and *p,p'*-DDT.

*p,p'*-DDE *p,p'*-DDD *o,p'*-DDT *p,p'*-DDT

μg/g 0.0001 0.0004 0.0004 0.0004

μg/g 0.0005 0.001 0.0006 0.001


Table 4. Consumption of DDT in kg per year in agriculture and forestry in Republic of Croatia (adapted from NIP, 2004).

Today, list of active substances permitted for use in the Republic of Croatia is synchronized with the official list of active substances permitted in the means of the European Union. The regulations require from pesticide manufacturers to provide data on all possible risks to human health and the environment, as well as data on the effectiveness of pesticides and information on possible contamination in order to obtain licenses for the production and use.

## **2.2 Methodology**

Residues of DDT and its metabolites were analyzed in samples of surface water, soil and food. One of the most sensitive techniques for measuring the rest of DDT and its metabolites in samples from the environment is gas chromatography with electron capture detector (GC/ECD). DDT and its metabolites were determined by applying analytical methods or modifications of the method: International Organization for Standardisation [ISO] 6468 (2002), ISO 10382 (2002), Reference Methods for Marine Pollution (United Nations Environment Programme/International Atomic Energy Agency [UNEP/IAEA], 1982) and EN 1528 1-4 (1996), according to the scheme; extraction, purification of the extracts and quantitative analysis.

#### **2.2.1 Sample extraction and cleanup**

Water samples, two to four litres, were extracted with methylene chloride (HPLC grade for spectroscopy, Merck, Darmstadt, Germany) as solvent using an Ultra Turrax system. Extracts were dried over granular anhydrous sodium sulphate and concentrated using rotary evaporator (ISO 6468, 2002). The soil sample was sieved (<2 mm) and stored at room temperature for two days before the experiments. 10 g soil sample (dry matter) was extracted in an ultrasonic bath (ISO 10382, 2002) and the extract was dried by passing them through anhydrous sodium sulphate. Determination of DDT in food samples was based on method EN 1528 1-4 (1996). Approximately 10-20 g of food were homogenized and extracted with hexane, the extract was evaporated in a rotary evaporator and in nitrogen stream to ensure dryness. Contents of fat were determined weighing a dry sample. Milk fat portions were extracted from each individual sample of milk, cheese, cream and yoghurt according to the method by Sannino et al. (1996). Butter does not normally require extraction procedures. An automated gel permeation chromatographic (GPC) procedure was used to determine DDT residues in fatty foods. About three grams of the fat or less was dissolved into methylene chloride and cleaned up by GPC with a Biobeads SX3 column (OI Analytical, College Station, TX, USA) and a methylene chloride as eluant. About 10 g of fish tissue was weighed, homogenized with anhydrous sodium sulphate and extracted with pesticide grade hexane (Merck, UNEP/IAEA, 1982). The extract was condensed in a rotary flask vaporator to a specific aliquot (5 ml). The aliquot was then subjected to acid treatment by adding concentrated sulphuric acid (Merck). All samples were cleaned up with florisil, commercially available cartridges, 3-6 mL, 500-1000 mg (Kodba & Voncina, 2007). Extracts were concentrated using evaporator in nitrogen stream. An aliquot of each extract was transferred to vials for the quantitative analysis.

## **2.2.2 Gas chromatograph analysis**

122 Insecticides – Pest Engineering

Today, list of active substances permitted for use in the Republic of Croatia is synchronized with the official list of active substances permitted in the means of the European Union. The regulations require from pesticide manufacturers to provide data on all possible risks to human health and the environment, as well as data on the effectiveness of pesticides and information on possible contamination in order to obtain licenses for the production and

Residues of DDT and its metabolites were analyzed in samples of surface water, soil and food. One of the most sensitive techniques for measuring the rest of DDT and its metabolites in samples from the environment is gas chromatography with electron capture detector (GC/ECD). DDT and its metabolites were determined by applying analytical methods or modifications of the method: International Organization for Standardisation [ISO] 6468 (2002), ISO 10382 (2002), Reference Methods for Marine Pollution (United Nations Environment Programme/International Atomic Energy Agency [UNEP/IAEA], 1982) and EN 1528 1-4 (1996), according to the scheme; extraction, purification of the extracts and

Water samples, two to four litres, were extracted with methylene chloride (HPLC grade for spectroscopy, Merck, Darmstadt, Germany) as solvent using an Ultra Turrax system. Extracts were dried over granular anhydrous sodium sulphate and concentrated using rotary evaporator (ISO 6468, 2002). The soil sample was sieved (<2 mm) and stored at room temperature for two days before the experiments. 10 g soil sample (dry matter) was extracted in an ultrasonic bath (ISO 10382, 2002) and the extract was dried by passing them through anhydrous sodium sulphate. Determination of DDT in food samples was based on

Consumption of DDT in agriculture (kg/year)

in forestry (kg/year)

1963. 2312 - 1964. - 1784 1965. 53428 1196 1966. 280 14051 1967. - 16323 1968. 600 4183 1969. - 6051 1970. - 5450 1971. 2363 4296 1972. 4912 1078 1973. 884 0 1974. 8437 0 1975. 6907 0 1976. 8437 0 1979. – 1987. 18658 0 Table 4. Consumption of DDT in kg per year in agriculture and forestry in Republic of

Year Consumption of DDT

Croatia (adapted from NIP, 2004).

use.

**2.2 Methodology** 

quantitative analysis.

**2.2.1 Sample extraction and cleanup** 

The samples were analyzed by gas chromatograph (GC) Shimadzu (Models GC 2010 and GC 17A series, Tokyo, Japan) equipped with autosampler and electron capture detector (ECD) on two fused silica capillary columns of different polarity. Nitrogen was used as the carrier gas with a flow rate of about 1.4 mL/min and as the makeup gas. The injection was set at splitless mode. The injection port and detector temperature were 250 and 300 0C, respectively. The compounds *p,p'*-DDE, *p,p'*-DDD, *o,p'*-DDT and *p,p'*-DDT were identified by comparing peak retention times between samples and known standards. The standard samples of the 18 pesticides were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany, and AccuStandard Inc., New Haven, CT, USA) with the purities of 97-99%. Calibration standard curves were created and DDT residues were quantitatively determined by comparison of the retention times and peak areas of the sample chromatogram with those of standard solutions run under the same operating conditions. Peak confirmation was done by running the samples and the standard on another column and comparing. The concentrations of DDT residues in each sample were reported as ng/L or μg/kg. Limit of quantification for every matrix is presented in Table 5. The laboratory has participated (annually) in intercalibration study proficiency test.


Table 5. Limit of quantification for *p,p'*-DDE, *p,p'*-DDD, *o,p'*-DDT and *p,p'*-DDT.

Presence of

samples of lamb meat.

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 125

Lamb meat results (Table 7) demand special attention, since the concentrations of *p,p'*-DDE were generally high. Similar results were found by Tompić et al. (2011) in samples of lamb imported from Bulgaria. *p,p'*-DDE are found in every examined sample of lamb meat, which indicates the need for continuous monitoring of concentrations of this metabolites in

Lamb meat 739.0 - 0.4 74.3 743.0 Lamb meat 173.0 - 0.5 17.8 178.0 Lamb meat 609.0 0.6 - 1.5 615.0 Lamb meat 964.0 6.1 1.2 103.7 1037.0 Lamb meat 354.0 1.4 0.5 37.3 373.0

Generally, it was observed that the total DDT or its metabolites residues were bellow acceptable and legally prescribed boundaries. These results highlight the need for regular analyzing of a larger number of samples from the environment to DDT residues and other

Pesticide exposure is ubiquitous, due not only to agricultural pesticide use and contamination of foods, but also to the extensive use of these products in and around residences. Because of their biological activity, the use of pesticides may cause undesired effects to human health. Pesticides tend to be very reactive compounds that can form covalent bonds with various nucleophilic centers of cellular biomolecules, including DNA (Crosby, 1982). For instance, the induction of DNA damage can potentially lead to adverse reproductive outcomes, the induction of cancer and many other chronic diseases (Ribas et al., 1996; Lander et al., 2000; Meinert et al., 2000; Ji et al., 2001). A great variety of tests and test systems based on microbes, plant and animals have been developed in order to asses the genotoxic effects of xenobiotic agents, including pesticides. Biomonitoring studies on human populations exposed to pesticides are employing circulating lymphocytes as biomarkers of exposure (and perhaps of effect). Those studies have essentially focused on cytogenetic endpoints such as chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) frequency. Genetic damage at the chromosomal level entails an alternation in either chromosome number or chromosome structure, and such alternations can be measured as CA or MN frequency. The SCE analysis was also adopted as an indicator of genotoxicity, although the exact mechanism that leads to an increased exchange

p,p'-DDT (ng/g)

p,p'-DDD (ng/g)

Table 7. Concentrations of DDT compounds in samples of lamb meat.

**3. Cytogenetic methods for detection of pesticide genotoxicity** 

Surface and ground water (ng/L) 0.6 – 20.5 2.6 1.6 Soil (μg/g; dry matter) 0 – 0.005 0.002 0.002 Fish and fish products (μg/kg; wet weight) 0.2 – 31.0 3.8 4.7 Meat and meat products (μg/kg; fat) 1.2 – 74.0 51.7 16.2 Milk and milk products (μg/kg; milk fat) 11.3 – 79.9 29.6 25.2

Table 6. Concentrations of DDT compounds in different samples.

p,p'-DDE (μg/kg)

chemicals of POPs, especially in imported food.

Value range Mean Median

Total DDT (ng/g)

Total DDT (μg/kg)

#### **2.3 Results**

During his lifetime a human being has been exposed to DDT and other pesticides through food, water and the environment. A level of these exposures can be established by analysing of samples from the environment. All food samples come from the market in Croatia while samples of surface water and soil were from different areas of Croatia. Food, particularly dairy products, meat and fish, has been identified as the primary immediate intake route of DDT and other organochlorine pesticides for the population (Johansen et al., 2004).

Samples of surface water of the rivers Sava, Krka, Mrežnica, Kupa, Zrmanja and Cetina were analyzed. The concentration of total DDT in surface waters in Croatia for the period 2009-2010 amounted from below detectable limit to 0.021 μg/L, with median value 0.0016 μg/L (Table 6). The average concentrations of total DDT in surface waters were 0.0026 μg/L. These results comply with those reported by Drevenkar et al. (1994), Drevenkar & Fingler (2000), and Fingler et al. (1992) in their papers. DDT may reach surface waters primarily by runoff, atmospheric transport and drift, or by direct application (e.g. to control mosquitoborne malaria). DDT is practically insoluble in water; but some DDT may be adsorbed onto the small amount of particulate matter present in water (ATSDR, 2002).

The concentration of DDT and metabolites in analyzed samples of soil were below 0.5 μg/g of dry matter (legally prescribed limit values). These analyses were conducted on the samples collected over the last two years and only from a few locations and therefore cannot be found sufficient to make any kind of general conclusions about the current situation. There is no systematic monitoring of DDT in soil in Croatia, resulting with little relevant data. Studies such as Picer et al. (2004) are rare examples of organized research in this area in Croatia, conducted as a part of post war damages in areas were there were concerns that soil was contaminated.

Data on levels and distribution of DDT and other persistent organic pollutant (POPs) in surface waters and soils in Croatia is insufficient, despite these hydrophobic substances being extremely important for assessing environmental contamination. DDT compound in sample of fish tissue were present in very low concentrations, although it is well known that DDT bioaccumulates in marine species. The mean value of DDT in fish tissue, analyzed in year 2007, was 3.8 μg/kg wet weights, with median value of 4.7 μg/kg . Similar results are reported by Krautchaker & Reiner (2001), and Bošnir et al. (2007). The five dairy products have been examined for the rest of DDT: milk, butter, cheese, cream and yoghurt. The mean values of the residual concentrations of total DDT in the examined dairy products were 29.6 μg/kg fat, respectively, with median 25.2 μg/kg (Table 6). The presence of DDT in milk and milk products has also been reported by Krautchaker & Reiner (2001) and Bošnir et al. (2010) as well as in other countries (Nevein et al., 2009; Dawood et al., 2004; Bulut et al., 2010). These studies have found that DDT complex were the most frequent contaminants in dairy products. Heck et al. (2006) concluded there is no difference in DDT in raw and pasteurized milk. The concentration of DDT in meat products sampled at food markets was 1.2-740.0 μg/kg fat, with median value 16.2 μg/kg fat (Table 6). These results should be taken with caution since the origin of meat is unknown; whether the meat is from domestic production or imported from other countries of the world. Meat is imported primarily from developing countries where there is limited or no control over the use and/or control of pesticide residues in foods. These results comply with those reported by Krautchaker & Reiner (2001), Covaci et al. (2004), and Tompić et al. (2011).

During his lifetime a human being has been exposed to DDT and other pesticides through food, water and the environment. A level of these exposures can be established by analysing of samples from the environment. All food samples come from the market in Croatia while samples of surface water and soil were from different areas of Croatia. Food, particularly dairy products, meat and fish, has been identified as the primary immediate intake route of DDT and other organochlorine pesticides for the population

Samples of surface water of the rivers Sava, Krka, Mrežnica, Kupa, Zrmanja and Cetina were analyzed. The concentration of total DDT in surface waters in Croatia for the period 2009-2010 amounted from below detectable limit to 0.021 μg/L, with median value 0.0016 μg/L (Table 6). The average concentrations of total DDT in surface waters were 0.0026 μg/L. These results comply with those reported by Drevenkar et al. (1994), Drevenkar & Fingler (2000), and Fingler et al. (1992) in their papers. DDT may reach surface waters primarily by runoff, atmospheric transport and drift, or by direct application (e.g. to control mosquitoborne malaria). DDT is practically insoluble in water; but some DDT may be adsorbed onto

The concentration of DDT and metabolites in analyzed samples of soil were below 0.5 μg/g of dry matter (legally prescribed limit values). These analyses were conducted on the samples collected over the last two years and only from a few locations and therefore cannot be found sufficient to make any kind of general conclusions about the current situation. There is no systematic monitoring of DDT in soil in Croatia, resulting with little relevant data. Studies such as Picer et al. (2004) are rare examples of organized research in this area in Croatia, conducted as a part of post war damages in areas were there were concerns that

Data on levels and distribution of DDT and other persistent organic pollutant (POPs) in surface waters and soils in Croatia is insufficient, despite these hydrophobic substances being extremely important for assessing environmental contamination. DDT compound in sample of fish tissue were present in very low concentrations, although it is well known that DDT bioaccumulates in marine species. The mean value of DDT in fish tissue, analyzed in year 2007, was 3.8 μg/kg wet weights, with median value of 4.7 μg/kg . Similar results are reported by Krautchaker & Reiner (2001), and Bošnir et al. (2007). The five dairy products have been examined for the rest of DDT: milk, butter, cheese, cream and yoghurt. The mean values of the residual concentrations of total DDT in the examined dairy products were 29.6 μg/kg fat, respectively, with median 25.2 μg/kg (Table 6). The presence of DDT in milk and milk products has also been reported by Krautchaker & Reiner (2001) and Bošnir et al. (2010) as well as in other countries (Nevein et al., 2009; Dawood et al., 2004; Bulut et al., 2010). These studies have found that DDT complex were the most frequent contaminants in dairy products. Heck et al. (2006) concluded there is no difference in DDT in raw and pasteurized milk. The concentration of DDT in meat products sampled at food markets was 1.2-740.0 μg/kg fat, with median value 16.2 μg/kg fat (Table 6). These results should be taken with caution since the origin of meat is unknown; whether the meat is from domestic production or imported from other countries of the world. Meat is imported primarily from developing countries where there is limited or no control over the use and/or control of pesticide residues in foods. These results comply with those reported by Krautchaker & Reiner (2001), Covaci et al. (2004),

the small amount of particulate matter present in water (ATSDR, 2002).

**2.3 Results** 

(Johansen et al., 2004).

soil was contaminated.

and Tompić et al. (2011).


Table 6. Concentrations of DDT compounds in different samples.

Lamb meat results (Table 7) demand special attention, since the concentrations of *p,p'*-DDE were generally high. Similar results were found by Tompić et al. (2011) in samples of lamb imported from Bulgaria. *p,p'*-DDE are found in every examined sample of lamb meat, which indicates the need for continuous monitoring of concentrations of this metabolites in samples of lamb meat.


Table 7. Concentrations of DDT compounds in samples of lamb meat.

Generally, it was observed that the total DDT or its metabolites residues were bellow acceptable and legally prescribed boundaries. These results highlight the need for regular analyzing of a larger number of samples from the environment to DDT residues and other chemicals of POPs, especially in imported food.

## **3. Cytogenetic methods for detection of pesticide genotoxicity**

Pesticide exposure is ubiquitous, due not only to agricultural pesticide use and contamination of foods, but also to the extensive use of these products in and around residences. Because of their biological activity, the use of pesticides may cause undesired effects to human health. Pesticides tend to be very reactive compounds that can form covalent bonds with various nucleophilic centers of cellular biomolecules, including DNA (Crosby, 1982). For instance, the induction of DNA damage can potentially lead to adverse reproductive outcomes, the induction of cancer and many other chronic diseases (Ribas et al., 1996; Lander et al., 2000; Meinert et al., 2000; Ji et al., 2001). A great variety of tests and test systems based on microbes, plant and animals have been developed in order to asses the genotoxic effects of xenobiotic agents, including pesticides. Biomonitoring studies on human populations exposed to pesticides are employing circulating lymphocytes as biomarkers of exposure (and perhaps of effect). Those studies have essentially focused on cytogenetic endpoints such as chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) frequency. Genetic damage at the chromosomal level entails an alternation in either chromosome number or chromosome structure, and such alternations can be measured as CA or MN frequency. The SCE analysis was also adopted as an indicator of genotoxicity, although the exact mechanism that leads to an increased exchange

Presence of

Wong et al., 2005).

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 127

neutral or mildly alkaline comet assay has the same limit of detection of DNA damage (SSB) as the alkaline comet assay, although the use of neutral pH does effect the comet image obtained (Collins, 2004). The comet tails are less pronounced at netural pH, and this can be an advantage when a less sensitive method is needed, for example when investigating cells that have large amount of background, or induced damage is high (Angelis et al., 1999). Alkaline version of the comet assay was presented by Singh et al. (1988) in which DNA is allowed to unwind at pH>13. In their paper DNA damage was measured as the migrating distance of DNA from the nucleoid. In 1990, Olive et al. (1990) also under alkaline conditions developed the concept of the tail "moment", a combination of tail length and DNA content, as a measure of DNA damage. Also, in 1990, the name "Comet assay" was introduced and the application of the first image analysis program was described (Olive, 1989; Olive et al., 1990; Sviežená et al., 2004). Image analysis has become essential for objective measurement of low–dose effects, or for distinguishing small differences among sub–populations of cells. Strong alkaline conditions enabled clearer images, and besides SSBs other types of DNA damage could be detected, such as alkaline labile sites (Tice et al., 2000). Olive et al. (1990) revealed that employing milder alkaline (pH 12.3) conditions prevents conversion of alkaline labile sites into breaks. Under the pH>13 alkali labile sites are formed into SSBs, thus revealing otherwise hidden damage. Therefore, by modifying the pH of lysis and/or electrophoresis over the range of 9.5–13.5, one can apply a comet assay of different sensitivity, but of similar limits of detection (Collins et al., 1997; Angelis et al., 1999;

In its basic form, comet assay gives limited information on the type of DNA damage being measured. Single strand breaks detected by standard alkaline method are not the most interesting of lesions, because they are quickly repaired, and are not regarded as a significant lethal or mutagenic lesion. Many genotoxic agents do not induce strand breaks directly. They may create apurinic/pyrimidinic (AP) sites, which are alkali labile and are probably converted to breaks while DNA is in the electrophoresis solution at high pH. Furthermore, it is not possible to determine whether the high level of breaks in the comet assay is the indicator of high damage or efficient repair, due to temporary presence of breaks in the lesions repair via base excision or nucleotide excision (Collins et al., 1997). More recently, the assay was modified further to enable the detection of specific kinds of DNA damage by combining the assay with the use of a purified DNA repair enzymes, which recognize the lesions along the DNA and convert them into the breaks expressed as an increase in comet DNA migration. Briefly, the DNA in the gel, following lysis, is digested with a lesion–specific repair endonuclease, which introduces breaks at sites of damage. In principle, any lesion for which a repair endonuclease exists can be detected in this way. To date, endonucleases most commonly used in the modified comet assay are the bacterial enzymes which recognize different types of oxidative damage. The first enzyme to be used was endonuclease III, a glycosylase which recognizes a variety of oxidized pyrimidines in DNA and removes them, leaving an AP site (Doetsch et al., 1987). Formamidopyrimidine DNA glycosylase (FPG) has the ability to convert altered purines, including 8-oxoguanine, into DNA breaks (Collins, 2007; Dušinská & Collins, 1996). When using these enzymes to measure oxidative DNA damage the usual practice is to incubate a slide with buffer alone in parallel with the enzyme slide. Slide with buffer would be a valid control slide, due to small increase in strand breaks on incubation without enzyme (Collins, 2009; Gajski et al., 2008). Recently, a mammalian analogue of FPG, 8–oxoguanidine DNA glycosylase, or OGG1, has been applied in the comet assay (Smith et al., 2006). OGG1 is the major base extension repair

of segments between sister chromatids is not known in detail at present (Palani-Kumur & Panneerselvam, 2008). Recent studies revealed the nucleotide pool imbalance can have severe consequences on DNA metabolism and it is critical in SCE formation. The modulation of SCE by DNA precursors raises the possibility that DNA changes are responsible for the induction of SCE and mutations in mammalian cells (Popescu, 1999; Ashman & Davidson, 1981). While increased levels of CA have been associated with increased cancer risk (Hagmar et al, 1994, 1998), a similar conclusion has not been reached for SCE or MN. However, high levels of SCE and MN frequency have been observed in persons at higher cancer risk due to occupational or environmental exposure to a wide variety of carcinogens (Fučić et al, 2000; Vaglenov et al, 1999; Fenech et al, 1997). Evidence of CA increases, mainly as structural chromosomal aberrations in occupationally exposed populations. The sensitivity of SCE is lower than that of the CA test in detecting genotoxic effects related to pesticide exposure and fewer data are therefore, available for MN than for the other cytogenetic endopoints (Bolognesi, 2003). Exposure to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation. Biomarkers of effect are generally pre-clinical indicators of abnormalities and the most frequently used in genotoxicity assessment are comet assay and cytokinesis-block micronucleus test that are being proposed as a useful biomarkers for early effects. The cytogenetic endpoints can give indication of genetic damage; hence they are used as effective biomarkers of exposure *in vivo* and *in vitro*. In recent years, the comet (single-cell gel) assay has been established as a useful technique for studying DNA damage and repair (Tice, 1995). The comet assay combines the single-cell approach typical of cytogenetic assays with the simplicity of biochemical techniques for detecting DNA single strand breaks. The advantages of the comet assay include its simple and rapid performance, its sensitivity for detecting DNA damage, and the use of extremely small cell samples (Hartmann et al., 1998). The advantage of micronucleus assay is its simplicity and speed over the assay of chromosomal aberration. Both techniques have become an important tool for genotoxicity testing because of their simplicity of scoring and wide applicability in different cell types. These techniques became the methods of choice for studies of environmental and occupational exposure to air pollutants, metals, radiation, pesticides, and other xenobiotics.

#### **3.1 Comet assay**

The comet assay, also known as the single-cell gel electrophoresis assay (SCGE), is a method for detecting DNA strand breakage (single-strand DNA breaks, alkali-labile sites, doublestrand DNA breaks, incomplete repair sites, and inter-strand cross-links) in virtually any nucleated cell (Collins et al., 2004, 2008; Shaposhnikov et al., 2008).

First quantification of DNA damage in individual cells was done by Rydberg & Johanson (1978). After gamma–irradiation they embedded cells in agarose on microscopic slides and lysed under mild alkali conditions. Upon neutralization, the cells were stained with acridin orange and the extent of DNA damage was measured by the ratio of green (indicating double–stranded DNA) to red (indicating single–strand DNA) fluorescence. To enhance the sensitivity for the DNA damage detection, Östling & Johanson (1984) proposed that strand breaks would enable DNA loops to stretch out upon electrophoresis, so the microelectrophoretic procedure under pH of 9.5 was developed. As reported by Singh et al. (1988) this pH of 9.5 is below the limit for DNA unwinding, and was notify to detect only double strand breaks (DSB), with more strongly alkaline conditions (pH 10 or above) needed for unwinding and detection of single strand breaks (SSB). It has been shown that

of segments between sister chromatids is not known in detail at present (Palani-Kumur & Panneerselvam, 2008). Recent studies revealed the nucleotide pool imbalance can have severe consequences on DNA metabolism and it is critical in SCE formation. The modulation of SCE by DNA precursors raises the possibility that DNA changes are responsible for the induction of SCE and mutations in mammalian cells (Popescu, 1999; Ashman & Davidson, 1981). While increased levels of CA have been associated with increased cancer risk (Hagmar et al, 1994, 1998), a similar conclusion has not been reached for SCE or MN. However, high levels of SCE and MN frequency have been observed in persons at higher cancer risk due to occupational or environmental exposure to a wide variety of carcinogens (Fučić et al, 2000; Vaglenov et al, 1999; Fenech et al, 1997). Evidence of CA increases, mainly as structural chromosomal aberrations in occupationally exposed populations. The sensitivity of SCE is lower than that of the CA test in detecting genotoxic effects related to pesticide exposure and fewer data are therefore, available for MN than for the other cytogenetic endopoints (Bolognesi, 2003). Exposure to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation. Biomarkers of effect are generally pre-clinical indicators of abnormalities and the most frequently used in genotoxicity assessment are comet assay and cytokinesis-block micronucleus test that are being proposed as a useful biomarkers for early effects. The cytogenetic endpoints can give indication of genetic damage; hence they are used as effective biomarkers of exposure *in vivo* and *in vitro*. In recent years, the comet (single-cell gel) assay has been established as a useful technique for studying DNA damage and repair (Tice, 1995). The comet assay combines the single-cell approach typical of cytogenetic assays with the simplicity of biochemical techniques for detecting DNA single strand breaks. The advantages of the comet assay include its simple and rapid performance, its sensitivity for detecting DNA damage, and the use of extremely small cell samples (Hartmann et al., 1998). The advantage of micronucleus assay is its simplicity and speed over the assay of chromosomal aberration. Both techniques have become an important tool for genotoxicity testing because of their simplicity of scoring and wide applicability in different cell types. These techniques became the methods of choice for studies of environmental and occupational exposure to air pollutants, metals, radiation, pesticides, and other xenobiotics.

The comet assay, also known as the single-cell gel electrophoresis assay (SCGE), is a method for detecting DNA strand breakage (single-strand DNA breaks, alkali-labile sites, doublestrand DNA breaks, incomplete repair sites, and inter-strand cross-links) in virtually any

First quantification of DNA damage in individual cells was done by Rydberg & Johanson (1978). After gamma–irradiation they embedded cells in agarose on microscopic slides and lysed under mild alkali conditions. Upon neutralization, the cells were stained with acridin orange and the extent of DNA damage was measured by the ratio of green (indicating double–stranded DNA) to red (indicating single–strand DNA) fluorescence. To enhance the sensitivity for the DNA damage detection, Östling & Johanson (1984) proposed that strand breaks would enable DNA loops to stretch out upon electrophoresis, so the microelectrophoretic procedure under pH of 9.5 was developed. As reported by Singh et al. (1988) this pH of 9.5 is below the limit for DNA unwinding, and was notify to detect only double strand breaks (DSB), with more strongly alkaline conditions (pH 10 or above) needed for unwinding and detection of single strand breaks (SSB). It has been shown that

nucleated cell (Collins et al., 2004, 2008; Shaposhnikov et al., 2008).

**3.1 Comet assay** 

neutral or mildly alkaline comet assay has the same limit of detection of DNA damage (SSB) as the alkaline comet assay, although the use of neutral pH does effect the comet image obtained (Collins, 2004). The comet tails are less pronounced at netural pH, and this can be an advantage when a less sensitive method is needed, for example when investigating cells that have large amount of background, or induced damage is high (Angelis et al., 1999).

Alkaline version of the comet assay was presented by Singh et al. (1988) in which DNA is allowed to unwind at pH>13. In their paper DNA damage was measured as the migrating distance of DNA from the nucleoid. In 1990, Olive et al. (1990) also under alkaline conditions developed the concept of the tail "moment", a combination of tail length and DNA content, as a measure of DNA damage. Also, in 1990, the name "Comet assay" was introduced and the application of the first image analysis program was described (Olive, 1989; Olive et al., 1990; Sviežená et al., 2004). Image analysis has become essential for objective measurement of low–dose effects, or for distinguishing small differences among sub–populations of cells. Strong alkaline conditions enabled clearer images, and besides SSBs other types of DNA damage could be detected, such as alkaline labile sites (Tice et al., 2000). Olive et al. (1990) revealed that employing milder alkaline (pH 12.3) conditions prevents conversion of alkaline labile sites into breaks. Under the pH>13 alkali labile sites are formed into SSBs, thus revealing otherwise hidden damage. Therefore, by modifying the pH of lysis and/or electrophoresis over the range of 9.5–13.5, one can apply a comet assay of different sensitivity, but of similar limits of detection (Collins et al., 1997; Angelis et al., 1999; Wong et al., 2005).

In its basic form, comet assay gives limited information on the type of DNA damage being measured. Single strand breaks detected by standard alkaline method are not the most interesting of lesions, because they are quickly repaired, and are not regarded as a significant lethal or mutagenic lesion. Many genotoxic agents do not induce strand breaks directly. They may create apurinic/pyrimidinic (AP) sites, which are alkali labile and are probably converted to breaks while DNA is in the electrophoresis solution at high pH. Furthermore, it is not possible to determine whether the high level of breaks in the comet assay is the indicator of high damage or efficient repair, due to temporary presence of breaks in the lesions repair via base excision or nucleotide excision (Collins et al., 1997).

More recently, the assay was modified further to enable the detection of specific kinds of DNA damage by combining the assay with the use of a purified DNA repair enzymes, which recognize the lesions along the DNA and convert them into the breaks expressed as an increase in comet DNA migration. Briefly, the DNA in the gel, following lysis, is digested with a lesion–specific repair endonuclease, which introduces breaks at sites of damage. In principle, any lesion for which a repair endonuclease exists can be detected in this way. To date, endonucleases most commonly used in the modified comet assay are the bacterial enzymes which recognize different types of oxidative damage. The first enzyme to be used was endonuclease III, a glycosylase which recognizes a variety of oxidized pyrimidines in DNA and removes them, leaving an AP site (Doetsch et al., 1987). Formamidopyrimidine DNA glycosylase (FPG) has the ability to convert altered purines, including 8-oxoguanine, into DNA breaks (Collins, 2007; Dušinská & Collins, 1996). When using these enzymes to measure oxidative DNA damage the usual practice is to incubate a slide with buffer alone in parallel with the enzyme slide. Slide with buffer would be a valid control slide, due to small increase in strand breaks on incubation without enzyme (Collins, 2009; Gajski et al., 2008). Recently, a mammalian analogue of FPG, 8–oxoguanidine DNA glycosylase, or OGG1, has been applied in the comet assay (Smith et al., 2006). OGG1 is the major base extension repair

Presence of

Kirsch-Volders & Fenech, 2001).

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 129

Heddle (1973) who showed that this assay provided a simple method to detect the genotoxic potential of mutagens after *in vivo* exposure of animals using dividing cell population such as bone marrow erythrocytes. A few years later it was shown by Countryman & Heddle (1976) that peripheral blood lymphocytes could also be used for the *in vivo* micronucleus approach and they recommended using micronuclei as a biomarker in testing schemes. As only dividing cells could express micronuclei, for the *in vitro* micronucleus studies it was necessary to establish cell proliferation and micronucleus induction at the same time. The decisive breakthrough of micronuclei as assay for *in vitro* genotoxicity testing came with work of Fenech & Morley (1986) that developed the CBMN assay. In the CBMN assay, cells that have completed nuclear division are blocked from performing cytokinesis using cytochalasin–B and are consequently readily identified by their binucleated appearance (Fenech & Morley, 1985, 1986). Whereas micronuclei originate from chromosome fragments or whole chromosomes that leg behind at anaphase during nuclear division, their occurrence is proven in binucleated cells. As a consequence, the CBMN assay has been shown to be more accurate and more sensitive than the conventional methods that do not distinguish between dividing and nondividing cells (Fenech & Morley, 1986; Fenech, 1991;

Baseline or spontaneous micronucleus frequencies in culture human lymphocytes provide an indicator of accumulated genetic damage occurred during the lifespan of circulating lymphocytes. The half-life and mean lifespan of T-lymphocytes has been estimated to be three to four years, respectively (Natarajan & Obe, 1982; Buckton et al., 1967). The observed genetic instability may also reflect accumulated mutations in the stem cell lineage from which the mature lymphocytes originate. The type of mutations that could contribute to spontaneous micronuclei include: 1) mutations to kinetochore proteins, centromeres, and spindle apparatus that could lead to unequal chromosome distribution or whole chromosome loss at anaphase, and 2) unrepaired DNA strand breaks induced endogenously or as a result of environmental mutagens, which may result in acentric chromosome fragments. Studies using kinetochore antibodies to identify whole chromosomes suggest that approximately 50% of spontaneously occurring micronuclei are the consequence of whole chromosome loss and the rest are presumably derived from acentric chromosome fragments (Thompson & Perry, 1988; Fenech & Morley, 1989; Eastmond & Tucker, 1989). The spontaneous micronucleus frequency refers to the incidence of micronucleus observed in the absence of the environmental risk or exposure that is being assessed. The spontaneous micronucleus frequency of a population has to be established to determine acceptable normal values as well as providing baseline data for those situations when spontaneous micronucleus frequencies for individuals is not known before exposure. Micronuclei harbouring whole chromosomes are primarily formed from failure of the mitotic spindle, kinetochore, or other parts of the mitotic apparatus or by damage to chromosomal substructures, alterations in cellular physiology, and mechanical disruption. An increased number of micronucleated cells is a biomarker of genotoxic effects and can reflect exposure to agents with clastogenic modes of action (chromosome breaking; DNA as target) or aneugenic ones (aneuplodigenic; effect on chromosome number; mostly non-DNA target) (Albertini, 2000). The advantage of the CBMN assay is its ability to detect both clastogenic and aneugenic events, leading to structural and numerical chromosomal aberrations, respectively (Kirsch-Volders et al., 2002; Mateuca et al., 2006). Micronuclei observed in cultured lymphocytes are believed to arise primarily *in vitro* from: 1) chromatid-type chromosomal aberrations formed during DNA replication on a damaged template, 2)

enzyme that initiates the repair of oxidative base lesion. It is a bifunctional DNA glycosylase possessing both DNA glycosylase and AP lyase activities (Boiteux & Radicella, 2000). In human it is named hOGG1. hOGG1 recognizes both 8-oxoguanine (8-oxodG) and 8 oxoadenine (8-oxodA) and removes these oxidized bases from double-stranded DNA, initiating the base lesion repair process (Smith et al., 2006). 8-hidroxy-2-deoxyguanosine (8- OHdG) lesion causes G→T and A→C transversions (Moriya, 1993) that have been reported as the sites of spontaneous oncogene expression and ultimately cancer manifestation (Valko et al., 2004; Bartsch, 1996; Shinmura & Yokota, 2001). Deletion of the hOGG1 gene was shown to be associated with accumulation of 8-OHdG lesion and increase in mutational risk (Hansen & Kelley, 2000; El-Zein et al., 2010).

For evaluation of DNA specific damage the comet assay has also been coupled with the method of fluorescent in situ hybridization (Comet–FISH). Since its initial development, Comet–FISH has been used to handle a number of quite different questiones. First, it was used to identify chromosome-specific areas on electrostretched DNA fibres and to determine their special distribution (Santos et al., 1997). Further applications were then to detect region–specific repair activities (Horvathova et al., 2004; McKenna et al., 2003; Mellon et al., 1986), genotoxic effects in total DNA and in telomeres (Arutyunyan et al., 2005), or in tumor relevant genes, like TP53 (Schaeferhenrich et al., 2003). Also specific chromosomal alternations (Harreus et al., 2004) were studied as were genetic instabilities (Tirukalikundram et al., 2005). Comet–FISH has also been used to discriminate between DNA double-and single-strand breaks (Fernandez et al., 2001). Whereas results from the Comet assay alone reflect only the level of overall DNA damage, the combination with the FISH-technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively). If two fluorescence signals are obtained with a probe for a particular gene in the head of a comet, this indicates that the gene is in an undamaged region of DNA, whereas the appearance of a spot or several spots in the tail of a comet indicates that a break or breaks has/have occurred in the proximity of the probed gene.

#### **3.2 Micronucleus assay**

Human exposure to environmental mutagens can be monitored using cytokinesis–block micronucleus (CBMN) assay (Natarajan et al., 1996) which is an efficient biomarker for diagnosing genetic damage and/or genome instability at the chromosome/molecular level in animal and/or human cells. It provides a comprehensive measure of chromosome breakage, chromosome loss, chromosome rearrangments, non-disjuction, gene amplification, necrosis and apoptosis (Fenech, 2000, 2006; Kirsch-Volders et al., 2000). In the classical cytogenetic techniques, chromosomes are studied directly by observing and counting aberrations in methaphases (Natarajan & Obe, 1982). The complexity and laboriousness of enumerating aberrations in methaphase and the confounding effect of artefactual loss of chromosomes from methaphase preparations has stimulated the development of a simpler system of measuring chromosome damage. More than a century ago micronuclei were described in the cytoplasm of erythrocytes and were called "fragment of nuclear material" by Howell or "corpuscules intraglobulaires" in the terminology of Jolly in the late 1800s and early 1900s. To the hematologists these structures are known as "Howell-Jolly bodies". Similar structures were described in mouse and rat embryos and in *Vicia faba* (Thoday, 1951) and called "fragment nuclei" or "micronuclei". In the early 1970s the term micronucleus test was suggested for the first time by Boller & Schmidt (1970) and

enzyme that initiates the repair of oxidative base lesion. It is a bifunctional DNA glycosylase possessing both DNA glycosylase and AP lyase activities (Boiteux & Radicella, 2000). In human it is named hOGG1. hOGG1 recognizes both 8-oxoguanine (8-oxodG) and 8 oxoadenine (8-oxodA) and removes these oxidized bases from double-stranded DNA, initiating the base lesion repair process (Smith et al., 2006). 8-hidroxy-2-deoxyguanosine (8- OHdG) lesion causes G→T and A→C transversions (Moriya, 1993) that have been reported as the sites of spontaneous oncogene expression and ultimately cancer manifestation (Valko et al., 2004; Bartsch, 1996; Shinmura & Yokota, 2001). Deletion of the hOGG1 gene was shown to be associated with accumulation of 8-OHdG lesion and increase in mutational risk

For evaluation of DNA specific damage the comet assay has also been coupled with the method of fluorescent in situ hybridization (Comet–FISH). Since its initial development, Comet–FISH has been used to handle a number of quite different questiones. First, it was used to identify chromosome-specific areas on electrostretched DNA fibres and to determine their special distribution (Santos et al., 1997). Further applications were then to detect region–specific repair activities (Horvathova et al., 2004; McKenna et al., 2003; Mellon et al., 1986), genotoxic effects in total DNA and in telomeres (Arutyunyan et al., 2005), or in tumor relevant genes, like TP53 (Schaeferhenrich et al., 2003). Also specific chromosomal alternations (Harreus et al., 2004) were studied as were genetic instabilities (Tirukalikundram et al., 2005). Comet–FISH has also been used to discriminate between DNA double-and single-strand breaks (Fernandez et al., 2001). Whereas results from the Comet assay alone reflect only the level of overall DNA damage, the combination with the FISH-technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively). If two fluorescence signals are obtained with a probe for a particular gene in the head of a comet, this indicates that the gene is in an undamaged region of DNA, whereas the appearance of a spot or several spots in the tail of a comet indicates that a break or breaks has/have occurred in the proximity of

Human exposure to environmental mutagens can be monitored using cytokinesis–block micronucleus (CBMN) assay (Natarajan et al., 1996) which is an efficient biomarker for diagnosing genetic damage and/or genome instability at the chromosome/molecular level in animal and/or human cells. It provides a comprehensive measure of chromosome breakage, chromosome loss, chromosome rearrangments, non-disjuction, gene amplification, necrosis and apoptosis (Fenech, 2000, 2006; Kirsch-Volders et al., 2000). In the classical cytogenetic techniques, chromosomes are studied directly by observing and counting aberrations in methaphases (Natarajan & Obe, 1982). The complexity and laboriousness of enumerating aberrations in methaphase and the confounding effect of artefactual loss of chromosomes from methaphase preparations has stimulated the development of a simpler system of measuring chromosome damage. More than a century ago micronuclei were described in the cytoplasm of erythrocytes and were called "fragment of nuclear material" by Howell or "corpuscules intraglobulaires" in the terminology of Jolly in the late 1800s and early 1900s. To the hematologists these structures are known as "Howell-Jolly bodies". Similar structures were described in mouse and rat embryos and in *Vicia faba* (Thoday, 1951) and called "fragment nuclei" or "micronuclei". In the early 1970s the term micronucleus test was suggested for the first time by Boller & Schmidt (1970) and

(Hansen & Kelley, 2000; El-Zein et al., 2010).

the probed gene.

**3.2 Micronucleus assay** 

Heddle (1973) who showed that this assay provided a simple method to detect the genotoxic potential of mutagens after *in vivo* exposure of animals using dividing cell population such as bone marrow erythrocytes. A few years later it was shown by Countryman & Heddle (1976) that peripheral blood lymphocytes could also be used for the *in vivo* micronucleus approach and they recommended using micronuclei as a biomarker in testing schemes. As only dividing cells could express micronuclei, for the *in vitro* micronucleus studies it was necessary to establish cell proliferation and micronucleus induction at the same time. The decisive breakthrough of micronuclei as assay for *in vitro* genotoxicity testing came with work of Fenech & Morley (1986) that developed the CBMN assay. In the CBMN assay, cells that have completed nuclear division are blocked from performing cytokinesis using cytochalasin–B and are consequently readily identified by their binucleated appearance (Fenech & Morley, 1985, 1986). Whereas micronuclei originate from chromosome fragments or whole chromosomes that leg behind at anaphase during nuclear division, their occurrence is proven in binucleated cells. As a consequence, the CBMN assay has been shown to be more accurate and more sensitive than the conventional methods that do not distinguish between dividing and nondividing cells (Fenech & Morley, 1986; Fenech, 1991; Kirsch-Volders & Fenech, 2001).

Baseline or spontaneous micronucleus frequencies in culture human lymphocytes provide an indicator of accumulated genetic damage occurred during the lifespan of circulating lymphocytes. The half-life and mean lifespan of T-lymphocytes has been estimated to be three to four years, respectively (Natarajan & Obe, 1982; Buckton et al., 1967). The observed genetic instability may also reflect accumulated mutations in the stem cell lineage from which the mature lymphocytes originate. The type of mutations that could contribute to spontaneous micronuclei include: 1) mutations to kinetochore proteins, centromeres, and spindle apparatus that could lead to unequal chromosome distribution or whole chromosome loss at anaphase, and 2) unrepaired DNA strand breaks induced endogenously or as a result of environmental mutagens, which may result in acentric chromosome fragments. Studies using kinetochore antibodies to identify whole chromosomes suggest that approximately 50% of spontaneously occurring micronuclei are the consequence of whole chromosome loss and the rest are presumably derived from acentric chromosome fragments (Thompson & Perry, 1988; Fenech & Morley, 1989; Eastmond & Tucker, 1989). The spontaneous micronucleus frequency refers to the incidence of micronucleus observed in the absence of the environmental risk or exposure that is being assessed. The spontaneous micronucleus frequency of a population has to be established to determine acceptable normal values as well as providing baseline data for those situations when spontaneous micronucleus frequencies for individuals is not known before exposure. Micronuclei harbouring whole chromosomes are primarily formed from failure of the mitotic spindle, kinetochore, or other parts of the mitotic apparatus or by damage to chromosomal substructures, alterations in cellular physiology, and mechanical disruption. An increased number of micronucleated cells is a biomarker of genotoxic effects and can reflect exposure to agents with clastogenic modes of action (chromosome breaking; DNA as target) or aneugenic ones (aneuplodigenic; effect on chromosome number; mostly non-DNA target) (Albertini, 2000). The advantage of the CBMN assay is its ability to detect both clastogenic and aneugenic events, leading to structural and numerical chromosomal aberrations, respectively (Kirsch-Volders et al., 2002; Mateuca et al., 2006). Micronuclei observed in cultured lymphocytes are believed to arise primarily *in vitro* from: 1) chromatid-type chromosomal aberrations formed during DNA replication on a damaged template, 2)

Presence of

**4.1 Methodology 4.1.1 Chemicals** 

included.

mg/L at different time points.

**4.1.2 Blood sampling and treatment** 

**4.1.3 Cell viability (cytotoxicity) assay** 

**4.1.4 The alkaline comet (SCGE) assay** 

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 131

and nuclear dysfunctions caused by *in vitro* or *in vivo* exposure to toxic substances (Fenech,

Chromosome kit P was from Euroclone, Milan, Italy; RPMI 1640, was from Invitrogen, Carlsbad, CA, USA; cytochalasin B, histopaque-1119, ethidium bromide, low melting point (LMP) and normal melting point (NMP) agaroses were from Sigma, St Louis, MO, USA; heparinised vacutainer tubes from Becton Dickinson, Franklin Lakes, NJ, USA; acridine orange form Heidelberg, Germany; Giemsa from Merk, Darmastad, Germany; All other reagents used were laboratory-grade chemicals from Kemika, Zagreb, Croatia. DDT was administered as *p,p'*-DDT (Sulpeco, Bellefonte, PA, USA) in final concentration of 0.025

Whole blood samples were taken from a healthy female donor who had not been exposed to ionizing radiation, vaccinated or treated with drugs for a year before blood sampling. Whole venous blood was collected under sterile conditions in heparinised vacutainer tubes containing lithium heparin as anticoagulant. The comet assay and the micronucleus assay were conducted on whole blood cultivated at 37 °C in an atmosphere with 5% CO2 (Heraeus Heracell 240 incubator, Langenselbold, Germany). The whole blood was treated with 0.025 mg/L *p,p'*-DDT for 1, 2, 4, 8, 24 and 48 h for the cytotoxicity assay and alkaline comet assay and 24 and 48 h for the CBMN assay. In each experiment, a non treated control was

The indices of cell viability and necrosis were established by differential staining of human peripheral blood lymphocytes (HPBLs) with acridine orange and ethidium bromide using fluorescence microscopy (Duke & Cohen, 1992). Lymphocytes were isolated using a modified Ficoll-Histopaque centrifugation method (Singh, 2000). The slides were prepared using 200 μL of HPBLs and 2 μL of stain (acridine orange and ethidium bromide). The suspension mixed with dye was covered with a cover slip and analyzed under the epifluorescence microscope (Olympus AX 70, Tokyo, Japan) using a 60× objective and fluorescence filters of 515–560 nm. A total of 100 cells per repetition were examined. The nuclei of vital cells emitted a green fluorescence and necrotic cells emitted red fluorescence.

The alkaline comet assay was carried out as described by Singh et al. (1988). Briefly, after the exposure to *p,p'*-DDT, 5 L of whole blood was mixed with 100 L of 0.5% LMP agarose and added to fully frosted slides pre-coated with 0.6% NMP agarose. After solidifiying, the slides were covered with 0.5% LMP agarose, and the cells were lysed (2.5 M NaCl, 100 mM EDTANa2, 10mM Tris, 1% sodium sarcosinate, 1% Triton X-100, 10% dimethyl sulfoxide, pH 10) overnight at 4°C. After the lysis the slides were placed into alkaline solution (300 mM NaOH, 1 mM EDTANa2, pH 13) for 20 min at 4°C to allow DNA unwinding and subsequently electrophoresed for 20 min at 1 V/cm. Finally, the slides were neutralized in

**4. Genotoxicity of** *p,p'***-DDT in human peripheral blood lymphocytes** 

2006; Garaj- Vrhovac et al., 2008; Thomas & Fenech, 2011).

chromosome-type aberrations initiated before the mitosis and duplicated at replication, or 3) disturbances of mitotic apparatus leading to chromosome lagging. Micronuclei arising *in vivo*, inducible by both clastogenic and aneugenic mechanisms, can be scored in exfoliated epithelial cells (Salama et al., 1999) sampled, e.g., from buccal or nasal mucosa or urine, or in peripheral blood mononuclear cells e.g., isolated lymphocytes (Surrallés et al., 1996; Albertini, 2000). The CBMN assay is the preferred method for measuring micronuclei (MNi) in cultured human and/or mammalian cells because scoring is specifically restricted to once–divided cells. For scoring MNi uniform criteria should be used. Only MNi not exceeding 1/3 of the main nucleus diameter, clearly separable from the main nucleus and with distinct borders and of the same color as the nucleus, should be scored. In practice, 1000–2000 cells are often scored per subject in lymphocyte studies utilising the CBMN technique, while more cells (3000–5000 per subject) are evaluated in epithelial cells due to the lower baseline MNi frequency. In the CBMN method, only binucleate cells should be analysed for MNi; further divisions of a binucleate cell, usually resulting in cells with 3–4 nuclei, are highly irregular and show high MNi rates (Fenech et al., 2003).

The discovery of kinetochore-specific antibodies in the serum of scleroderma CREST (*Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly and Telangiectasia*) patients (Moroy et al., 1980) has made it possible to determine the contents of micronuclei. Kinetochore immunofluorescence has been rapidly developed for the *in situ* detection of aneuploidy and chromosome breakage in human micronuclei (Degrassi & Tanzarella, 1988; Hennig et al., 1988; Eastmond & Tucker, 1989). Also, the FISH technique using chromosomespecific DNA probes has improved the detection and evaluation of structural chromosomal aberrations. The combination of the micronucleus assay with FISH using a DNA probe specific to the centromeric regions, or with antibodies that specifically stain kinetochore proteins, provides the methodology to distinguish between micronuclei containing either one or several whole chromosomes, which are positively labeled (centromere positive micronucleus), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus) (Natarajan et al., 1996; Mateuca et al., 2006; Benameur et al., 2011). Except for cytogenetic damage measured by the number and distribution of micronuclei, according to the new criteria for micronuclei scoring, the CBMN assay also detects the nucleoplasmic bridges (NPBs), as well as nuclear buds (NBUDs). Current evidence suggests that NPBs derive from dicentric chromosomes which the centromeres have been pulled to the opposite poles of the cell during the anaphase stage, and are therefore indicative of the DNA mis-repair, chromosome rearrangement or telomere end-fusion. According to the new criteria applicable to the CBMN assay, NBUDs arise from the elimination of the amplified DNA and possibly from the elimination of the DNA-repair complexes, which therefore, may be considered a marker of gene amplification and altered gene dosage (Fenech, 2006; Fenech & Crott, 2002; Fenech et al., 2011; Garaj- Vrhovac et al., 2008; Lindberg et al., 2006; Thomas et al., 2003).

The significance of the CBMN assay lies in the fact that every cell in the system studied is scored cytologically for its viability status (necrosis, apoptosis), its mitotic status (mononucleated, metaphase, anaphase, binucleated, multinucleated) and its chromosomal instability or damage status (presence of MNi, NPBs, NBUDs and number of centromere probe signals amongst nuclei of binucleated cell if such molecular tools are used in combination with the assay). In this respect, the micronucleus assay has evolved into a comprehensive method employed in measuring chromosomal instability of the phenotype and altered cell viability and represents an effective tool to be used in research of cellular and nuclear dysfunctions caused by *in vitro* or *in vivo* exposure to toxic substances (Fenech, 2006; Garaj- Vrhovac et al., 2008; Thomas & Fenech, 2011).

## **4. Genotoxicity of** *p,p'***-DDT in human peripheral blood lymphocytes**

## **4.1 Methodology**

130 Insecticides – Pest Engineering

chromosome-type aberrations initiated before the mitosis and duplicated at replication, or 3) disturbances of mitotic apparatus leading to chromosome lagging. Micronuclei arising *in vivo*, inducible by both clastogenic and aneugenic mechanisms, can be scored in exfoliated epithelial cells (Salama et al., 1999) sampled, e.g., from buccal or nasal mucosa or urine, or in peripheral blood mononuclear cells e.g., isolated lymphocytes (Surrallés et al., 1996; Albertini, 2000). The CBMN assay is the preferred method for measuring micronuclei (MNi) in cultured human and/or mammalian cells because scoring is specifically restricted to once–divided cells. For scoring MNi uniform criteria should be used. Only MNi not exceeding 1/3 of the main nucleus diameter, clearly separable from the main nucleus and with distinct borders and of the same color as the nucleus, should be scored. In practice, 1000–2000 cells are often scored per subject in lymphocyte studies utilising the CBMN technique, while more cells (3000–5000 per subject) are evaluated in epithelial cells due to the lower baseline MNi frequency. In the CBMN method, only binucleate cells should be analysed for MNi; further divisions of a binucleate cell, usually resulting in cells with 3–4

The discovery of kinetochore-specific antibodies in the serum of scleroderma CREST (*Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly and Telangiectasia*) patients (Moroy et al., 1980) has made it possible to determine the contents of micronuclei. Kinetochore immunofluorescence has been rapidly developed for the *in situ* detection of aneuploidy and chromosome breakage in human micronuclei (Degrassi & Tanzarella, 1988; Hennig et al., 1988; Eastmond & Tucker, 1989). Also, the FISH technique using chromosomespecific DNA probes has improved the detection and evaluation of structural chromosomal aberrations. The combination of the micronucleus assay with FISH using a DNA probe specific to the centromeric regions, or with antibodies that specifically stain kinetochore proteins, provides the methodology to distinguish between micronuclei containing either one or several whole chromosomes, which are positively labeled (centromere positive micronucleus), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus) (Natarajan et al., 1996; Mateuca et al., 2006; Benameur et al., 2011). Except for cytogenetic damage measured by the number and distribution of micronuclei, according to the new criteria for micronuclei scoring, the CBMN assay also detects the nucleoplasmic bridges (NPBs), as well as nuclear buds (NBUDs). Current evidence suggests that NPBs derive from dicentric chromosomes which the centromeres have been pulled to the opposite poles of the cell during the anaphase stage, and are therefore indicative of the DNA mis-repair, chromosome rearrangement or telomere end-fusion. According to the new criteria applicable to the CBMN assay, NBUDs arise from the elimination of the amplified DNA and possibly from the elimination of the DNA-repair complexes, which therefore, may be considered a marker of gene amplification and altered gene dosage (Fenech, 2006; Fenech & Crott, 2002; Fenech et al., 2011; Garaj- Vrhovac et al.,

The significance of the CBMN assay lies in the fact that every cell in the system studied is scored cytologically for its viability status (necrosis, apoptosis), its mitotic status (mononucleated, metaphase, anaphase, binucleated, multinucleated) and its chromosomal instability or damage status (presence of MNi, NPBs, NBUDs and number of centromere probe signals amongst nuclei of binucleated cell if such molecular tools are used in combination with the assay). In this respect, the micronucleus assay has evolved into a comprehensive method employed in measuring chromosomal instability of the phenotype and altered cell viability and represents an effective tool to be used in research of cellular

nuclei, are highly irregular and show high MNi rates (Fenech et al., 2003).

2008; Lindberg et al., 2006; Thomas et al., 2003).

## **4.1.1 Chemicals**

Chromosome kit P was from Euroclone, Milan, Italy; RPMI 1640, was from Invitrogen, Carlsbad, CA, USA; cytochalasin B, histopaque-1119, ethidium bromide, low melting point (LMP) and normal melting point (NMP) agaroses were from Sigma, St Louis, MO, USA; heparinised vacutainer tubes from Becton Dickinson, Franklin Lakes, NJ, USA; acridine orange form Heidelberg, Germany; Giemsa from Merk, Darmastad, Germany; All other reagents used were laboratory-grade chemicals from Kemika, Zagreb, Croatia. DDT was administered as *p,p'*-DDT (Sulpeco, Bellefonte, PA, USA) in final concentration of 0.025 mg/L at different time points.

## **4.1.2 Blood sampling and treatment**

Whole blood samples were taken from a healthy female donor who had not been exposed to ionizing radiation, vaccinated or treated with drugs for a year before blood sampling. Whole venous blood was collected under sterile conditions in heparinised vacutainer tubes containing lithium heparin as anticoagulant. The comet assay and the micronucleus assay were conducted on whole blood cultivated at 37 °C in an atmosphere with 5% CO2 (Heraeus Heracell 240 incubator, Langenselbold, Germany). The whole blood was treated with 0.025 mg/L *p,p'*-DDT for 1, 2, 4, 8, 24 and 48 h for the cytotoxicity assay and alkaline comet assay and 24 and 48 h for the CBMN assay. In each experiment, a non treated control was included.

## **4.1.3 Cell viability (cytotoxicity) assay**

The indices of cell viability and necrosis were established by differential staining of human peripheral blood lymphocytes (HPBLs) with acridine orange and ethidium bromide using fluorescence microscopy (Duke & Cohen, 1992). Lymphocytes were isolated using a modified Ficoll-Histopaque centrifugation method (Singh, 2000). The slides were prepared using 200 μL of HPBLs and 2 μL of stain (acridine orange and ethidium bromide). The suspension mixed with dye was covered with a cover slip and analyzed under the epifluorescence microscope (Olympus AX 70, Tokyo, Japan) using a 60× objective and fluorescence filters of 515–560 nm. A total of 100 cells per repetition were examined. The nuclei of vital cells emitted a green fluorescence and necrotic cells emitted red fluorescence.

### **4.1.4 The alkaline comet (SCGE) assay**

The alkaline comet assay was carried out as described by Singh et al. (1988). Briefly, after the exposure to *p,p'*-DDT, 5 L of whole blood was mixed with 100 L of 0.5% LMP agarose and added to fully frosted slides pre-coated with 0.6% NMP agarose. After solidifiying, the slides were covered with 0.5% LMP agarose, and the cells were lysed (2.5 M NaCl, 100 mM EDTANa2, 10mM Tris, 1% sodium sarcosinate, 1% Triton X-100, 10% dimethyl sulfoxide, pH 10) overnight at 4°C. After the lysis the slides were placed into alkaline solution (300 mM NaOH, 1 mM EDTANa2, pH 13) for 20 min at 4°C to allow DNA unwinding and subsequently electrophoresed for 20 min at 1 V/cm. Finally, the slides were neutralized in

Presence of

**4.2.2 Induction of DNA strand breaks** 

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 133

The whole blood was exposed to aqueous solution of *p,p'*-DDT (0.025 mg/L) and the DNA damage in HPBLs was determined with the alkaline comet assay. Figure 3 represents different levels of DNA fragmentation between non-exposed sample and sample exposed to aqueous solution of *p,p'*-DDT. Statistically significant (*P*<0.05) increase in the amount of

DNA strand breaks was observed after all exposure times to *p,p'*-DDT (Figure 4).

Fig. 3. Comet assay microphotographs represent undamaged lymphocytes from the unexposed sample (A). Image (B) represents damaged lymphocytes that have comet

Fig. 4. Tail length (length of DNA migration) as comet assay parameter in human peripheral blood lymphocytes after exposure to low concentration of aqueous *p,p'*-DDT. \* Statistically

The genotoxic activity of *p,p'*-DDT (0.025 mg/L) was further evaluated using the CBMN assay. Figure 5 represents binucleated lymphocytes from the non-exposed sample and samples exposed to aqueous solution of *p,p'*-DDT. Following *p,p'*-DDT treatment for 24 and 48 h, increase in the frequency of MNi was detected for both exposure times (Table 8). Additionally, statistically significant induction (*P*<0.05) of NBPs and NBUDs was also

**4.2.3 Induction of micronuclei, nucleoplasmic bridges and nuclear buds** 

A B

significant compared to corresponding control (*P*<0.05).

observed following *p,p'*-DDT treatment for 24 and 48 h (Table 9).

appearance after the treatment with aqueous solution of *p,p'*-DDT.

0.4 M Tris buffer (pH 7.5) for 5 minutes 3 times, stained with EtBr (20 g/mL) and analyzed at 250× magnification using an epifluorescence microscope (Zeiss, Göttingen, Germany) connected through camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd., Haverhill, Suffolk, UK). The tail length parameter was used to measure the level of DNA damage and a total of 100 randomly captured nuclei were examined from each slide. Tail length (i.e. the length of DNA migration) is related directly to the DNA fragment size and is presented in micrometers (µm). It was calculated from the centre of the nucleus.

## **4.1.5 Cytokinesis-blocked micronucleus (CBMN) assay**

The micronucleus assay was performed in agreement with guidelines by Fenech & Morley, (1985). After the exposure to *p,p'*-DDT the whole blood (500 µL) was incubated in a Euroclone medium at 37 °C in an atmosphere of 5% CO2 in air. Cytochalasin-B was added at a final concentration of 3 µg/mL 44 h after the culture was started. The cultures were harvested at 72 h. The lymphocytes were fixed in methanol-acetic acid solution (3:1), airdried and stained with 5% Giemsa solution. All slides were randomised and coded prior to analysis. Binuclear lymphocytes were analyzed under a light microscope (Olympus CX41, Tokyo, Japan) at 400× magnification. Micronuclei, nucleoplasmic bridges and nuclear buds were counted in 1000 binucleated cells and were scored according to the HUMN project criteria published by Fenech et al. (2003).

## **4.1.6 Statistics**

For the results of the comet assay measured after treatment with *p,p'*-DDT statistical evaluation was performed using Statistica 5.0 package (StaSoft, Tulsa, OK, USA). Multiple comparisons between groups were done by means of ANOVA on log-transformed data. Post hoc analyses of differences were done by using the Scheffé test. Differences in the frequency of micronuclei, nucleoplasmic bridges, and nuclear buds were assessed using the chi-square test. *P*<0.05 was considered significant.

## **4.2 Results**

## **4.2.1 Vital staining using ethidium bromide and acridine orange**

The viability of HPBLs exposed to aqueous *p,p'*-DDT (0.025 mg/L) for different lengths of time as determined by acridine orange and ethidium bromide staining, using fluorescence microscopy was not significantly affected (data not shown). Changes were determined according to the different staining of the nucleus (Figure 2).

Fig. 2. Cell viability microphotographs represent viable lymphocyte from the un-exposed sample (A; green), and dead lymphocyte (B; red) from sample treated with aqueous solution of *p,p'*-DDT.

## **4.2.2 Induction of DNA strand breaks**

132 Insecticides – Pest Engineering

0.4 M Tris buffer (pH 7.5) for 5 minutes 3 times, stained with EtBr (20 g/mL) and analyzed at 250× magnification using an epifluorescence microscope (Zeiss, Göttingen, Germany) connected through camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd., Haverhill, Suffolk, UK). The tail length parameter was used to measure the level of DNA damage and a total of 100 randomly captured nuclei were examined from each slide. Tail length (i.e. the length of DNA migration) is related directly to the DNA fragment size and is presented in micrometers (µm). It was calculated from the centre of the nucleus.

The micronucleus assay was performed in agreement with guidelines by Fenech & Morley, (1985). After the exposure to *p,p'*-DDT the whole blood (500 µL) was incubated in a Euroclone medium at 37 °C in an atmosphere of 5% CO2 in air. Cytochalasin-B was added at a final concentration of 3 µg/mL 44 h after the culture was started. The cultures were harvested at 72 h. The lymphocytes were fixed in methanol-acetic acid solution (3:1), airdried and stained with 5% Giemsa solution. All slides were randomised and coded prior to analysis. Binuclear lymphocytes were analyzed under a light microscope (Olympus CX41, Tokyo, Japan) at 400× magnification. Micronuclei, nucleoplasmic bridges and nuclear buds were counted in 1000 binucleated cells and were scored according to the HUMN project

For the results of the comet assay measured after treatment with *p,p'*-DDT statistical evaluation was performed using Statistica 5.0 package (StaSoft, Tulsa, OK, USA). Multiple comparisons between groups were done by means of ANOVA on log-transformed data. Post hoc analyses of differences were done by using the Scheffé test. Differences in the frequency of micronuclei, nucleoplasmic bridges, and nuclear buds were assessed using the

The viability of HPBLs exposed to aqueous *p,p'*-DDT (0.025 mg/L) for different lengths of time as determined by acridine orange and ethidium bromide staining, using fluorescence microscopy was not significantly affected (data not shown). Changes were determined

Fig. 2. Cell viability microphotographs represent viable lymphocyte from the un-exposed sample (A; green), and dead lymphocyte (B; red) from sample treated with aqueous solution

**4.1.5 Cytokinesis-blocked micronucleus (CBMN) assay** 

criteria published by Fenech et al. (2003).

chi-square test. *P*<0.05 was considered significant.

**4.2.1 Vital staining using ethidium bromide and acridine orange** 

according to the different staining of the nucleus (Figure 2).

A B

**4.1.6 Statistics** 

**4.2 Results** 

of *p,p'*-DDT.

The whole blood was exposed to aqueous solution of *p,p'*-DDT (0.025 mg/L) and the DNA damage in HPBLs was determined with the alkaline comet assay. Figure 3 represents different levels of DNA fragmentation between non-exposed sample and sample exposed to aqueous solution of *p,p'*-DDT. Statistically significant (*P*<0.05) increase in the amount of DNA strand breaks was observed after all exposure times to *p,p'*-DDT (Figure 4).

Fig. 3. Comet assay microphotographs represent undamaged lymphocytes from the unexposed sample (A). Image (B) represents damaged lymphocytes that have comet appearance after the treatment with aqueous solution of *p,p'*-DDT.

Fig. 4. Tail length (length of DNA migration) as comet assay parameter in human peripheral blood lymphocytes after exposure to low concentration of aqueous *p,p'*-DDT. \* Statistically significant compared to corresponding control (*P*<0.05).

## **4.2.3 Induction of micronuclei, nucleoplasmic bridges and nuclear buds**

The genotoxic activity of *p,p'*-DDT (0.025 mg/L) was further evaluated using the CBMN assay. Figure 5 represents binucleated lymphocytes from the non-exposed sample and samples exposed to aqueous solution of *p,p'*-DDT. Following *p,p'*-DDT treatment for 24 and 48 h, increase in the frequency of MNi was detected for both exposure times (Table 8). Additionally, statistically significant induction (*P*<0.05) of NBPs and NBUDs was also observed following *p,p'*-DDT treatment for 24 and 48 h (Table 9).

Presence of

**5. Discussion** 

Turusov et al., 2002).

Garaj-Vrhovac et al., 2008).

*p,p'*-DDT treatment.

exact mechanisms of action of this chemical.

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 135

Due to uncontrolled use for several decades, many pesticides, among them DDT, probably the best known and the most useful insecticide in the world has damaged wild life and might have adverse effects on human health. Because of its current use in countries of the Third World, DDT still enters environment and in that way it can still represent health risk for human population, even in countries that have banned its use almost 40 years ago (ATSDR, 2002; Crinnion, 2009; Ecobichon, 2000, 2001; Eskenazi et al., 2009; Gajski et al., 2007;

Large number of epidemiological studies regarding health risk of DDT confirmed that it represents major threat for wild life and human health (Aronson et al., 2010; Demers et al., 2000; Donato & Zani, 2010; Ecobichon, 1995; Martin et al., 2002; van Wendel de Joode et al., 2001; Wojtowicz et al., 2004, 2007; Woolcoot et al., 2001). Although tested in several genotoxicity studies on bacterial and animal models (Amer et al., 1996; Binneli et al., 2007, 2008a, 2008b; Canales-Aquirre, et al., 2011; Donnato et al., 1997a, 1997b; Galindo Reyes et al., 2002; Gauthier et al., 1999; Uppala et al., 2005) there is still necessity for conducting cytogenetic research regarding its genotoxicity employing sensitive methods to reveal the

Combining different cytogenetic methods may play an important role in assessing genotoxic damage from different environmental chemical or physical agents. With these methods it is possible to evaluate the level of primary DNA damage or the dynamics of its repair even after short-term exposure to these agents (Garcia-Sagredo, 2008; Gajski & Garaj-Vrhovac, 2008). The comet assay is a sensitive method for measuring and analyzing DNA damage at the single cell level, and can be used both in *in vivo* and *in vitro* (Collins, 2004; Collins et al., 2008; Dušinská & Collins, 2008). The comet assay detects single and double stranded breaks at the level of DNA molecule, sites of incomplete repair, alkali labile sites, and DNA-DNA and DNA-protein cross-links (Piperakis, 2009). Furthermore, micronucleus assay can indicate cellular and nuclear dysfunction caused by *in vitro* or *in vivo* exposure to toxic substances. It is a reliable method for measuring chromosomal instability and altered cellular viability (Fenech, 2009; Fenech et al., 2003). It includes micronuclei, which are biomarkers of chromosome breakage and whole chromosome loss, nucleoplasmic bridges, which are biomarkers of DNA misrepair and telomere end-fusions, and nuclear buds, which are biomarkers of elimination of amplified DNA and DNA repair complexes (Fenech, 2007;

Considering the lack of data on the effect of DDT on the cellular genome, and taking into account its usage in some countries of the Third World and its environmental persistence, the aim of this study was to evaluate the genotoxic potential of a low concentration of aqueous *p,p'*-DDT upon *in vitro* exposure of HPBLs of different duration, by using alkaline comet assay and CBMN assay. Our results showed that exposure of HPBLs to aqueous *p,p'*- DDT increased DNA damage in time dependent fashion as measured by the comet assay. In addition, CBMN assay parameters revealed a wider scale of chromosomal alterations after

Cytogenetic studies of DDT are mainly based on *in vitro* research on the animal models and its genotoxicity was evaluated in a variety of test systems. Results obtained by studying cytogenetic effects of DDT on DNA of shrimp larvae (*Litopenaeus stylirostris*) indicated that DDT causes DNA adducts and/or breaks (Galindo Reyes et al., 2002). DDT and its

Fig. 5. Cytokinesis-block miconucleus assay microphotographs represent binucleated lymphocyte from the un-exposed sample (A). Image (B) represents binucleated lymphocyte with two micronuclei, image (C) binucleated lymphocyte with nucleoplasmic bridge (right), and image (D) binucleated lymphocyte with nucler bud after the treatment with aqueous solution of *p,p'*-DDT.


Table 8. Incidence of micronuclei (MNi) as cytokinesis-block micrunucleus assay parameter in human peripheral blood lymphocytes after exposure to low concentration of aqueous *p,p'*-DDT, \* Statistically significant compared to corresponding control (*P*<0.05).


Table 9. Incidence of nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) as cytokinesis-block micrunucleus assay parameters in human peripheral blood lymphocytes after exposure to low concentration of aqueous *p,p'*-DDT, \* Statistically significant compared to corresponding control (*P*<0.05).

## **5. Discussion**

134 Insecticides – Pest Engineering

A B

C D

solution of *p,p'*-DDT.

Exposure time (h)

to corresponding control (*P*<0.05).

Exposure time (h)

Fig. 5. Cytokinesis-block miconucleus assay microphotographs represent binucleated lymphocyte from the un-exposed sample (A). Image (B) represents binucleated lymphocyte with two micronuclei, image (C) binucleated lymphocyte with nucleoplasmic bridge (right), and image (D) binucleated lymphocyte with nucler bud after the treatment with aqueous

Sample 1 MN 2 MNi 3 MNi Total no.

*p,p'*-DDT 28.50±13.44\* 1.50±2.12 0.50±0.71 33.00±19.80\*

*p,p'*-DDT 30.50±9.19\* 1.50±2.12 0.50±0.71 35.00±15.56\*

of NPBs

*p,p'*-DDT 10.50±0.71\* 10.50±4.95\*

*p,p'*-DDT 14.00±11.31\* 20.50±3.54\*

24 Control 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

48 Control 6.00±1.41 0.00±0.00 0.00±0.00 6.00±1.41

Table 8. Incidence of micronuclei (MNi) as cytokinesis-block micrunucleus assay parameter in human peripheral blood lymphocytes after exposure to low concentration of aqueous

Sample Total no.

24 Control 0.00±0.00 0.00±0.00

48 Control 0.00±0.00 2.50±0.71

cytokinesis-block micrunucleus assay parameters in human peripheral blood lymphocytes after exposure to low concentration of aqueous *p,p'*-DDT, \* Statistically significant compared

*p,p'*-DDT, \* Statistically significant compared to corresponding control (*P*<0.05).

Table 9. Incidence of nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) as

of MNi

Total no. of NBUDs Due to uncontrolled use for several decades, many pesticides, among them DDT, probably the best known and the most useful insecticide in the world has damaged wild life and might have adverse effects on human health. Because of its current use in countries of the Third World, DDT still enters environment and in that way it can still represent health risk for human population, even in countries that have banned its use almost 40 years ago (ATSDR, 2002; Crinnion, 2009; Ecobichon, 2000, 2001; Eskenazi et al., 2009; Gajski et al., 2007; Turusov et al., 2002).

Large number of epidemiological studies regarding health risk of DDT confirmed that it represents major threat for wild life and human health (Aronson et al., 2010; Demers et al., 2000; Donato & Zani, 2010; Ecobichon, 1995; Martin et al., 2002; van Wendel de Joode et al., 2001; Wojtowicz et al., 2004, 2007; Woolcoot et al., 2001). Although tested in several genotoxicity studies on bacterial and animal models (Amer et al., 1996; Binneli et al., 2007, 2008a, 2008b; Canales-Aquirre, et al., 2011; Donnato et al., 1997a, 1997b; Galindo Reyes et al., 2002; Gauthier et al., 1999; Uppala et al., 2005) there is still necessity for conducting cytogenetic research regarding its genotoxicity employing sensitive methods to reveal the exact mechanisms of action of this chemical.

Combining different cytogenetic methods may play an important role in assessing genotoxic damage from different environmental chemical or physical agents. With these methods it is possible to evaluate the level of primary DNA damage or the dynamics of its repair even after short-term exposure to these agents (Garcia-Sagredo, 2008; Gajski & Garaj-Vrhovac, 2008). The comet assay is a sensitive method for measuring and analyzing DNA damage at the single cell level, and can be used both in *in vivo* and *in vitro* (Collins, 2004; Collins et al., 2008; Dušinská & Collins, 2008). The comet assay detects single and double stranded breaks at the level of DNA molecule, sites of incomplete repair, alkali labile sites, and DNA-DNA and DNA-protein cross-links (Piperakis, 2009). Furthermore, micronucleus assay can indicate cellular and nuclear dysfunction caused by *in vitro* or *in vivo* exposure to toxic substances. It is a reliable method for measuring chromosomal instability and altered cellular viability (Fenech, 2009; Fenech et al., 2003). It includes micronuclei, which are biomarkers of chromosome breakage and whole chromosome loss, nucleoplasmic bridges, which are biomarkers of DNA misrepair and telomere end-fusions, and nuclear buds, which are biomarkers of elimination of amplified DNA and DNA repair complexes (Fenech, 2007; Garaj-Vrhovac et al., 2008).

Considering the lack of data on the effect of DDT on the cellular genome, and taking into account its usage in some countries of the Third World and its environmental persistence, the aim of this study was to evaluate the genotoxic potential of a low concentration of aqueous *p,p'*-DDT upon *in vitro* exposure of HPBLs of different duration, by using alkaline comet assay and CBMN assay. Our results showed that exposure of HPBLs to aqueous *p,p'*- DDT increased DNA damage in time dependent fashion as measured by the comet assay. In addition, CBMN assay parameters revealed a wider scale of chromosomal alterations after *p,p'*-DDT treatment.

Cytogenetic studies of DDT are mainly based on *in vitro* research on the animal models and its genotoxicity was evaluated in a variety of test systems. Results obtained by studying cytogenetic effects of DDT on DNA of shrimp larvae (*Litopenaeus stylirostris*) indicated that DDT causes DNA adducts and/or breaks (Galindo Reyes et al., 2002). DDT and its

Presence of

**6. Conclusion** 

chemicals on human genome and health.

ATSDR, Atlanta, USA

2133-2138, ISSN 0173–0835

0260-437X

Sports of the Republic of Croatia (Grant no. 022-0222148-2125).

**7. Acknowledgment** 

**8. References** 

Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity 137

Significant levels of DDT and its metabolites can still be found in biological samples of serum, adipose tissue and maternal milk of populations that are not occupationally exposed. People are usually exposed to DDT through food, inhalation or dermal contact. Also, there are evidences on damages to the health, specially related to the reproductive area, and more recently damages at cellular level, as well as, alteration in the psychomotor development of children exposed in uterus. Although there are studies dealing with adverse effects of pesticide exposure there is still great need for elucidating the exact mechanism and health consequences related to DDT exposure and its metabolites. Our data in conjunction with other available data regarding pesticide genotoxicity have identified that DDT induces DNA strand breaks in human peripheral blood lymphocytes *in vitro* as well *in vivo*. In our study, this effect was noted even after the treatment with very low concentration of aqueous DDT. These results also confirms previous findings that DDT induces alterations of the ultrastructure of cells and DNA damage by causing single strand breakage and adducts in DNA molecule. Present study also confirms that combinations of sensitive techniques like alkaline comet assay and cytokinesys-block micronucleus assay are useful for the assessment of cellular and DNA alterations after exposure to mutagens and carcinogens from the environment. Results obtained in this research indicate the need for further environmental and food monitoring, and cytogenetic research using sensitive methods in detection of primary genome damage after exposure to DDT to establish the impact of such

The study is a part of a research project supported by the Ministry of Science, Education and

Agency for Toxic Substances and Disease Service. (2002). *Toxicological profile for DDT, DDE* 

Albertini, R.J.; Anderson, D.; Douglas, G.R.; Hagmar, L.; Hemminki, K.; Merlo, F.; Natarajan,

Amer, S.M.; Fahmy, M.A. & Donya, S.M. (1996). Cytogenetic effect of some insecticides in

Angelis, K.J.; Dušinská, M. & Collins, A.R. (1999). Single cell gel electrophoresis: detection of

Aronson, K.J.; Wilson, J.W.; Hamel, M.; Diarsvitri, W.; Fan, W.; Woolcott, C.; Heaton, J.P.;

*Mutation Research*, Vol.463, No.2, pp. 111–172, ISSN 1383-5742

*and DDD*, U.S. Department of Health and Human Services, Public Health Service,

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Nickel, J.C.; Macneily, A. & Morales, A. (2010). Plasma organochlorine levels and

metabolites DDE and DDD showed a clear genotoxic effect on haemocytes of zebra mussel (*Dreissena polymorpha*) specimens in different concentrations that have been found in several aquatic ecosystems worldwide, with a greater genotoxic potential of the DDE in respect to the other two chemicals (Binneli et al., 2008a, 2008b). DDT has also the ability to induce chromosomal aberrations in mouse spleen indicating its genotoxicity (Amer at al., 1996). In addition, DDT was genotoxic towards lymphocytes and mammary epithelial cells of female rats showing an increase in lipid peroxidation, the outcome of the growth level of free oxygen radicals, which lead to an oxidative stress (Canales-Aquirre et al., 2011). DDT also induces cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that it can induce early events in mammary carcinogenesis (Uppala et al., 2005). Additionally, beluga whales (*Delphinapterus leucas*) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants including DDT which can induced significant increases of micronucleated cells in skin fibroblasts of an Arctic beluga whale (Gauthier et al., 1999).

Regarding human test system, the cytogenetic effect of DDT was investigated both *in vitro* and *in vivo*. *In vitro*, certain DDT concentrations have the effects on human leukocyte functions (Lee et al., 1979), are causing chromosomal aberrations (Lessa et al., 1976), DNA strand breaks (Yáñez et al., 2004), and apoptosis induction which is preceded by an increase in the levels of reactive oxygen species (Pérez-Maldonado et al., 2004, 2005). *In vivo*, DDT is able to induce chromatid lesions (Rabello et al., 1975), increase in chromosomal aberrations and sister chromatid exchanges (Rupa et al., 1989, 1991), DNA strand breaks (Yáñez et al., 2004; Pérez-Maldonado et al., 2006), apoptosis (Pérez-Maldonado et al., 2004) as well as cell cycle delay and decrease in mitotic index (Rupa et al., 1991).

Before 1973 when it was banned, DDT entered the air, water and soil during its production and use as an insecticide. DDT is present at many waste sites and from these sites it might continue to contaminate the environment. DDT still enters the environment because of its current use in other areas of the world. DDT may be released into atmosphere in countries where it is still manufactured and used; it can also enter the air by evaporation from contaminated water and soil and than it can be deposited on land or surface water. This cycle of evaporation and deposition may be repeated many times and as a result, DDT can be carried long distances in the atmosphere (ATSDR, 2002; Crinnion, 2009; Donato & Zani, 2010; Gajski et al., 2007; Torres-Sánchez & López-Carrillo 2007). These chemicals have been found in bogs, snow and animals even in the Artic and Antarctic regions, far from where they were ever used. DDT can last in the soil for a very long time, potentially for hundreds of years. Most DDT breaks down slowly into DDE and DDD, generally by the action of microorganisms and can be deposited in other places like in the surface layers of soil; it may get into rivers and lakes in runoff or get into groundwater. In surface waters, DDT will bind to particles in the water, settle and be deposited in the sediment. DDT is than taken up by small organisms and fish in the water. It accumulates to high levels in adipose tissue of fish and marine mammals, reaching levels many thousands of times higher than in water. DDT can also be absorbed by some plants and by the animals that can directly impact human population and like that represent a major health threat (ATSDR, 2002; Beard, 2006; Gajski et al., 2007; Gauthier et al., 1999).

All of these findings suggests that DDT is still present not only in poorly developed countries of the Third World but it can still be found in other countries that have banned its use almost 40 years ago due to its stability and long persistence in the environment.

## **6. Conclusion**

136 Insecticides – Pest Engineering

metabolites DDE and DDD showed a clear genotoxic effect on haemocytes of zebra mussel (*Dreissena polymorpha*) specimens in different concentrations that have been found in several aquatic ecosystems worldwide, with a greater genotoxic potential of the DDE in respect to the other two chemicals (Binneli et al., 2008a, 2008b). DDT has also the ability to induce chromosomal aberrations in mouse spleen indicating its genotoxicity (Amer at al., 1996). In addition, DDT was genotoxic towards lymphocytes and mammary epithelial cells of female rats showing an increase in lipid peroxidation, the outcome of the growth level of free oxygen radicals, which lead to an oxidative stress (Canales-Aquirre et al., 2011). DDT also induces cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that it can induce early events in mammary carcinogenesis (Uppala et al., 2005). Additionally, beluga whales (*Delphinapterus leucas*) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants including DDT which can induced significant increases of micronucleated cells in skin fibroblasts of an Arctic beluga

Regarding human test system, the cytogenetic effect of DDT was investigated both *in vitro* and *in vivo*. *In vitro*, certain DDT concentrations have the effects on human leukocyte functions (Lee et al., 1979), are causing chromosomal aberrations (Lessa et al., 1976), DNA strand breaks (Yáñez et al., 2004), and apoptosis induction which is preceded by an increase in the levels of reactive oxygen species (Pérez-Maldonado et al., 2004, 2005). *In vivo*, DDT is able to induce chromatid lesions (Rabello et al., 1975), increase in chromosomal aberrations and sister chromatid exchanges (Rupa et al., 1989, 1991), DNA strand breaks (Yáñez et al., 2004; Pérez-Maldonado et al., 2006), apoptosis (Pérez-Maldonado et al., 2004) as well as cell

Before 1973 when it was banned, DDT entered the air, water and soil during its production and use as an insecticide. DDT is present at many waste sites and from these sites it might continue to contaminate the environment. DDT still enters the environment because of its current use in other areas of the world. DDT may be released into atmosphere in countries where it is still manufactured and used; it can also enter the air by evaporation from contaminated water and soil and than it can be deposited on land or surface water. This cycle of evaporation and deposition may be repeated many times and as a result, DDT can be carried long distances in the atmosphere (ATSDR, 2002; Crinnion, 2009; Donato & Zani, 2010; Gajski et al., 2007; Torres-Sánchez & López-Carrillo 2007). These chemicals have been found in bogs, snow and animals even in the Artic and Antarctic regions, far from where they were ever used. DDT can last in the soil for a very long time, potentially for hundreds of years. Most DDT breaks down slowly into DDE and DDD, generally by the action of microorganisms and can be deposited in other places like in the surface layers of soil; it may get into rivers and lakes in runoff or get into groundwater. In surface waters, DDT will bind to particles in the water, settle and be deposited in the sediment. DDT is than taken up by small organisms and fish in the water. It accumulates to high levels in adipose tissue of fish and marine mammals, reaching levels many thousands of times higher than in water. DDT can also be absorbed by some plants and by the animals that can directly impact human population and like that represent a major health threat (ATSDR, 2002; Beard, 2006; Gajski et

All of these findings suggests that DDT is still present not only in poorly developed countries of the Third World but it can still be found in other countries that have banned its

use almost 40 years ago due to its stability and long persistence in the environment.

cycle delay and decrease in mitotic index (Rupa et al., 1991).

whale (Gauthier et al., 1999).

al., 2007; Gauthier et al., 1999).

Significant levels of DDT and its metabolites can still be found in biological samples of serum, adipose tissue and maternal milk of populations that are not occupationally exposed. People are usually exposed to DDT through food, inhalation or dermal contact. Also, there are evidences on damages to the health, specially related to the reproductive area, and more recently damages at cellular level, as well as, alteration in the psychomotor development of children exposed in uterus. Although there are studies dealing with adverse effects of pesticide exposure there is still great need for elucidating the exact mechanism and health consequences related to DDT exposure and its metabolites. Our data in conjunction with other available data regarding pesticide genotoxicity have identified that DDT induces DNA strand breaks in human peripheral blood lymphocytes *in vitro* as well *in vivo*. In our study, this effect was noted even after the treatment with very low concentration of aqueous DDT. These results also confirms previous findings that DDT induces alterations of the ultrastructure of cells and DNA damage by causing single strand breakage and adducts in DNA molecule. Present study also confirms that combinations of sensitive techniques like alkaline comet assay and cytokinesys-block micronucleus assay are useful for the assessment of cellular and DNA alterations after exposure to mutagens and carcinogens from the environment. Results obtained in this research indicate the need for further environmental and food monitoring, and cytogenetic research using sensitive methods in detection of primary genome damage after exposure to DDT to establish the impact of such chemicals on human genome and health.

## **7. Acknowledgment**

The study is a part of a research project supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grant no. 022-0222148-2125).

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quantitation of low levels of DNA damage in individual cells. *Experimental Cell* 

using the comet assay with greater specificity than FPG or ENDOIII. *Mutagenesis,* 

analysis of micronuclei in uncultured human T lymphocytes. *Cytogenetics and Cell* 

Comet assay and the troubles with its application in the green alga *Chlamydomonas* 

*polychlorinated biphenyls (PCBs), CEN/TC 275 N0360 Food analysis – Horizontal* 

Chromosome breakage and the lethality of ionizing radiations to the root meristem,

lymphocytes. *Methods in Molecular Biology*, Vol.682, No.3, pp. 217-234, ISSN 1940-

measure of chromosome rearrangement in the cytokinesis-block micronucleus

possible assay for aneuploidy. *Mutagenesis,* Vol.3, No.5, pp. 415-418, ISSN 0267-

genotoxic pollutants, In: *Biomonitors and Biomarkers as Indicators of Environmental Change,* F.M. Butterworth, (Ed.), 314-327, Plenum Press, ISBN 9780306451904, New

Y.; Rojas, E.; Ryu, J.C. & Sasaki, Y.F. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. *Environmental and Molecular* 


**Part 2** 

**Vector Management** 


## **Part 2**

**Vector Management** 

150 Insecticides – Pest Engineering

World Health Organization. (2007). *Insecticide-treated mosquito nets: a WHO Position* 

World Health Organization. (2009a). *The WHO recommended classification of pesticides by* 

World Health Organization. (2009b). *Global insecticide use for vector - borne disease control* (4th

Yáñez, L.; Borja-Aburto, V.H.; Rojas, E.; de la Fuente, H.; González-Amaro, R.; Gómez, H.;

atoz/itnspospaperfinal/en/index.html

ed), WHO, ISBN 9789241598781, Lyon, France

*Research*, Vol.94, No.1, pp. 18-24, ISSN 0013-9351

Switzerland

*Statement*, WHO, Available from http://www.who.int/malaria/publications/

*hazard and guidlines to classification*, WHO, ISBN 9789241547963, Geneva

Jongitud, A.A. & Díaz-Barriga, F. (2004). DDT induces DNA damage in blood cells. Studies in vitro and in women chronically exposed to this insecticide. *Environmental* 

**7** 

Alhaji Aliyu

*Nigeria* 

*Department of Community Medicine, Ahmadu Bello University, Zaria* 

**Vector Control Using Insecticides** 

At the end of the 19th century, it was discovered that certain species of insects, other arthropods and fresh water snails were responsible for the transmission of some diseases of pubic health importance. Since effective vaccines or drugs were not always available for the prevention or treatment of these diseases, control of transmission then had to rely mainly on control of vectors. The control programmes included among others, use of mosquito nets, drainage of gutters, filling of potholes and other water bodies used by insects for breeding. The 1940s sawed the discovery of DDT insecticide (dichlorodiphenyl tricloroethene) which was a major breakthrough in the control of vector-borne diseases. DDT also appeared to be effective and economical in the control of other biting flies (tsetse fly, simulium, and sand fly) and midges and of infestations with fleas, lice,

The initial large scale success achieved in the control programme was short-lived as the vectors developed resistance to the insecticides in use, thereby creating a need for new more expensive chemicals. Interest in alternatives to the use of insecticides such as environmental management (source reduction) and biological control, has been revived because of increasing resistance to the commonly used insecticides among important vector species e.g. (malaria) and also because of concerns about the effects of DDT and certain other insecticides on the environment. For many vector species, environmental sanitation1 through source reduction and health education is the fundamental means of control; other methods should serve as a supplement, not as a substitute. Thus, in recent years, the practices of vector control have evolved, and environmental management and modification have come to the fore, both for disease control and for agricultural and other economic purposes2, this is a complex and multi-disciplinary field3. Effective application of any control measure must be based on a fundamental understanding of the ecology, bionomics and behaviour of the target vector species and its relation to its host and the environment. Effective vector control also requires careful training and supervision of pest control

In more recent years, less reliance has been placed on the use of a single method of chemical control; there is a shift towards more integrated vector control involving several types of environmental management supplemented by more than one method of chemical control and the use of drugs. More attention has been paid to community participation (a key component of primary health care (PHC) in eliminating breeding sites of vectors (including

operations and periodic evaluation of the impacts of the control measures.

clearing of weeds/bushes near residential houses) and reducing vector densities.

**1. Introduction** 

bedbugs and triatomine bugs.

## Alhaji Aliyu

*Department of Community Medicine, Ahmadu Bello University, Zaria Nigeria* 

## **1. Introduction**

At the end of the 19th century, it was discovered that certain species of insects, other arthropods and fresh water snails were responsible for the transmission of some diseases of pubic health importance. Since effective vaccines or drugs were not always available for the prevention or treatment of these diseases, control of transmission then had to rely mainly on control of vectors. The control programmes included among others, use of mosquito nets, drainage of gutters, filling of potholes and other water bodies used by insects for breeding. The 1940s sawed the discovery of DDT insecticide (dichlorodiphenyl tricloroethene) which was a major breakthrough in the control of vector-borne diseases. DDT also appeared to be effective and economical in the control of other biting flies (tsetse fly, simulium, and sand fly) and midges and of infestations with fleas, lice, bedbugs and triatomine bugs.

The initial large scale success achieved in the control programme was short-lived as the vectors developed resistance to the insecticides in use, thereby creating a need for new more expensive chemicals. Interest in alternatives to the use of insecticides such as environmental management (source reduction) and biological control, has been revived because of increasing resistance to the commonly used insecticides among important vector species e.g. (malaria) and also because of concerns about the effects of DDT and certain other insecticides on the environment. For many vector species, environmental sanitation1 through source reduction and health education is the fundamental means of control; other methods should serve as a supplement, not as a substitute. Thus, in recent years, the practices of vector control have evolved, and environmental management and modification have come to the fore, both for disease control and for agricultural and other economic purposes2, this is a complex and multi-disciplinary field3. Effective application of any control measure must be based on a fundamental understanding of the ecology, bionomics and behaviour of the target vector species and its relation to its host and the environment. Effective vector control also requires careful training and supervision of pest control operations and periodic evaluation of the impacts of the control measures.

In more recent years, less reliance has been placed on the use of a single method of chemical control; there is a shift towards more integrated vector control involving several types of environmental management supplemented by more than one method of chemical control and the use of drugs. More attention has been paid to community participation (a key component of primary health care (PHC) in eliminating breeding sites of vectors (including clearing of weeds/bushes near residential houses) and reducing vector densities.

and Japanese encephalitis. For practical purposes and entomologically, it is useful to be able to differentiate between these three types of mosquitoes; so the main distinguishing features

in each stage of the life cycle are shown below in the diagram

Fig. 1. Life cycles of different mosquito genera (online, 2011).

Finally, there is also need to provide on continuous basis information on single, effective and acceptable methods for vector source reduction and personal protection to individuals and families in the community at a reasonable cost.

In a chapter of this nature only a few, but more important species of vectors could be discussed and even such a discussion could only be brief, emphasizing only the important features of the arthropod which serve to illustrate how the arthropod affects public health.

## **2. Malaria and its vectors**

Human malaria is a number one public enemy and an illness caused by the bite of an infective female anopheles mosquito which transfers parasites called plasmodium from person to person. Four plasmodium parasites exists (P. falciparum, P. ovale, P. malariae and P. vivax) but only one (P. falciparum) is of vital importance in disease transmission in Nigeria. The important vectors in Nigeria are anopheles gambiae and A. funestus. These mosquitoes breed readily in ditches and collections of water in empty receptacles around the houses. The disease is endemic in Nigeria and over 90% of the population is at risk. Fifty percent of the population will have at least one attack per year, 300, 000 children and 11% of pregnant women die of malaria each year respectively and millions of dollars is lost each year in treatment of malaria.


The table below shows some of these vectors and diseases transmitted by them.

Table 1. Pathogens and vectors of some human diseases (sg) indicates definite involvement of salivary gland.

The results of several surveys showed that the most prevalent species of malaria parasites is p. falciparum with a prevalent rate of between 80% and 100% of all positive blood films. P. vivax is conspicuously absent in West Africans who because of their Duffy negative erythrocyte membrane are immune to P. vivax4, 5.

It is estimated that there are more than 400 species of anopheles in the world, but only 40 of these are important vectors (as transmitters or carriers) of malaria. Only the female anopheles mosquito transmits malaria to humans. Some anopheles mosquitoes can also carry other human diseases like Filariasis and some viruses. There are two other genera of mosquitoes which are important carriers of other mosquito-borne diseases: Aedes is a vector of viral diseases- Yellow fever and Dengue while culex mosquito is the vector of Filariasis

Finally, there is also need to provide on continuous basis information on single, effective and acceptable methods for vector source reduction and personal protection to individuals

In a chapter of this nature only a few, but more important species of vectors could be discussed and even such a discussion could only be brief, emphasizing only the important features of the arthropod which serve to illustrate how the arthropod affects public health.

Human malaria is a number one public enemy and an illness caused by the bite of an infective female anopheles mosquito which transfers parasites called plasmodium from person to person. Four plasmodium parasites exists (P. falciparum, P. ovale, P. malariae and P. vivax) but only one (P. falciparum) is of vital importance in disease transmission in Nigeria. The important vectors in Nigeria are anopheles gambiae and A. funestus. These mosquitoes breed readily in ditches and collections of water in empty receptacles around the houses. The disease is endemic in Nigeria and over 90% of the population is at risk. Fifty percent of the population will have at least one attack per year, 300, 000 children and 11% of pregnant women die of malaria each year respectively and millions of dollars is lost each

The table below shows some of these vectors and diseases transmitted by them.

Type Designation Disease Vectors Mode of

Malaria

sickness

Filariasis

Viruses YF Yellow Fever Aedes Bite (sg)

Cholera Typhoid fever Dysentery

Table 1. Pathogens and vectors of some human diseases (sg) indicates definite involvement

The results of several surveys showed that the most prevalent species of malaria parasites is p. falciparum with a prevalent rate of between 80% and 100% of all positive blood films. P. vivax is conspicuously absent in West Africans who because of their Duffy negative

It is estimated that there are more than 400 species of anopheles in the world, but only 40 of these are important vectors (as transmitters or carriers) of malaria. Only the female anopheles mosquito transmits malaria to humans. Some anopheles mosquitoes can also carry other human diseases like Filariasis and some viruses. There are two other genera of mosquitoes which are important carriers of other mosquito-borne diseases: Aedes is a vector of viral diseases- Yellow fever and Dengue while culex mosquito is the vector of Filariasis

African sleeping

Onchocerciasis

transmission

Bite (sg) Bite (sg)

Bite

Bite

carriers

Cockroaches Mechanical

Mosquitoes Tsetse fly

Mosquitoes

Black flies

and families in the community at a reasonable cost.

**2. Malaria and its vectors** 

year in treatment of malaria.

Protozoa Plasmodium spp

Wucheria Bancrofti Onchocerca Volvula

Filarial nematodes

Bacteria

of salivary gland.

Trypanosome spp

Cholera/diarrhea Typhoid fever Bacillary dysentery

erythrocyte membrane are immune to P. vivax4, 5.

and Japanese encephalitis. For practical purposes and entomologically, it is useful to be able to differentiate between these three types of mosquitoes; so the main distinguishing features in each stage of the life cycle are shown below in the diagram

Fig. 1. Life cycles of different mosquito genera (online, 2011).

This is currently unaffordable to most patients surviving barely on less than a US\$1 per day. The treatment of malaria has become quite challenging, and the emergence of resistant strains of the parasite. In Nigeria where at least 80%9,10 of the people live in rural areas and are supperstitious illiterates, early recognition and the right treatment are not adhered to.

The Abuja declaration on Roll Back Malaria on 25 April 2000 and agreed to by African Heads of State sets an ambitious goals to reduce the burden of malaria (insecticide-treated nets, prompt access to treatment and prevention of malaria in pregnancy) by the year 20108. Achieving high coverage in both IPT and use of ITNs among the vulnerable groups and the general population has remained elusive for many countries in sub-Saharan Africa11. A major barrier to net ownership is poverty as the price of a net represents a large proportion

Environmental control (source reduction) offers the best practical and easy measure to control disease vector as it eliminates the breeding places. The filling of mosquito breeding sites with soil, ash or rubbish and is most suitable for reducing breeding in small depressions, water holes or pools, which does not require much filling material. On the other hand, drainage of water can be achieved by constructing open ditches; however, the drainage systems used in agriculture or for the transportation of sewage and rainwater in

The first house-spraying campaigns after the Second World War, showed the capacity of this interventions to produce profound reductions in malaria transmission in a wide variety of circumstances. In Africa, the intervention was used in 1960s and 1970s but later abandoned except in some countries in southern and eastern Africa where residual insecticide spraying (IRS) remained the cornerstone of malaria control strategy. Since 2005, however, there has been renewed interest in large scale IRS programmes. To date, 25 out of the 42 malaria endemic countries in the WHO Africa region have included IRS in their control strategy for malaria control. Of these, 17 countries routinely implement IRS as a major malaria control intervention, six including Nigeria are piloting the IRS in a few districts while 2 are planning to pilot. At the end of 2006, the National Malaria Control Programme (NMCP) and its partners initiated a pilot IRS project in 3 local government areas/districts in 3 states (Lagos, Plateau, Borno states) using the WHOPES approved

The evaluation of the local context of the 3 chemicals confirmed that residual effectiveness of

of the income of a poor household, this has been reported in various studies12, 13, 14.

cities often promote breeding because of poor design and maintenance.

**Drug US\$**  Chloroquine 0.13 SP 0.14 Amodiaquine 0.20 Artemisinin-based combinations (ACTs) 1-3

This encourages drug resistance.

**4. The use of insecticides** 

a. Lambdacyhalothrin CS 10% (0.030g/m2) b. Alphacypermethrin WP10% (0.030g/m2)

the insecticides lasted for at least 4 months.

c. Bifenthrin WP 10% (0.050g/m2)

insecticides:

Table 3. Average cost of a full course of adult outpatient treatment8.

Basically, the mosquito has four stages in its life cycle: eggs → larva→ pupa → adult. It is useful to understand the life cycle and natural history of the anopheles mosquito not only for epidemiological reasons but most importantly for the fact that all the four stages are targets to control mosquito vector. There are several features of the behaviour of anopheles mosquitoes which are important in understanding malaria epidemiology and also for planning mosquito control.

Malaria is holo-endemic in most regions of Nigeria; ideal climatic conditions for the propagation and transmission of the infection are a temperature between 26oC – 30oC and relative humidity of over 60 percent.

## **3. Disease control**

The general methods of malaria control can be grouped into three measures directed against the parasite in man, measures directed against the vector and measures designed to prevent mosquito-man contact, these are summarized in table 2


Table 2. Interventions to control malaria5.

The first involves the use of appropriate anti-malarial agents to treat clinical malaria and the use of chemoprophylaxis among the vulnerable groups. The problem facing most tropical countries is serious as majority of patients with clinical malaria are either untreated or treated inadequately by self-medication. Also, their governments cannot afford to buy sufficient anti-malarial drugs for their needs and most people cannot afford to purchase effective treatments7. The annual per capita expenditure on anti-malarial drugs in most of sub-Saharan Africa is still <US\$10. An adult (60kg) course of Chloroquine costs US\$0.08 but the new artemisinin based combinations (ACTs) cost more than five times as shown in table 3.

156 Insecticides – Pest Engineering

Basically, the mosquito has four stages in its life cycle: eggs → larva→ pupa → adult. It is useful to understand the life cycle and natural history of the anopheles mosquito not only for epidemiological reasons but most importantly for the fact that all the four stages are targets to control mosquito vector. There are several features of the behaviour of anopheles mosquitoes which are important in understanding malaria epidemiology and also for

Malaria is holo-endemic in most regions of Nigeria; ideal climatic conditions for the propagation and transmission of the infection are a temperature between 26oC – 30oC and

The general methods of malaria control can be grouped into three measures directed against the parasite in man, measures directed against the vector and measures designed to prevent

> Outpatient treatment of uncomplicated malaria Inpatient treatment of severe and complicated malaria

i) Source reduction (drainage, filing in ditches)

development to avoid breeding sites

i) IRS (indoor residual spraying)

ii) Repellants, sprays, coils, etc

The first involves the use of appropriate anti-malarial agents to treat clinical malaria and the use of chemoprophylaxis among the vulnerable groups. The problem facing most tropical countries is serious as majority of patients with clinical malaria are either untreated or treated inadequately by self-medication. Also, their governments cannot afford to buy sufficient anti-malarial drugs for their needs and most people cannot afford to purchase effective treatments7. The annual per capita expenditure on anti-malarial drugs in most of sub-Saharan Africa is still <US\$10. An adult (60kg) course of Chloroquine costs US\$0.08 but the new artemisinin based combinations (ACTs) cost more

ii) Chemoprophylaxis

iii) Management of agricultural, industrial and urban

ii) ITMs (insecticide treated materials – bed nets, curtains, etc)

i) IPT (intermittent preventive treatment of pregnant women)

planning mosquito control.

**3. Disease control** 

Treatment

Prevention

breeding

(b)Kill adult mosquitoes

contact

(a) Inhibit mosque ito

(c) Prevent mosquito

(d) Reduce malaria infection and

morbidity in humans

Table 2. Interventions to control malaria5.

than five times as shown in table 3.

relative humidity of over 60 percent.

mosquito-man contact, these are summarized in table 2

Home treatment

i) ITMs

ii) Chemical larviciding

**Principal goal Interventions** 


Table 3. Average cost of a full course of adult outpatient treatment8.

This is currently unaffordable to most patients surviving barely on less than a US\$1 per day. The treatment of malaria has become quite challenging, and the emergence of resistant strains of the parasite. In Nigeria where at least 80%9,10 of the people live in rural areas and are supperstitious illiterates, early recognition and the right treatment are not adhered to. This encourages drug resistance.

The Abuja declaration on Roll Back Malaria on 25 April 2000 and agreed to by African Heads of State sets an ambitious goals to reduce the burden of malaria (insecticide-treated nets, prompt access to treatment and prevention of malaria in pregnancy) by the year 20108. Achieving high coverage in both IPT and use of ITNs among the vulnerable groups and the general population has remained elusive for many countries in sub-Saharan Africa11. A major barrier to net ownership is poverty as the price of a net represents a large proportion of the income of a poor household, this has been reported in various studies12, 13, 14.

Environmental control (source reduction) offers the best practical and easy measure to control disease vector as it eliminates the breeding places. The filling of mosquito breeding sites with soil, ash or rubbish and is most suitable for reducing breeding in small depressions, water holes or pools, which does not require much filling material. On the other hand, drainage of water can be achieved by constructing open ditches; however, the drainage systems used in agriculture or for the transportation of sewage and rainwater in cities often promote breeding because of poor design and maintenance.

## **4. The use of insecticides**

The first house-spraying campaigns after the Second World War, showed the capacity of this interventions to produce profound reductions in malaria transmission in a wide variety of circumstances. In Africa, the intervention was used in 1960s and 1970s but later abandoned except in some countries in southern and eastern Africa where residual insecticide spraying (IRS) remained the cornerstone of malaria control strategy. Since 2005, however, there has been renewed interest in large scale IRS programmes. To date, 25 out of the 42 malaria endemic countries in the WHO Africa region have included IRS in their control strategy for malaria control. Of these, 17 countries routinely implement IRS as a major malaria control intervention, six including Nigeria are piloting the IRS in a few districts while 2 are planning to pilot. At the end of 2006, the National Malaria Control Programme (NMCP) and its partners initiated a pilot IRS project in 3 local government areas/districts in 3 states (Lagos, Plateau, Borno states) using the WHOPES approved insecticides:


The evaluation of the local context of the 3 chemicals confirmed that residual effectiveness of the insecticides lasted for at least 4 months.

other insects: bedbugs, flies and cockroaches, community and household mass effective which may be more important in some contexts22 and mosquito nuisance effect. There have been reports also of dead insects on the nets and on the floor, less mosquito noise and that

Most nets (and other materials) need to be treated and retreated with insecticides to increase their effectiveness. There are several insecticides that can be used which have proved to be safe. The insecticides recommended by the National Malaria and Vector Control Programme for treatment/retreatment of nets are those approved by WHOPES and registered by NAFDAC (National Foods and Drugs Administration Commission) are shown in table 4

**Generic name Trade name Dose per 1 net Manufacturer** 

For a detailed description of preparation of insecticide mixtures and treatment methods, the reader should consult guidelines on the use of insecticide-treated mosquito

A single impregnation of a cotton or nylon mosquito net will provide protection for 1 year24,25. Nylon tends to retain permethrin and deltamethrin better than cotton. The impregnated nets can be washed and can tolerate small tears/holes without markedly reducing the protective effect. Recently, long lasting nets (LLNs) have been developed and have the advantage of retaining insecticidal activity for years (so the nets will not lose its potency with repeated washings). Despite various government policies, cost of the nets remains a significant barrier and a long obstacle to the Roll Back Malaria goal of universal coverage – defined as one long lasting insecticide-treated nets for every two people in the household with 80% usage. ITN development is a public good. The development of insecticide resistance in the 1950s and recently by vectors has been a cause for concern. There have been reports of resistance to DDT in a wide range of sub-Saharan African countries26, but this has not reached an operationally significant level. With the exception of the Gezira region of Sudan27, widespread loss of vector susceptibility is not yet a big problem in Nigeria. A worrying new development is the emergence of knockdown resistance to pyrethroid insecticides in natural populations of anopheles mosquitoes in Cote d'Ivoire and Burkina Faso28, 29, 30 where insecticides are widely used in cotton

40ml Aventis

75ml Aventis

Alphacypermethrin 10%.SCa Fendona 6ml BASF

Etofenprox 10%.EWb Vectron 30ml Mitsui

Imperator

Cyfluthrin 5% EW Solfac EW 15ml Bayer Lambdacyhalothrin 2.5% CSd Icon 10ml Syngenta

Tab

ITNs providing a better night's sleep than a net alone16.

Deltamelthrin 1%.SC K-Othrine, Ko-

Permethrin 10%.ECc Peripel,

SCa = Suspension concentrate EWb = Emulsion oil in water ECc = Emulsifiable concentrate CSd = Capsule suspension

below:

Key:

Table 4.

nets23.

production.

Residual spraying of houses involves the treating of interior walls and ceilings using a handheld compression spraying and is effective against mosquitoes that favour indoor resting before or after feeding. For a more detailed discourse of notable insecticide formulations, spray pumps, spraying techniques and maintenance of equipment, see Jan A. Rozendaal15.

The figure 2, below summarizes and describes the IRS management principles;

Fig. 2. IRS Management cycle.

Mosquito nets are an old technology and most people in sub-Saharan Africa are aware of the existence of nets. In some countries such as Nigeria and the Gambia nets have been in regular use for over hundred years. Similarly, in some parts of Africa, net ownership is a well established social norm and nets are widely available. For instance, recently, it was found that 70% of households (HHs) in Dar es Salaam (Tanzania) owned a mosquito net16 and around 35% of HHs were found to own a net in urban Burkina Faso17.

Insecticide treated mosquito nets have had significant impact in reducing morbidity and mortality among children under-five years old and pregnant women where ITNs have been appropriately and extensively used in malaria endemic areas. The potential epidemiological advantages and public health benefits of treating nets with insecticide for protection against malaria were recognized in the mid-1980s. Specifically, the efficacy of insecticide treated nets (ITNs) for the control of malaria in children under-5 years of age has recently been demonstrated by several large scale studies18, 19, 20, 21 which find reductions in all cause – mortality, ranging from 16% to 63%. These insecticides which have been approved by WHOPES (World Health Organization Pesticides Evaluation Scheme) are safe. They have the following properties; provide personal protection from mosquito bites, effective against

Residual spraying of houses involves the treating of interior walls and ceilings using a handheld compression spraying and is effective against mosquitoes that favour indoor resting before or after feeding. For a more detailed discourse of notable insecticide formulations, spray pumps, spraying techniques and maintenance of equipment, see Jan A.

Mosquito nets are an old technology and most people in sub-Saharan Africa are aware of the existence of nets. In some countries such as Nigeria and the Gambia nets have been in regular use for over hundred years. Similarly, in some parts of Africa, net ownership is a well established social norm and nets are widely available. For instance, recently, it was found that 70% of households (HHs) in Dar es Salaam (Tanzania) owned a mosquito net16

Insecticide treated mosquito nets have had significant impact in reducing morbidity and mortality among children under-five years old and pregnant women where ITNs have been appropriately and extensively used in malaria endemic areas. The potential epidemiological advantages and public health benefits of treating nets with insecticide for protection against malaria were recognized in the mid-1980s. Specifically, the efficacy of insecticide treated nets (ITNs) for the control of malaria in children under-5 years of age has recently been demonstrated by several large scale studies18, 19, 20, 21 which find reductions in all cause – mortality, ranging from 16% to 63%. These insecticides which have been approved by WHOPES (World Health Organization Pesticides Evaluation Scheme) are safe. They have the following properties; provide personal protection from mosquito bites, effective against

and around 35% of HHs were found to own a net in urban Burkina Faso17.

The figure 2, below summarizes and describes the IRS management principles;

Rozendaal15.

Fig. 2. IRS Management cycle.

other insects: bedbugs, flies and cockroaches, community and household mass effective which may be more important in some contexts22 and mosquito nuisance effect. There have been reports also of dead insects on the nets and on the floor, less mosquito noise and that ITNs providing a better night's sleep than a net alone16.

Most nets (and other materials) need to be treated and retreated with insecticides to increase their effectiveness. There are several insecticides that can be used which have proved to be safe. The insecticides recommended by the National Malaria and Vector Control Programme for treatment/retreatment of nets are those approved by WHOPES and registered by NAFDAC (National Foods and Drugs Administration Commission) are shown in table 4 below:


Key:


#### Table 4.

For a detailed description of preparation of insecticide mixtures and treatment methods, the reader should consult guidelines on the use of insecticide-treated mosquito nets23.

A single impregnation of a cotton or nylon mosquito net will provide protection for 1 year24,25. Nylon tends to retain permethrin and deltamethrin better than cotton. The impregnated nets can be washed and can tolerate small tears/holes without markedly reducing the protective effect. Recently, long lasting nets (LLNs) have been developed and have the advantage of retaining insecticidal activity for years (so the nets will not lose its potency with repeated washings). Despite various government policies, cost of the nets remains a significant barrier and a long obstacle to the Roll Back Malaria goal of universal coverage – defined as one long lasting insecticide-treated nets for every two people in the household with 80% usage. ITN development is a public good. The development of insecticide resistance in the 1950s and recently by vectors has been a cause for concern. There have been reports of resistance to DDT in a wide range of sub-Saharan African countries26, but this has not reached an operationally significant level. With the exception of the Gezira region of Sudan27, widespread loss of vector susceptibility is not yet a big problem in Nigeria. A worrying new development is the emergence of knockdown resistance to pyrethroid insecticides in natural populations of anopheles mosquitoes in Cote d'Ivoire and Burkina Faso28, 29, 30 where insecticides are widely used in cotton production.

[13] Aliyu A A, Alti-Mu'azu M, Insecticide-treated net usage and malaria episodes among

[14] Aniefiok M. Acceptance and use of insecticide treated bed nets (ITN in the Roll Back

[15] Insecticide-treated nets. New findings from Kenya, published by Centers for Disease

[16] Jan A. Rozendaal: Vector control (Methods used by individuals and communities)

[19] Alonso P L, Lindsay SW, Schellenberg J. et al. A malaria control net trial using

[20] D'Alessandro U, Olaleye BO, McGuire W et al. Mortality and Morbidity from malaria

[21] Binka F N, Kubaje A, Adjuik M et al. Impact of permethrin impregnated bed nets on

[22] Nevill CG, Same ES, Mung'ala VO et al. Insecticide-treated bed nets reduce mortality

[23] Nicholas J. White. Malaria Gordon C. Cook and Alimuddin I Zumla (Eds). Manson's Tropical Diseases! 22nd edition, China, Saunders Elsevier: 2009; 1201-1300 [24] WHO Guidelines on the use of insecticide-treated nets for the prevention and control

[25] Lindsay SW, Gibson MF. Bed nets revisited – old idea, new angle. Parasitology Today

[26] Alonso PI, Lindsay SW, Armstrong IRM, et al. The effects of insecticide-treated bed

[27] WHO Vector resistance to pesticides, Fifteenth Report of the WHO Expert Committee

[28] El Gaddal A A, Haridi A A M, Hassan F T, Hussein H. Malaria Control in Gezira-

Managil Irrigated Scheme of the Sudan. Journal of Tropical Medicine and Hygiene

nets on mortality of Gambian children. Lancet 1991;337:1499-1502

trial. Tropical Medicine and International health 1996; 1(2):147-54.

[17] Jones C. Bed nets and malaria. Postgraduate Doctor. May 2002; vol. 24 No. 2:23-25. [18] Guiguemde T R et al. Household expenditure on Malaria prevention and treatment of

intense transmission. Africa Health (Mera) July 2003; Issue 6

Tropical Medicine and Hygiene 1994: 88: (3)285- 287

Medicine and International Health 1996: 1(2) 139-46

of Vector Biology and Control, Geneva; 1992

8:2:85-89.

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1988:4:270-272

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Lancet 1995:345 (8948).479-83

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boarding students in Zaria, Northern Nigeria. Annals of African Medicine, 2009;

Malaria Programme in Kuje Area Council, Abuja. Journal of Environmental health

Control and Prevention (CDC), Atlanta, USA, Confirm effectiveness in areas of

families in the town of Bobo-Diolasso, Burkina Faso. Trans. of the Royal Society of

insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of the Gambia, West Africa. The impact of the interventions on mortality and morbidity from malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene,

in Gambian children after introduction of an impregnated bed net programme.

child mortality in Kassena-Nankana district, Ghana: A randomized controlled

and severe morbidity from malaria among children on the Kenyan Coast. Tropical

Currently, pyrethroids are the only insecticides used for net treatment and are also increasingly used for spraying, so there is threat that if widespread resistance develops, the interventions will gradually become less cost-effective over time.

Finally, there is need to describe importantly the WHO integrated vector management (IVM) concept. Vector control is well suited for integrated approaches because vectors are responsible for multiple diseases and since interventions are effective against several vectors (use of insecticides) the concept of IVM was developed as a result of lessons learnt from integrated pest management which was used in Agriculture.

Integrated vector management (IVM) is a major component of the global campaign against malaria. The revised strategic plan for RBM recommended that from 2006 – 2010, 80% of the population at risk need to be protected using effective vector control measures. IVM creates synergies between various vector-borne disease control programmes. Utilization of single method could be optimized to control more than one vector-borne disease, eg ITNs can control malaria, lymphatic filariasis and to some extend leishmaniasis. IVM operates in the context of inter-sectoral collaboration. The application of IVM principles to vector control will contribute to the judicious use of insecticides and extend their useful life.

### **5. References**


Currently, pyrethroids are the only insecticides used for net treatment and are also increasingly used for spraying, so there is threat that if widespread resistance develops, the

Finally, there is need to describe importantly the WHO integrated vector management (IVM) concept. Vector control is well suited for integrated approaches because vectors are responsible for multiple diseases and since interventions are effective against several vectors (use of insecticides) the concept of IVM was developed as a result of lessons learnt from

Integrated vector management (IVM) is a major component of the global campaign against malaria. The revised strategic plan for RBM recommended that from 2006 – 2010, 80% of the population at risk need to be protected using effective vector control measures. IVM creates synergies between various vector-borne disease control programmes. Utilization of single method could be optimized to control more than one vector-borne disease, eg ITNs can control malaria, lymphatic filariasis and to some extend leishmaniasis. IVM operates in the context of inter-sectoral collaboration. The application of IVM principles to vector control

[1] World Health Organization. Chemical methods for the control of arthropod vectors and

[2] Najera J A. Malaria control: achievements, problems and strategies. Parasitologica 2001,

[5] Kara Hanson, Catherine Goodman, Jolines, Sylvia Meek, David Bradley and Anne Mills.

[7] Miller, L.H., Mason, S J, Clyde, DF and McGinnis M H. Resistance factors to plasmodium vivax: Duffy genotype Fyfy. New England Journal of Medicine: 1976; 255:302-304 [8] Denis MB, Tsuyuoka R, Poravuth Y et al. Surveillance of the efficacy artersunate and

[10] Salako A A. Environmental health education in schools and communities in Nigeria: The essence. The Nigerian Clinical Review Journal (2008): 14; 12:17-25 [11] The Challenges of Malaria Infection in Nigeria: The Nigerian clinical Review.

[12] Anna Maria van Eijk, Jenny Hill, Victor A. Alegana et al. Coverage of Malaria

National survey data. Medi-Link Journal. April 2011: 12:4:20-42

Global Forum for health Research: The Economics of Malaria Control

mefloquine combination for the treatment of uncomplicated malaria in Cambodia.

protection in pregnant women in sub-Saharan Africa: A synthesis and analysis of

will contribute to the judicious use of insecticides and extend their useful life.

pests of Public Health importance. WHO, Geneva; 1984.

[4] Garnham P.c.C. (1971).Progress in parasitology. The Atlone Press, London

www.who.int/docstore/water\_control\_htlm (cited 11/05/2011)

Tropical Medicine International health 2006; 11:1160-1366 [9] WHO. The Africa Malaria Report 2003, WHO/CDS/MAL/2003/1903:31-37

[3] Molineaux I, Grammiccia G: The Garki project; WHO 1980

[6] Stages of life cycle of vector parasites, available at

interventions will gradually become less cost-effective over time.

integrated pest management which was used in Agriculture.

**5. References** 

43:1-89

Interventions, 2004.

March/April 2011; 89:3-6.


**8** 

**Susceptibility Status of** *Aedes aegypti* **to** 

Dengue is a disease of public health interest for Colombia due to its impact on morbidity and mortality. This disease mantains an endemic behavior in the departments of the country located below 2200 meters above sea level. Between the years 1978 to 2010 it was recorded approximately a number of 1,011,852 cases of dengue in Colombia. Vector populations of *Aedes aegypti* in the country have been continuously pressed for over five decades with insecticides, being the most widely used tool for interrupting transmission of dengue virus during outbreaks and epidemics in the absence of an available vaccine (Figure 1). The selection pressure excercised with these chemicals has led to the emergence of resistant populations of the vector to organochlorine, organophosphate, pyrethroid and carbamate-

Fig. 1. Application of insecticides in the home by fogging durin outbreaks or epidemics.

**1. Introduction** 

like molecules.

**Insecticides in Colombia** 

Ronald Maestre Serrano

*Cartagena University* 

*Colombia* 

*Doctoral Student in Tropical Medicine* 


## **Susceptibility Status of** *Aedes aegypti* **to Insecticides in Colombia**

## Ronald Maestre Serrano

*Doctoral Student in Tropical Medicine Cartagena University Colombia* 

## **1. Introduction**

162 Insecticides – Pest Engineering

[29] Martinez Torres D, Chandre F, Willamson M S et al. Molecular characterization of

[30] Elissa N, Morchet J, Riviere F, Mevnier J-Y, Yaok. Resistance of Anopheles gambiae SS

Gambiae SS. Insect Molecular Biology 1998; 7(2):174-184

1993; 73:291-294.

pyrethroid knockdown resistance (kdr) in the major malaria vector anopheles

to pyrethroids in Cote d'Ivoire. Annales de la Societe Belge de Medecine Tropicale

Dengue is a disease of public health interest for Colombia due to its impact on morbidity and mortality. This disease mantains an endemic behavior in the departments of the country located below 2200 meters above sea level. Between the years 1978 to 2010 it was recorded approximately a number of 1,011,852 cases of dengue in Colombia. Vector populations of *Aedes aegypti* in the country have been continuously pressed for over five decades with insecticides, being the most widely used tool for interrupting transmission of dengue virus during outbreaks and epidemics in the absence of an available vaccine (Figure 1). The selection pressure excercised with these chemicals has led to the emergence of resistant populations of the vector to organochlorine, organophosphate, pyrethroid and carbamatelike molecules.

Fig. 1. Application of insecticides in the home by fogging durin outbreaks or epidemics.

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 165

Resistance is defined as the development of the ability to tolerate doses of toxics, which can be lethal to most individuals in a normal population of the same species and is the result of positive selection pressure exerted by the insecticidal over genes initially in low frequency

The two main resistance mechanisms are alterations in the target site and metabolic resistance, also called increase in the rate of detoxification of insecticides. However, there are other less frequent as there are the resistance behavior and decreased penetration.

This is generated when no silent mutations occur in structural genes that generate a

In bugs this is an hetero multi numeric channel acting as a site of action for cyclodiene and avermectins. The resistance is given by *Rdl* gene (Resistance to dieldrin), which encodes for RDL, a GABA receptor subunit. This type of resistance was first identified in *Drosophila* first

They are the target of action of pyrethroids and DDT. The sodium para-channel protein is a complex of over 2000 amino acids, composed of 4 homologous domains separated by hydrophilic links, each domain contains six segments. The first mutation in the sodium channel that conferred resistance was detected in *Musca domestica*. subsequently mutations in other insects have been identified including Culicidae resistant to pyrethroids (Martínez et al, 1998; Brengues et al, 2003; Anstead, 2005; Saavedra et al, 2007; Brooke, 2008; Chang et

Insecticide resistance, attributed to Ache insensitivity is found in a number of *Anopheles* and *Culex* species. In general, this mechanism produces a broad spectrum resistance to most organophosphates and carbamates, although more pronounced to carbamatos. (Bisset, 2002).

It is conferred by an increase in detoxification insecticide or an inability to metabolize the toxic compound. The most important form of metabolic resistance is given by detoxificating enzymes

They Catalyze the hydrolysis of carboxylic esters and changes in their expression levels, this is the resistance mechanism that occurs most often in insects. In mosquitoes, high levels of these enzymes have been associated with resistance to organophosphate and pyrethroid insecticides (Cui et al, 2007; Rodriguez et al, 2007). Esterases is a family of six proteins

of the type glutathione S-transferase, mixed function oxidases and esterases (Bisset 2002).

disturbance of amino acids responsible of anchor of the insecticide at a specific site.

**4. Resistance to insecticides** 

**4.2 Mechanisms of resistance** 

**4.2.1 Altered target site** 

**4.2.1.1 GABA Receptor** 

al 2009; Yanola et al, 2010).

**4.2.2 Metabolic resistance** 

**4.2.2.1 Carboxylesterases** 

**4.2.1.2 Voltage-gated sodium channel** 

**4.2.1.3 Insensitive acethylcholinesterase** 

(Miller, 1988; Bisset, 2002; Fonseca and Quiñones, 2005)

identified this type of resistance (Fonseca and Quiñones, 2005).

**4.1 Definition** 

(WHO, 1957).

The records of vector resistance in the country have increased considerably since 2007 when in Colombia was created the national network for the surveillance of susceptibility to insecticides used in public health to *A. aegypti* and major vector of malaria. With this information a base line has been defined for some Colombian departments in the region and basic knowledge has been generated for decision making from the operating standpoint of vector control programs. However, more research on issues related to the enzymatic mechanisms and molecular causes of resistance in vector populations is required as most results to date have been from diagnostic doses through standardized techniques by the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). In this chapter these results are socialized and we discuss some ideas related to the topic.

## **2. Study area**

The continental territory of the Republic of Colombia is located in the northwest corner of South America on the equator. Colombia is located in the north to 12°26'46'' north latitude at the place called Punta Gallinas in the peninsula of Guajira. To the south, is located at 4° 12' 30'' de latitude south, the East is located at 60°50'54'' west longitude from Greenwich, by the West is located at 79°02' 33'' west longitude from Greenwich.

Colombia territory also includes the archipelago of San Andres and Providencia in the Caribbean sea between 12° and 16°30´north latitude and 78° and 82° west longitude of Greenwich. Colombia has a land area of 1.141.748 km2 ranking fourth among the countries of South America.

Colombia is a country with a wide variety of climates, from the coldest to 0°C to temperatures exceeding 28°C especially in the Caribbean regions, Pacific, Amazon and the Orinoco, accounting for nearly 80% of Colombia territory. Colombia is divided administratively into 32 states, these in turn are divided into 1102 municipalities.

## **3. The situation of dengue in Colombia**

In Colombia, dengue is a disease of public health interest for its impact on morbidity and mortality. Its endemic behavior is due to endemic multiple factors including the re-emergence and intense viral transmission with a rising trend, the behavior of epidemic cycles getting shorter, the increased frequency of outbreaks and serious of the disease, the simultaneous circulation of the four serotypes, infestation by *A. aegypti* in over 90% of the national territory located below the 2,200 meters, and other social factors such as uncontrolled increase and lack of basic sanitation in urban centers (Escobar, 2009). The epidemiological pattern of disease in the last decades has been upward, characterized by exponential increase in endemic areas during the different decades. Cyclical behavior has been characterized by epidemic peaks every three or four years, dealing with the reentry of new serotypes to the country.

In Colombia from 1978 to 2010, has officially been recorded a total of 1.011.852 cases of dengue. For this time period there is record history of the following epidemics in the country:

1977: First recorded epidemic of dengue in Colombia.

2002: Epidemic in 76,996 cases, of which 5269 corresponded to Dengue hemorrhagic fever.

2007: Epidemic in 43,227 cases, of which 4665 corresponded to Dengue hemorrhagic fever.

2010: It is recorded in the country the worst epidemic of this disease with 157,152 confirmed cases and 217 deaths.

The departments that have historically had more transmission of dengue in the country are: Atlántico, Santander, Norte de Santander, Valle del Cauca, Antioquia, Tolima, Huila, Cundinamarca, and Casanare.

## **4. Resistance to insecticides**

## **4.1 Definition**

164 Insecticides – Pest Engineering

The records of vector resistance in the country have increased considerably since 2007 when in Colombia was created the national network for the surveillance of susceptibility to insecticides used in public health to *A. aegypti* and major vector of malaria. With this information a base line has been defined for some Colombian departments in the region and basic knowledge has been generated for decision making from the operating standpoint of vector control programs. However, more research on issues related to the enzymatic mechanisms and molecular causes of resistance in vector populations is required as most results to date have been from diagnostic doses through standardized techniques by the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). In this chapter these results are socialized and we discuss some ideas related to the topic.

The continental territory of the Republic of Colombia is located in the northwest corner of South America on the equator. Colombia is located in the north to 12°26'46'' north latitude at the place called Punta Gallinas in the peninsula of Guajira. To the south, is located at 4° 12' 30'' de latitude south, the East is located at 60°50'54'' west longitude from Greenwich, by the

Colombia territory also includes the archipelago of San Andres and Providencia in the Caribbean sea between 12° and 16°30´north latitude and 78° and 82° west longitude of Greenwich. Colombia has a land area of 1.141.748 km2 ranking fourth among the countries

Colombia is a country with a wide variety of climates, from the coldest to 0°C to temperatures exceeding 28°C especially in the Caribbean regions, Pacific, Amazon and the Orinoco, accounting for nearly 80% of Colombia territory. Colombia is divided

In Colombia, dengue is a disease of public health interest for its impact on morbidity and mortality. Its endemic behavior is due to endemic multiple factors including the re-emergence and intense viral transmission with a rising trend, the behavior of epidemic cycles getting shorter, the increased frequency of outbreaks and serious of the disease, the simultaneous circulation of the four serotypes, infestation by *A. aegypti* in over 90% of the national territory located below the 2,200 meters, and other social factors such as uncontrolled increase and lack of basic sanitation in urban centers (Escobar, 2009). The epidemiological pattern of disease in the last decades has been upward, characterized by exponential increase in endemic areas during the different decades. Cyclical behavior has been characterized by epidemic peaks

administratively into 32 states, these in turn are divided into 1102 municipalities.

every three or four years, dealing with the reentry of new serotypes to the country.

For this time period there is record history of the following epidemics in the country:

In Colombia from 1978 to 2010, has officially been recorded a total of 1.011.852 cases of dengue.

2002: Epidemic in 76,996 cases, of which 5269 corresponded to Dengue hemorrhagic fever. 2007: Epidemic in 43,227 cases, of which 4665 corresponded to Dengue hemorrhagic fever. 2010: It is recorded in the country the worst epidemic of this disease with 157,152 confirmed

The departments that have historically had more transmission of dengue in the country are: Atlántico, Santander, Norte de Santander, Valle del Cauca, Antioquia, Tolima, Huila,

West is located at 79°02' 33'' west longitude from Greenwich.

**3. The situation of dengue in Colombia** 

1977: First recorded epidemic of dengue in Colombia.

**2. Study area** 

of South America.

cases and 217 deaths.

Cundinamarca, and Casanare.

Resistance is defined as the development of the ability to tolerate doses of toxics, which can be lethal to most individuals in a normal population of the same species and is the result of positive selection pressure exerted by the insecticidal over genes initially in low frequency (WHO, 1957).

## **4.2 Mechanisms of resistance**

The two main resistance mechanisms are alterations in the target site and metabolic resistance, also called increase in the rate of detoxification of insecticides. However, there are other less frequent as there are the resistance behavior and decreased penetration. (Miller, 1988; Bisset, 2002; Fonseca and Quiñones, 2005)

## **4.2.1 Altered target site**

This is generated when no silent mutations occur in structural genes that generate a disturbance of amino acids responsible of anchor of the insecticide at a specific site.

## **4.2.1.1 GABA Receptor**

In bugs this is an hetero multi numeric channel acting as a site of action for cyclodiene and avermectins. The resistance is given by *Rdl* gene (Resistance to dieldrin), which encodes for RDL, a GABA receptor subunit. This type of resistance was first identified in *Drosophila* first identified this type of resistance (Fonseca and Quiñones, 2005).

## **4.2.1.2 Voltage-gated sodium channel**

They are the target of action of pyrethroids and DDT. The sodium para-channel protein is a complex of over 2000 amino acids, composed of 4 homologous domains separated by hydrophilic links, each domain contains six segments. The first mutation in the sodium channel that conferred resistance was detected in *Musca domestica*. subsequently mutations in other insects have been identified including Culicidae resistant to pyrethroids (Martínez et al, 1998; Brengues et al, 2003; Anstead, 2005; Saavedra et al, 2007; Brooke, 2008; Chang et al 2009; Yanola et al, 2010).

## **4.2.1.3 Insensitive acethylcholinesterase**

Insecticide resistance, attributed to Ache insensitivity is found in a number of *Anopheles* and *Culex* species. In general, this mechanism produces a broad spectrum resistance to most organophosphates and carbamates, although more pronounced to carbamatos. (Bisset, 2002).

## **4.2.2 Metabolic resistance**

It is conferred by an increase in detoxification insecticide or an inability to metabolize the toxic compound. The most important form of metabolic resistance is given by detoxificating enzymes of the type glutathione S-transferase, mixed function oxidases and esterases (Bisset 2002).

## **4.2.2.1 Carboxylesterases**

They Catalyze the hydrolysis of carboxylic esters and changes in their expression levels, this is the resistance mechanism that occurs most often in insects. In mosquitoes, high levels of these enzymes have been associated with resistance to organophosphate and pyrethroid insecticides (Cui et al, 2007; Rodriguez et al, 2007). Esterases is a family of six proteins

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 167

Technique standardized by the World Health Organization (WHO). In adult mosquitoes the technique consists of exposing individuals of the populations we intent to evaluate to papers impregnated with a single and specific dose of an insecticide during a determined time (1 hour with the exception of the organophosphate fenitrothion, to which will be exposed for 2 hours). The mosquitoes are then transfered to a paper that hasn't been impregnated, where they rest under controlled conditions of temperature, relative humidity and an energy source consisting of a sugar solution (Figure 2A). The mortality reading is

The susceptibility of larvae is measured by exposing individuals to a diagnostic concentration or several temephos concentrations to determine the lethal concentration (LC50 or CL95) to calculate resistance factor using water as solvent (Figure 2B). Individuals

**4.4 Techniques used to detect resistance insecticides in mosquitoes** 

**4.4.1 Biological test of the World Health Organization (WHO)** 

done after 24 hours of exposure to the insecticide (WHO 1981a).

remain exposed for 24 hours. (WHO 1981b) (Santacoloma, 2008).

Fig. 2A. Biological test of the World Health Organization (WHO) (mosquitoes).

The impregnated bottles technique was standardized by the Center for Diseases Control and Prevention (CDC) (Brogdon and McAllister, 1998). The objective of this technique is to measure the time it takes dose of an insecticide to reach the target site of action in the

**4.4.2 Test for biological control center and prevention (CDC)** 

Esterases are a family of six proteins grouped in α and β hydrolases superfamily. In the Diptera these enzymes are encoded by a gene cluster on the same chromosome, each gene may suffer modification that confers resistance to the insecticide (Fonseca and Quiñones, 2005; Santacoloma, 2008).

#### **4.2.2.2 P450 Enzymes or mixed function oxidases**

P450 enzymes are encoded by cytochrome P450 genes. P450 enzymes also have other names such as: cytochrome P450 monooxygenases, mixed function oxidases, monooxygenases with polisustrato, microsomal oxidases and heme-thiolate proteins. In insects these enzymes are involved in growth and development through the processing of fatty acids, hormones and pheromones; in the metabolism of secondary plants products and synthetic chemicals such as insecticides. The cytochrome P-450 is implicated as the major factor in many cases of metabolic resistance to carbamates and detoxify organophosphates, pyrethroids and DDT as well. It expresses in tissues of the gut, fat, reproductive tract and Malpighian tubes (Feyereisen, 1999; Poupardin et al, 2008).

## **4.2.2.3 Glutatione s-transferase**

The glutathione s-transferases (GSTs, EC 2.5.1.18) are phase II enzymes involved in xenobiotics detoxification in many organisms. These enzymes metabolize a wide range of toxic hydrophobic compounds such as drugs, insecticides and endogenous toxic substrates, catalyzing the conjugation of glutathione to the hydrophilic center of toxic substances, allowing the increase in compounds solubility. The GSTs are divided into three main groups: the cytosolic, microsomal and mitochondrial. The mitochondrial GSTs have not been found in insects species including mosquitoes. The cytosolic GSTs in insects are grouped into six different kinds: Delta, Epsilon, Omega, Sigma, Theta and Zeta. Most GSTs involved in the metabolism of xenobiotics in insects belong to the class Delta Epsilon. GSTs in insects have been implicated in the insecticide resistance through its direct metabolism, sequestration or protection against secondary toxic effects such as increased lipid peroxidation induced by exposure to insecticides. Glutathione S- transferase mediated detoxification has been reported for insecticides of the organophosphate type and DDT. The main role of GSTs in the resistance to organophosphates is the detoxification of the insecticide by a conjugation reaction. The specific GSTs also catalyze the metabolism of DDT to a non-toxic substance: 1,1-dichloro-2 ,2-bis-(p-chlorophenyl) ethane (DDE), through the process of dehydrochlorination. Epsilon class of GST has been involved in resistance to DDT in *Anopheles gambiae* and *A. aegypti* (Lumjuan et al, 2007).

## **4.3 Type of resistance**

### **4.3.1 Cross resistance**

It occurs when a single gene confers resistance to a number of chemicals in the same group, as it is the case of phosphotriesterases that provides resistance to several organophosphates or *kdr* gene that confers resistance DDT and the pyrethroids (WHO, 1957).

## **4.3.2 Multiple resistance**

It occurs when two or more resistance mechanisms are operating in the same insect. The multiple resistance term does not necessarily involve the term cross-resistance, because a bug may be resistant to 2 or more insecticides and each resistance can be attributed to different mechanisms (Bisset, 2002).

Esterases are a family of six proteins grouped in α and β hydrolases superfamily. In the Diptera these enzymes are encoded by a gene cluster on the same chromosome, each gene may suffer modification that confers resistance to the insecticide (Fonseca and Quiñones,

P450 enzymes are encoded by cytochrome P450 genes. P450 enzymes also have other names such as: cytochrome P450 monooxygenases, mixed function oxidases, monooxygenases with polisustrato, microsomal oxidases and heme-thiolate proteins. In insects these enzymes are involved in growth and development through the processing of fatty acids, hormones and pheromones; in the metabolism of secondary plants products and synthetic chemicals such as insecticides. The cytochrome P-450 is implicated as the major factor in many cases of metabolic resistance to carbamates and detoxify organophosphates, pyrethroids and DDT as well. It expresses in tissues of the gut, fat, reproductive tract and Malpighian tubes

The glutathione s-transferases (GSTs, EC 2.5.1.18) are phase II enzymes involved in xenobiotics detoxification in many organisms. These enzymes metabolize a wide range of toxic hydrophobic compounds such as drugs, insecticides and endogenous toxic substrates, catalyzing the conjugation of glutathione to the hydrophilic center of toxic substances, allowing the increase in compounds solubility. The GSTs are divided into three main groups: the cytosolic, microsomal and mitochondrial. The mitochondrial GSTs have not been found in insects species including mosquitoes. The cytosolic GSTs in insects are grouped into six different kinds: Delta, Epsilon, Omega, Sigma, Theta and Zeta. Most GSTs involved in the metabolism of xenobiotics in insects belong to the class Delta Epsilon. GSTs in insects have been implicated in the insecticide resistance through its direct metabolism, sequestration or protection against secondary toxic effects such as increased lipid peroxidation induced by exposure to insecticides. Glutathione S- transferase mediated detoxification has been reported for insecticides of the organophosphate type and DDT. The main role of GSTs in the resistance to organophosphates is the detoxification of the insecticide by a conjugation reaction. The specific GSTs also catalyze the metabolism of DDT to a non-toxic substance: 1,1-dichloro-2 ,2-bis-(p-chlorophenyl) ethane (DDE), through the process of dehydrochlorination. Epsilon class of GST has been involved in resistance to DDT

It occurs when a single gene confers resistance to a number of chemicals in the same group, as it is the case of phosphotriesterases that provides resistance to several organophosphates

It occurs when two or more resistance mechanisms are operating in the same insect. The multiple resistance term does not necessarily involve the term cross-resistance, because a bug may be resistant to 2 or more insecticides and each resistance can be attributed to

or *kdr* gene that confers resistance DDT and the pyrethroids (WHO, 1957).

2005; Santacoloma, 2008).

**4.2.2.2 P450 Enzymes or mixed function oxidases**

(Feyereisen, 1999; Poupardin et al, 2008).

in *Anopheles gambiae* and *A. aegypti* (Lumjuan et al, 2007).

**4.2.2.3 Glutatione s-transferase** 

**4.3 Type of resistance 4.3.1 Cross resistance** 

**4.3.2 Multiple resistance** 

different mechanisms (Bisset, 2002).

## **4.4 Techniques used to detect resistance insecticides in mosquitoes 4.4.1 Biological test of the World Health Organization (WHO)**

Technique standardized by the World Health Organization (WHO). In adult mosquitoes the technique consists of exposing individuals of the populations we intent to evaluate to papers impregnated with a single and specific dose of an insecticide during a determined time (1 hour with the exception of the organophosphate fenitrothion, to which will be exposed for 2 hours). The mosquitoes are then transfered to a paper that hasn't been impregnated, where they rest under controlled conditions of temperature, relative humidity and an energy source consisting of a sugar solution (Figure 2A). The mortality reading is done after 24 hours of exposure to the insecticide (WHO 1981a).

The susceptibility of larvae is measured by exposing individuals to a diagnostic concentration or several temephos concentrations to determine the lethal concentration (LC50 or CL95) to calculate resistance factor using water as solvent (Figure 2B). Individuals remain exposed for 24 hours. (WHO 1981b) (Santacoloma, 2008).

Fig. 2A. Biological test of the World Health Organization (WHO) (mosquitoes).

## **4.4.2 Test for biological control center and prevention (CDC)**

The impregnated bottles technique was standardized by the Center for Diseases Control and Prevention (CDC) (Brogdon and McAllister, 1998). The objective of this technique is to measure the time it takes dose of an insecticide to reach the target site of action in the

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 169

These are chemicals that specifically inhibit insecticide-metabolizing enzymes, enhancing their action. Among the synergists more used to detect resistance mechanisms in Insects are the S, S, S - tributilfosforotioato (DJF) and esterases inhibitor and of the enzyme glutathione transferase (GST), the triphenyl phosphate (TFF) specific esterase inhibitor, piperonyl butoxide (PB), an inhibitor of monooxygenases, and ethacrynic acid (EA), a specific inhibitor

The biochemical assays are used to define metabolic mechanisms that may be responsible for the physiological resistance in an insect population (WHO, 1992). The metabolic mechanisms include tests to determine the target enzyme decreased sensitivity or the increased enzyme activity. For the first mechanism in particular, measures the change in acetylcholinesterase associated with resistance to carbamates and organophosphates. For the second, evaluates the increased activity of esterases, mixed function oxidases and glutathione s-transferase for kidnapping or increased detoxification of insecticides

These tests consist in the amplification of specific gene sequences through polymerase chain

Since the late forties, when first reported resistance to DDT in *Aedes tritaeniorhynchus*  (Weidemann) and *Aedes solicitans* (Walker) resistance has been recorded in over a hundred species for one or more insecticides of public health use worldwide (Brown, 1986; Fonseca & Quiñones, 2005). For *A. aegypti* in America resistance has been reported to organochlorines, organophosphates, pyrethroids and carbamates in Argentina, Brazil, Mexico, El Salvador, Peru, Panamá, Venezuela, Cuba, Puerto Rico, among other Caribbean countries, whose resistance mechanism in some of these stocks has been associated with altered levels of alpha esters, beta esterases, mixed function oxidases, glutathione s- transferase, as well as mutations in the voltage-gated sodium channel. (Rawlins, 1998; Bisset et al, 2001; Brengues et al, 2003; Macoris et al, 2003; Aparecida et al, 2004; Rodríguez et al, 2004; Chavez et al; 2005, Flores et al, 2005; Pereira da-Cunha et al, 2005, Alvarez et al, 2006, Pereira-Lima et al, 2006; Beserra et al, 2007; Saavedra et al, 2007; Bisset et al, 2009; Martins et al, 2009; Albrieu-

Colombia has applied insecticides for control of vector insects for over five decades. The DDT was the first applied to control malaria and during the campaign for the eradication of *A. aegypti* conducted in the early 1950. This insecticide was banned in the late 60's, due to the findings of resistance worldwide (Brown, 1986). Since 1970, organophosphates including temephos were applied and from the early 90's the use of pyrethroids was started. From that time on, the country has been rotating the application of molecules for mosquito control such as: deltamethrin, lambda-cyhalothrin, malathion, fenitrothion and in the last three years cyfluthrin and pirimiphos-methyl. However, the resistance to these insecticides has been documented gradually, making it difficult to take control actions within programs of

**4.5 Current state of susceptibility to insecticides** *Aedes aegypti* **in Colombia** 

of the enzyme glutathione transferase (GST) (Rodríguez, 2008).

**4.4.3 Synergists** 

**4.4.4 Biochemical test** 

(Santacoloma, 2008).

**4.4.5 Molecular tests** 

reaction technique (PCR) to detect mutations.

Llinas et al, 2010; Polson et al, 2010).

Vector Borne Diseases in different regions of the country.

Fig. 2B. Biological test of the World Health Organization (WHO) (Larvae).

mosquito. Involves subjecting a sample of mosquitoes between 15 and 25 individuals of the population to be evaluated on a glass surface pre-impregnated with the dose of insecticide. (Santacoloma, 2008) (Figure 3).

Fig. 3. Test for biological control center and prevention (CDC).

## **4.4.3 Synergists**

168 Insecticides – Pest Engineering

Fig. 2B. Biological test of the World Health Organization (WHO) (Larvae).

Fig. 3. Test for biological control center and prevention (CDC).

(Santacoloma, 2008) (Figure 3).

mosquito. Involves subjecting a sample of mosquitoes between 15 and 25 individuals of the population to be evaluated on a glass surface pre-impregnated with the dose of insecticide.

These are chemicals that specifically inhibit insecticide-metabolizing enzymes, enhancing their action. Among the synergists more used to detect resistance mechanisms in Insects are the S, S, S - tributilfosforotioato (DJF) and esterases inhibitor and of the enzyme glutathione transferase (GST), the triphenyl phosphate (TFF) specific esterase inhibitor, piperonyl butoxide (PB), an inhibitor of monooxygenases, and ethacrynic acid (EA), a specific inhibitor of the enzyme glutathione transferase (GST) (Rodríguez, 2008).

## **4.4.4 Biochemical test**

The biochemical assays are used to define metabolic mechanisms that may be responsible for the physiological resistance in an insect population (WHO, 1992). The metabolic mechanisms include tests to determine the target enzyme decreased sensitivity or the increased enzyme activity. For the first mechanism in particular, measures the change in acetylcholinesterase associated with resistance to carbamates and organophosphates. For the second, evaluates the increased activity of esterases, mixed function oxidases and glutathione s-transferase for kidnapping or increased detoxification of insecticides (Santacoloma, 2008).

## **4.4.5 Molecular tests**

These tests consist in the amplification of specific gene sequences through polymerase chain reaction technique (PCR) to detect mutations.

## **4.5 Current state of susceptibility to insecticides** *Aedes aegypti* **in Colombia**

Since the late forties, when first reported resistance to DDT in *Aedes tritaeniorhynchus*  (Weidemann) and *Aedes solicitans* (Walker) resistance has been recorded in over a hundred species for one or more insecticides of public health use worldwide (Brown, 1986; Fonseca & Quiñones, 2005). For *A. aegypti* in America resistance has been reported to organochlorines, organophosphates, pyrethroids and carbamates in Argentina, Brazil, Mexico, El Salvador, Peru, Panamá, Venezuela, Cuba, Puerto Rico, among other Caribbean countries, whose resistance mechanism in some of these stocks has been associated with altered levels of alpha esters, beta esterases, mixed function oxidases, glutathione s- transferase, as well as mutations in the voltage-gated sodium channel. (Rawlins, 1998; Bisset et al, 2001; Brengues et al, 2003; Macoris et al, 2003; Aparecida et al, 2004; Rodríguez et al, 2004; Chavez et al; 2005, Flores et al, 2005; Pereira da-Cunha et al, 2005, Alvarez et al, 2006, Pereira-Lima et al, 2006; Beserra et al, 2007; Saavedra et al, 2007; Bisset et al, 2009; Martins et al, 2009; Albrieu-Llinas et al, 2010; Polson et al, 2010).

Colombia has applied insecticides for control of vector insects for over five decades. The DDT was the first applied to control malaria and during the campaign for the eradication of *A. aegypti* conducted in the early 1950. This insecticide was banned in the late 60's, due to the findings of resistance worldwide (Brown, 1986). Since 1970, organophosphates including temephos were applied and from the early 90's the use of pyrethroids was started. From that time on, the country has been rotating the application of molecules for mosquito control such as: deltamethrin, lambda-cyhalothrin, malathion, fenitrothion and in the last three years cyfluthrin and pirimiphos-methyl. However, the resistance to these insecticides has been documented gradually, making it difficult to take control actions within programs of Vector Borne Diseases in different regions of the country.

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 171

insecticide Bendiocarb to which there has been resistance registered in the populations in the department of Cauca, Valle del Cauca, Huila and Nariño (Figure 10 AB) (Ocampo et al, 2011). Currently it has not been registered for the country mutations in voltage-gated sodium channel gene. It is therefore recommended studies to perform studies to explain resistance observed to most of the pyrethroids evaluated in different country populations and may be related to a crossed resistance to DDT. It is also recommended to perform studies to determine cross resistance to other molecules such as organophosphate and carbamate, as

For Colombia it is recommended to maintain a system of permanent time and space surveillance that allows health authorities to use insecticides with technical criteria to

Fig. 4A. Susceptility status of *Aedes aegypti* populations to DDT in Colombia (CDC test).

maintain effective control interventions in vector populations.

well as multi resistance studies.

In Colombia until the 1990's there were few works assessing the state of susceptibility in Culicidae populations of interest in public health. Between 1959 and 1987 the first cases of DDT resistance were registered in populations of *Anopheles albimanus* (Wiedemann) in the municipalities of El Carmen (Bolivar); Codazzi, Robles and Valledupar (Cesar); Acandí (Choco) and *An. darlingi* (Root) in some locations of Quibdó municipality (Chocó) (Quiñones et al, 1987). Later Suarez et al, (1996) recorded in the 90's decade the first case of resistance to temephos in the *A. aegypti* species in Cali, Valle del Cauca, and Bisset et al, (1998) evaluated the susceptibility in a strain of *Culex quinquefasciatus* (Say) from Medellin, Antioquia, encountering resistance to organophosphates malathion, primifos-methyl, chlorpyrifos, temephos, fenthion and pyrethroids deltamethrin and permethrin.

In the absence of enough studies in Colombia on susceptibility status of populations of *A. aegypti* to several insecticides of use in public health and in compliance with public policies enshrined in the American continent resolutions CD39.R11 1996, CD43R4 2001 of the Pan American Health Organization, during the years of 2005 and 2007 a national project was conducted, this was funded by Colciencias (Colombian Science and Research Organization) and implemented by the Learning and Control of Tropical Diseases Program (PECET) of the University of Antioquia, the International Centre for Training and Medical Research (CIDEIM), the National University in Colombia, the National Institute of Health (NIH) and 12 departments of health seeking to generate baseline susceptibility of vector populations in Colombia. This multicentered project gave rise to the national surveillance network susceptibility to insecticides for *A. aegypti* and main vectors of malaria led by the National Institute of Health (INS). Since then the record of resistance to *A. aegypti* in Colombia expanded through biological tests by the CDC and WHO as well as the determination of impairment of enzymes involved in resistance.

With these results it has been observed for Colombia widespread resistance to DDT (Figure 4A-4B) and variability in susceptibility to the following insecticides: temephos, lambdacyhalothrin, deltamethrin, permethrin, cyfluthrin, etofenprox, malathion, fenitrothion, pirimiphos methyl, bendiocarb and propoxur in different regions, with deterioration in some populations in the levels of nonspecific esterases, mixed function oxidases and in smaller proportion to glutathione s-transferesas (Figure 5A, 5B, 5C, 5D) (Rojas et al, 2003; Cadavid et al, 2008; Fonseca et al, 2006; Fonseca et al, 2007; Orjuela et al; 2007, Salazar et al, 2007; Santacoloma et al, 2008; Fonseca et al, 2009; Maestre et al, 2009; Maestre et al, 2010; Ardila and Brochero, 2010, Gomez et al, 2010; Maestre et al, 2010, Fonseca et al, 2011).

For temephos resistance has been observed in Cundinamarca, Guaviare, Meta, Santander, Cauca, Valle del Cauca, Nariño, Huila, Caldas, Sucre, Atlantico, La Guajira. (Anaya et al, 2007; Santacoloma, 2008; Maestre et al, 2009; Ocampo et al, 2011) (Figure 6).

Pyrethroids that despite being used in Colombia more recently compared to organophosphates, have shown higher levels of resistance despite the increased use time of organophosphate (Figures 7A, 7B, 7C, 7D, 7E), (8A, 8B, 8C). Among the pyrethroids lambda is the insecticide wich displays higher frequency of resistance in vector populations in the country. However, there are pyrethroids such as permethrin and etofenprox that despite having no use in public health have resistance generated in populations of *A. aegypti* from Casanare, Antioquia, Chocó and Putumayo (Ardila and Brochero, 2010; Fonseca et al, 2011).

For the carbamate propoxur discordance in susceptibility results was observed between the WHO technique in which resistance and CDC susceptibility is recorded. Further studies are required to determine the state of populations' susceptibility to the insecticide (Fig. 9 AB). Furthermore, few studies in the country have evaluated the susceptibility of the vector to the

In Colombia until the 1990's there were few works assessing the state of susceptibility in Culicidae populations of interest in public health. Between 1959 and 1987 the first cases of DDT resistance were registered in populations of *Anopheles albimanus* (Wiedemann) in the municipalities of El Carmen (Bolivar); Codazzi, Robles and Valledupar (Cesar); Acandí (Choco) and *An. darlingi* (Root) in some locations of Quibdó municipality (Chocó) (Quiñones et al, 1987). Later Suarez et al, (1996) recorded in the 90's decade the first case of resistance to temephos in the *A. aegypti* species in Cali, Valle del Cauca, and Bisset et al, (1998) evaluated the susceptibility in a strain of *Culex quinquefasciatus* (Say) from Medellin, Antioquia, encountering resistance to organophosphates malathion, primifos-methyl, chlorpyrifos,

In the absence of enough studies in Colombia on susceptibility status of populations of *A. aegypti* to several insecticides of use in public health and in compliance with public policies enshrined in the American continent resolutions CD39.R11 1996, CD43R4 2001 of the Pan American Health Organization, during the years of 2005 and 2007 a national project was conducted, this was funded by Colciencias (Colombian Science and Research Organization) and implemented by the Learning and Control of Tropical Diseases Program (PECET) of the University of Antioquia, the International Centre for Training and Medical Research (CIDEIM), the National University in Colombia, the National Institute of Health (NIH) and 12 departments of health seeking to generate baseline susceptibility of vector populations in Colombia. This multicentered project gave rise to the national surveillance network susceptibility to insecticides for *A. aegypti* and main vectors of malaria led by the National Institute of Health (INS). Since then the record of resistance to *A. aegypti* in Colombia expanded through biological tests by the CDC and WHO as well as the determination of

With these results it has been observed for Colombia widespread resistance to DDT (Figure 4A-4B) and variability in susceptibility to the following insecticides: temephos, lambdacyhalothrin, deltamethrin, permethrin, cyfluthrin, etofenprox, malathion, fenitrothion, pirimiphos methyl, bendiocarb and propoxur in different regions, with deterioration in some populations in the levels of nonspecific esterases, mixed function oxidases and in smaller proportion to glutathione s-transferesas (Figure 5A, 5B, 5C, 5D) (Rojas et al, 2003; Cadavid et al, 2008; Fonseca et al, 2006; Fonseca et al, 2007; Orjuela et al; 2007, Salazar et al, 2007; Santacoloma et al, 2008; Fonseca et al, 2009; Maestre et al, 2009; Maestre et al, 2010; Ardila and Brochero, 2010, Gomez et al, 2010; Maestre et al, 2010, Fonseca et al, 2011). For temephos resistance has been observed in Cundinamarca, Guaviare, Meta, Santander, Cauca, Valle del Cauca, Nariño, Huila, Caldas, Sucre, Atlantico, La Guajira. (Anaya et al,

Pyrethroids that despite being used in Colombia more recently compared to organophosphates, have shown higher levels of resistance despite the increased use time of organophosphate (Figures 7A, 7B, 7C, 7D, 7E), (8A, 8B, 8C). Among the pyrethroids lambda is the insecticide wich displays higher frequency of resistance in vector populations in the country. However, there are pyrethroids such as permethrin and etofenprox that despite having no use in public health have resistance generated in populations of *A. aegypti* from Casanare, Antioquia, Chocó and Putumayo (Ardila and Brochero, 2010; Fonseca et al, 2011). For the carbamate propoxur discordance in susceptibility results was observed between the WHO technique in which resistance and CDC susceptibility is recorded. Further studies are required to determine the state of populations' susceptibility to the insecticide (Fig. 9 AB). Furthermore, few studies in the country have evaluated the susceptibility of the vector to the

2007; Santacoloma, 2008; Maestre et al, 2009; Ocampo et al, 2011) (Figure 6).

temephos, fenthion and pyrethroids deltamethrin and permethrin.

impairment of enzymes involved in resistance.

insecticide Bendiocarb to which there has been resistance registered in the populations in the department of Cauca, Valle del Cauca, Huila and Nariño (Figure 10 AB) (Ocampo et al, 2011). Currently it has not been registered for the country mutations in voltage-gated sodium channel gene. It is therefore recommended studies to perform studies to explain resistance observed to most of the pyrethroids evaluated in different country populations and may be related to a crossed resistance to DDT. It is also recommended to perform studies to determine cross resistance to other molecules such as organophosphate and carbamate, as well as multi resistance studies.

For Colombia it is recommended to maintain a system of permanent time and space surveillance that allows health authorities to use insecticides with technical criteria to maintain effective control interventions in vector populations.

Fig. 4A. Susceptility status of *Aedes aegypti* populations to DDT in Colombia (CDC test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 173

Fig. 5A. Non-specific esterases (NSE).

Fig. 4B. Susceptility status of *Aedes aegypti* populations to DDT in Colombia (OMS test).

Fig. 4B. Susceptility status of *Aedes aegypti* populations to DDT in Colombia (OMS test).

Fig. 5A. Non-specific esterases (NSE).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 175

Fig. 5C. Glutathione-*S*-transferases (GST).

Fig. 5B. Mixed-function oxidases (MFO).

Fig. 5B. Mixed-function oxidases (MFO).

Fig. 5C. Glutathione-*S*-transferases (GST).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 177

Fig. 6. Susceptility status of *Aedes aegypti* populations to Temephos in Colombia (OMS test).

Fig. 5. Biochemical mechanism of resistance in population of *Aedes aegypti* in Colombia: Non-specific esterases (NSE) (5A); Mixed-function oxidases (MFO) (5B); Glutathione-*S*transferases (GST) (5C); acethylcholinesterase (AChE) (5D).

5D

Fig. 5. Biochemical mechanism of resistance in population of *Aedes aegypti* in Colombia: Non-specific esterases (NSE) (5A); Mixed-function oxidases (MFO) (5B); Glutathione-*S*-

transferases (GST) (5C); acethylcholinesterase (AChE) (5D).

Fig. 6. Susceptility status of *Aedes aegypti* populations to Temephos in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 179

Fig. 7B. Susceptility status of *Aedes aegypti* populations to lambda-cyhalothrin in Colombia

(OMS test).

Fig. 7A. Susceptility status of *Aedes aegypti* populations to lambda-cyhalothrin in Colombia (CDC test).

Fig. 7A. Susceptility status of *Aedes aegypti* populations to lambda-cyhalothrin in Colombia

(CDC test).

Fig. 7B. Susceptility status of *Aedes aegypti* populations to lambda-cyhalothrin in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 181

Fig. 7D. Susceptility status of *Aedes aegypti* populations to Deltamethrin in Colombia (OMS

Fig. 7C. Susceptility status of *Aedes aegypti* populations to Deltamethrin in Colombia (CDC test).

Fig. 7C. Susceptility status of *Aedes aegypti* populations to Deltamethrin in Colombia (CDC

Fig. 7D. Susceptility status of *Aedes aegypti* populations to Deltamethrin in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 183

Fig. 7G. Susceptility status of *Aedes aegypti* populations to Permethrin in Colombia (CDC

Fig. 7F. Susceptility status of *Aedes aegypti* populations to cyfluthrin in Colombia (OMS test).

Fig. 7F. Susceptility status of *Aedes aegypti* populations to cyfluthrin in Colombia (OMS test).

Fig. 7G. Susceptility status of *Aedes aegypti* populations to Permethrin in Colombia (CDC test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 185

Fig. 7I. Susceptility status of *Aedes aegypti* populations to etofenprox in Colombia (OMS test).

Fig. 7H. Susceptility status of *Aedes aegypti* populations to Permethrin in Colombia (OMS test).

Fig. 7H. Susceptility status of *Aedes aegypti* populations to Permethrin in Colombia (OMS

Fig. 7I. Susceptility status of *Aedes aegypti* populations to etofenprox in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 187

Fig. 8B. Susceptility status of *Aedes aegypti* populations to Malathion in Colombia (OMS test).

Fig. 8A. Susceptility status of *Aedes aegypti* populations to Malathion in Colombia (CDC test).

Fig. 8A. Susceptility status of *Aedes aegypti* populations to Malathion in Colombia (CDC

Fig. 8B. Susceptility status of *Aedes aegypti* populations to Malathion in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 189

Fig. 8D. Susceptility status of *Aedes aegypti* populations to Fenitrothion in Colombia (OMS

Fig. 8C. Susceptility status of *Aedes aegypti* populations to Fenitrothion in Colombia (CDC test).

Fig. 8C. Susceptility status of *Aedes aegypti* populations to Fenitrothion in Colombia (CDC

Fig. 8D. Susceptility status of *Aedes aegypti* populations to Fenitrothion in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 191

Fig. 8F. Susceptility status of *Aedes aegypti* populations to pirimiphos methyl in Colombia

(OMS test).

Fig. 8E. Susceptility status of *Aedes aegypti* populations to pirimiphos methyl in Colombia (CDC test).

Fig. 8E. Susceptility status of *Aedes aegypti* populations to pirimiphos methyl in Colombia

(CDC test).

Fig. 8F. Susceptility status of *Aedes aegypti* populations to pirimiphos methyl in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 193

Fig. 9B. Susceptility status of *Aedes aegypti* populations to propoxur in Colombia (OMS test).

Fig. 9A. Susceptility status of *Aedes aegypti* populations to propoxur in Colombia (CDC test).

Fig. 9A. Susceptility status of *Aedes aegypti* populations to propoxur in Colombia (CDC test).

Fig. 9B. Susceptility status of *Aedes aegypti* populations to propoxur in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 195

Fig. 10B. Susceptility status of *Aedes aegypti* populations to bendiocarb in Colombia (OMS

Fig. 10A. Susceptility status of *Aedes aegypti* populations to bendiocarb in Colombia (CDC test).

Fig. 10A. Susceptility status of *Aedes aegypti* populations to bendiocarb in Colombia (CDC

Fig. 10B. Susceptility status of *Aedes aegypti* populations to bendiocarb in Colombia (OMS test).

Susceptibility Status of *Aedes aegypti* to Insecticides in Colombia 197

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## **5. Conclusion**

The selection pressure exert by insecticides for more than five decades on the populations of *A. aegypti* in Colombia, has generated widespread resistance to DDT and variability in susceptibility to phosphorated, carabamates and pyrethroids insecticides. As a possible cause of metabolic resistance to these insecticides, alteration in nonspecific esterase levels has been registered, mixed function oxidases and s-glutathione transferases. However, it is unknown whether there are alterations in target sites of action, especially in voltagedependent sodium channel genes that explains the generalized resistance to DDT and most pyrethroids in the country.

## **6. Acknowledgment**

Sponsored by the project Strengthening UNIMOL Group 2011 Vice-Rectory of Research - Res. 4357 (2010) Colombia.

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**5. Conclusion** 

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Res. 4357 (2010) Colombia.

**6. Acknowledgment** 

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Rojas-Alvarez, DP. (2010). Comportamineto epidemiologico del dengue en Colombia, año 2009. *Informe quincenal Epidemiologico Nacional*, Vol.15, No.9, pp.129-37. Saavedra-Rodriguez, K.; Urdaneta-Marquez, L.; Rajatileka, S.; Moulton, M.; Flores, AE.;

permetrina, propoxur y DDT. *Biomédica,* Vol.23, No.1, pp.56

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Resistance status of *Aedes aegypti* in 10 localities of Colombia. *Acta Trop.*, Vol.118

insecticidas del mosquito *Aedes aegypti* en lo municipios de Chinchiná y La Dorada,

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian *Aedes aegypti*  (Diptera: Culicidae) populations collected between 2001 and 2003. *Memorias do* 

Cavalcanti, L. & Soares-Pontes, R. (2006). *Aedes aegypti* resistance to temephos in counties of Ceará State. *Revista da Sociedade Brasileira de Medicina Tropical,* Vol.39,

involved in DDT and pyrethroid resistance in Trinidad and Tobago strains of *Aedes aegypti. The American Journal of tropical medicine and hygiene*, Vol.83, No.5, pp.51. Poupardin, R.; Reynaud, S.; Strode, C.; Ranson, H.; Vontas, J. & David JP. (2008). Cross-

induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: Impact on larval tolerance to chemical insecticides.

los principales vectores de malaria en Colombia y su implicación epidemiológica.

poblaciones de *Aedes aegypti* en el Caribe. *Revista Panamericana de Salud Pública,*

larvas y adultos de *Aedes aegypti*: prevalencia de esterasa A4 asociada con la resistencia a temefos. *Revista Cubana de Medicina Tropical*, Vol.56, No.1, pp.54-60. Rodríguez, MM.; Bisset, JA. & Fernández, D. (2007). Determinación *in vivo* del papel de las

enzimas esterasas y glutation transferasa en la resistencia a piretroides en *Aedes aegypti* (Diptera: Culicidae). *Revista Cubana de Medicina Tropical,* Vol.59, No.3,

*Culicidae*). Tesis presentada al grado científico de Doctor en Ciencias de la Salud. Laboratorio de Toxicología y genética. Instituto de Medicina Tropical "Pedro

susceptibilidad de Aedes aegypti del municipio de Barrancabermeja Santander a los insecticidas Malatión Fenitrotion, temefos, Lambdacihalotrina, deltametrina,

Fernandez-Salas, I.; Bisset, J.; Rodriguez, M.; Mccall, PJ.; Donnelly, MJ.; Ranson, H.;


Fonseca-González, I. (2008). Estatus de la resistencia a insecticidas de los vectores primarios

Fonseca-Gonzalez, I.; Quiñones, ML.; Lenhartc, A. & Brogdon, WG. (2011). Insecticide

Gómez-Camargo DE.; Maestre, RY.; Pisciotti, I.; Malambo, DI. & Gómez-Alegría, CJ. (2010).

Hemingway, J. & Black IV, WC. (2007). A mutation in the voltage-gated sodium channel

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Macoris, M.; Andrighetti, M.; Takaku, L.; Glasse, C.; Garbeloto, V. & Bracco, J. (2003).

Maestre-Serrano, R.; Goenaga, S.; Carrasquilla-Ferro MC.; Rey-Vega G.; Santacoloma-Baron

Maestre, RY.; Florez, Z.; Cabrera, C.; Goenaga, S.; Gómez, D. & Gómez, C. (2010).

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Miller TA. 1988. Mechanisms of resistance to pyrethroid insecticides. *Parasitology Today*,

*American Journal of Tropical Medicine and Hygiene,* Vol.83, No.5, pp.53. Maestre, R.; Rey, G.; De las Salas, J.; Vergara, C.; Santacoloma, L.; Goenaga, S. &

gambiae s.s. *Insect Molecular Biology,* Vol.7, pp.179–184.

insecticidas. *Memórias do Instituto Oswaldo Cruz*, Vol.98, No.5, pp.703-8. Maestre, R.; Rey, G.; De las Salas, J.; Vergara, C.; Santacoloma, L.; Goenaga, S. &

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Colombia. [Tesis de doctorado]. Medellín: Universidad de Antioquia. Fonseca, I. & Bolaños, D. (2009).Variación temporal en la susceptibilidad a malatión y

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de malaria y dengue en Antioquia, Chocó, Norte de Santander y Putumayo,

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resistance status of *Aedes aegypti* (L.) from Colombia. *Pest Management Science*,

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gene associated with pyrethroid resistance in Latin American *Aedes aegypti*. *Insect* 

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Resistance of *Aedes aegypti* from the state of Sao Paulo, Brazil to Organophosphates

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L. & Vergara-Sánchez C. (2009). Estado de la susceptibilidad/resistencia a insecticidas en cuatro poblaciones de *Aedes aegypti* del departamento del Atlántico.

Susceptibility status of *Aedes aegypti* to insecticides in La Guajira (Colombia). *The* 

Carrasquilla, MC. (2010). Estado de la susceptibilidad de *Aedes aegypti* a insecticidas en Atlántico (Colombia). *Revista Colombiana de Entomología*,Vol.36, No.2, pp.242-248.

mutation in the voltage-gated sodium channel gene of *Aedes aegypti* Brazilian populations. *Tropical Medicine and International Health*, Vol.14, No.11, pp.1351-55. Martinez-Torres, D.; Chandre, F.; Williamson, MS.; Darriet, F.; Berge, JB.; Devonshire, AL.;

Guillet, P.; Pasteur, N. & Pauron, D. (1998). Molecular characterization of pyrethroid knockdown resistance (kdr ) in the major malaria vector Anopheles


**1. Introduction** 

**9** 

*Thailand* 

**Behavioral Responses of** 

Theeraphap Chareonviriyaphap

*Kasetsart University, Bangkok,* 

**Mosquitoes to Insecticides** 

*Department of Entomology, Faculty of Agriculture,* 

Many people living in areas of the tropical and subtropical world are at serious risk of infection from a wide variety of vector-borne diseases, most notably malaria and dengue. Globally, approximately 50-100 million people are estimated to be at risk of infection with dengue viruses (the cause of dengue fever/dengue haemorrhagic fever) and between 100- 300 million live in malaria endemic areas (World Health Organization [WHO], 2009). The viruses responsible for dengue are transmitted primarily by *Aedes aegypti*, a predominately urban, day-biting mosquito that often resides in and around human dwellings and preferentially feeds on humans (Gubler, 1997); whereas the 4 human malaria parasites (*Plasmodium*) are transmitted by a wide variety of *Anopheles* species (Service & Townson, 2002 ). Dengue vector has proven extremely resilient to control measures because of its close association and exploitation of domestic and peridomestic human settings (Reiter & Gubler, 1997). On the other hand, malaria vectors display a more diverse array of host seeking behaviors and preference, biting patterns and larval breeding habitats (Pates & Curtis, 2005; Sinka et al., 2011). Despite decades of extensive research, efficacious and commercially viable vaccines for these 2 important vector-borne diseases are not yet available. Therefore, the prevention and control of dengue and malaria remains dependent on various vector control strategies to reduce risk of transmission; in some instances this requires the use of various chemical insecticides as larvicides, space spray and indoor residual spray (IRS) applications, and use of insecticide-impregnated bed nets to control adult mosquito blood feeding (Roberts &

Chemical insecticides, including organochlorines, organophosphates, carbamates, and synthetic pyrethroids, have long been used with great effect in public health vector control programs worldwide (Reiter & Gubler, 1997; Roberts & Andre, 1994; WHO, 1992). Although DDT used ceased in many countries several decades ago, the chemical has returned for use in malaria control IRS programs in Africa because of some its superior attributes (Roberts & Tren, 2010). The dramatic impact of DDT on mosquito populations in terms of both toxicity and behavior suppressing disease transmission is well known but in some instances the actual mechanisms at work remain unclear and poorly understood. Most studies on insecticides have placed attention exclusively on the direct toxicological (knockdown and killing) effects on mosquito populations; whereas far less research has focused on the

Andre, 1994; WHO, 1999; Reiter & Gubler, 1997; Grieco et al., 2007).

Salazar, M.; Carvajal, A.; Cuellar, ME.; Olaya, A.; Quiñones, J.; Velásquez, OL.; Viveros, A. & Ocampo, C. (2007). Resitencia a insecticidas en poblaciones de *Aedes aegypti* y *Anopheles* spp en los departamentos de Huila, valle Cauca y Nariño. *Biomédica*, Vol.27, No.2, pp.177.


http://www.todacolombia.com/geografia/ubicacion.html


## **Behavioral Responses of Mosquitoes to Insecticides**

Theeraphap Chareonviriyaphap

*Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand* 

## **1. Introduction**

200 Insecticides – Pest Engineering

Santacoloma, L. (2008). Estado de la susceptibilidad a insecticidas de poblaciones naturales

Colombia. [Tesis de maestría]. Bogotá: Universidad Nacional de Colombia. Santacoloma-Varon, L.; Chavez-Cordoba, B. & Brochero, HL. (2010). Susceptibilidad de

Valderrama-Eslava, EI.; Gomzalez, R. & Jaramillo, GI. (2008). Evaluación de la

World Health Organization (WHO). (1957). Seventh report Expert Committee on

World Health Organization (WHO). (1981a). Instructions for determining the susceptibility

carbamate insecticides. Establishment of the base-line. WHO/ VBC/81.805. World Health Organization (WHO). (1981b). Instructions for determining the susceptibility

Yanola, J.; Somboon, P.; Walton, C.; Nachaiwieng, W. & Prapanthadara L. (2010). A novel

*Panamericana de Salud Publica*, Vol.27, No.1, pp.66-73. http://www.todacolombia.com/geografia/ubicacion.html

insecticides *WHO Tech Report Ser*, Vol.125, pp.37.

*Biomédica*, Vol.27, No.2, pp.177.

*del Valle*, Vol.9, No.2, pp.1-11.

VBC/81.807.

Vol.96, pp.127-13.

Salazar, M.; Carvajal, A.; Cuellar, ME.; Olaya, A.; Quiñones, J.; Velásquez, OL.; Viveros, A. & Ocampo, C. (2007). Resitencia a insecticidas en poblaciones de *Aedes aegypti* y *Anopheles* spp en los departamentos de Huila, valle Cauca y Nariño.

de *Aedes aegypti* Linnaeus 1762 vector del dengue y *Anopheles darlingi* root 1926 vector primario de malaria (Diptera: Culicidae) en cinco departamentos de

*Aedes aegypti* a DDT, Deltametrina y Lambdacialotrina en Colombia. *Revista* 

susceptibilidad de *Aedes aegypti* (L) (Diptera: Culicidae) a un insecticida Organosfosforado y un piretroide en cuatro poblaciones del Valle del Cauca, mediante dos tipos de bioensayos. *Boletin del Museo de Entomología de la Universidad* 

or resistance of adult mosquitoes to organoclorine, organophosphorus and

or resistance of mosquitoes larvae to insecticide. Unpublished document.WHO/

F1552/C1552 point mutation in the *Aedes aegypti* voltage-gated sodium channel gene associated with permethrin resistance. *Pesticide Biochemistry and hysiology*, Many people living in areas of the tropical and subtropical world are at serious risk of infection from a wide variety of vector-borne diseases, most notably malaria and dengue. Globally, approximately 50-100 million people are estimated to be at risk of infection with dengue viruses (the cause of dengue fever/dengue haemorrhagic fever) and between 100- 300 million live in malaria endemic areas (World Health Organization [WHO], 2009). The viruses responsible for dengue are transmitted primarily by *Aedes aegypti*, a predominately urban, day-biting mosquito that often resides in and around human dwellings and preferentially feeds on humans (Gubler, 1997); whereas the 4 human malaria parasites (*Plasmodium*) are transmitted by a wide variety of *Anopheles* species (Service & Townson, 2002 ). Dengue vector has proven extremely resilient to control measures because of its close association and exploitation of domestic and peridomestic human settings (Reiter & Gubler, 1997). On the other hand, malaria vectors display a more diverse array of host seeking behaviors and preference, biting patterns and larval breeding habitats (Pates & Curtis, 2005; Sinka et al., 2011). Despite decades of extensive research, efficacious and commercially viable vaccines for these 2 important vector-borne diseases are not yet available. Therefore, the prevention and control of dengue and malaria remains dependent on various vector control strategies to reduce risk of transmission; in some instances this requires the use of various chemical insecticides as larvicides, space spray and indoor residual spray (IRS) applications, and use of insecticide-impregnated bed nets to control adult mosquito blood feeding (Roberts & Andre, 1994; WHO, 1999; Reiter & Gubler, 1997; Grieco et al., 2007).

Chemical insecticides, including organochlorines, organophosphates, carbamates, and synthetic pyrethroids, have long been used with great effect in public health vector control programs worldwide (Reiter & Gubler, 1997; Roberts & Andre, 1994; WHO, 1992). Although DDT used ceased in many countries several decades ago, the chemical has returned for use in malaria control IRS programs in Africa because of some its superior attributes (Roberts & Tren, 2010). The dramatic impact of DDT on mosquito populations in terms of both toxicity and behavior suppressing disease transmission is well known but in some instances the actual mechanisms at work remain unclear and poorly understood. Most studies on insecticides have placed attention exclusively on the direct toxicological (knockdown and killing) effects on mosquito populations; whereas far less research has focused on the

Behavioral Responses of Mosquitoes to Insecticides 203

modular system is substantially reduced in size compared to the previous excito-repellency box and minimizes the treated surface area and therefore the amount of chemical required for testing. In field observations, numerous attempts have been made to determine behavioal responses of mosquitoes using specially constructed experimental huts (Smith, 1965; Roberts et al., 1984, 1987; Roberts & Alecrim, 1991; Rozendaal et al., 1989; Bangs, 1999; Grieco et al., 2000; Grieco et al., 2007; Polsomboon et al., 2008; Malaithong et al., 2010). Most experimental hut studies have been conducted to observe the behavior of *Anopheles* mosquitoes with far fewer studies on other genera. Grieco et al., (2007) successfully demonstrated all 3 chemical actions using *Ae. aegypti* as a model system. The results obtained from both laboratory and field studies can help faciliate the choice of the most

Two standard systems used in the laboratory, the excito-repellency box (ERB) and the high throughput screening system (HITSS), and the experimental hut in the field to help characterize the behavioral responses of mosquitoes to chemical compounds are discussed

Given the complexities of insect behavioral research, the excito-repellency testing design and testing methodology along with methods of analyzing and interpreting test data, have yet to be universally accepted. However, tremendous improvement has occurred in recent years that effectively addressed a number of the previous drawbacks, opening up significance progress in behavioral studies involving innate responses to insecticides. The ERB test system (Fig.1) was developed to distinguish irritancy (Fig. 2) and repellency (Fig. 3) (Roberts, et al., 1997) and was first used to study the avoidance behavior of *Anopheles albimanus* to DDT and synthetic pyrethroids in Belize, Central America in which consistent and reliable results were generated measuring the escape behavior of female mosquitoes (Chareonviriyaphap et al., 1997). Over time, the initial system was modified into a collapsible test chamber with identical dimensions and operational attributes to help alleviate some of the previous handicaps (Chareonviriyaphap et al., 2002). This improved version has been used extensively in the evaluation of behavioral responses by laboratory

In 2006, a more field compatible device was designed with a substantial reduction in chamber size to minimize the amount of chemical and treated paper required, provide greater ease of test preparation and allow use of a small number of test specimens (15 vs. 25 females) compared to the larger version (Tanasinchayakul et al., 2006). Using this system, consistent and reliable results have been produced (Muenworn et al., 2006; Polsomboon et

The system comprises of 4 outer stainless steel sheet metal walls (Fig. 1). Each wall is constructed with an aluminum sliding rib on each end and socket providing a surface for the test paper holder in the middle. The test paper holder consists of 2 sides: a sheet of fine mesh iron screen attached on one side and a panel to hold the test papers in place on top on the opposite side. There is a 0.8 cm gap between the test paper and screen barrier to prevent mosquitoes from making physical contact ('noncontact') on the treated paper surface during the repellency assay. The paper holder simply has to be inverted to provide the proper conditions to expose the paper in the contact test. On one end of the chamber a portal is made of overlapping sheets of dental dam and is used for placing mosquitoes inside the chamber and later for removing them after the exposure test period. A Plexiglas door serves

effective chemical measures to control house-frequenting adult mosquitoes.

**2.1 Excito-Repellency Box (ERB) test system** 

and field mosquito populations in Thailand.

al., 2008; Thanispong et al., 2009; Mongkalagoon et al., 2009).

herein.

behavioral responses and outcomes as a result of chemical exposure (Roberts, 1993; Roberts et al., 1997; Chareonviriyaphap et al., 1997). Studies have shown that most chemical compounds influence insect locomotor (movement) behavior, often resulting in profound excitation and pre-mature movement away from treated surfaces or areas, one or more kinetic mechanisms resulting in so-called "avoidance behavior" (Kongmee et al., 2004; Miller et al., 2009). The term "excito-repellency" is often used to describe behavior that is stimulated by direct contact with a chemical that results in abnormal excitation (sometimes termed 'irritancy') and spatial repellency that results without the insect making physical contact with the chemical (Roberts et al., 1997). This chapter describes the various behavioral responses to insecticides in some important mosquito vectors of malaria (*Anopheles* species) (Table 1) and dengue (*Aedes aegypti*) (Table 2) and the tropical house mosquito, *Culex quinquefasciatus* (Table 3) using two types of laboratory systems and field assays using experimental huts.

### **2. History of test systems to study mosquito behaviors**

Behavioral responses of mosquitoes to chemical compounds can be directly demonstrated by using various laboratory devices and field assay systems. For laboratory assays, many of the variations have been reviewed (Roberts et al., 1997). The WHO developed the first test box using plywoood in its construction attempting to access the excitation ("irritability") of exposed mosquitoes following physical contact with insecticides (WHO, 1970). This system was subsequently referred to as an "excito-repellency" test box (Rachou et al., 1963). Subsequently, the test system was further modified by other investigators interested in behavioral avoidance responses exposed to DDT and some of the early synthetic pyrethroids (Charlwood & Paraluppi, 1978; Roberts et al., 1984; Bondareva et al., 1986; Rozendaal et al., 1989; Quinones & Suarez, 1989; Ree & Loong, 1989). Years later, a lightproof test chamber was designed to study the irritant response of *Anopheles gambiae*, an importent malaria vector in Africa, to several chemical compounds (Evans, 1993). One key concern with all these test systems was associated with the technical difficulties of the test boxes for introducing and removing test specimens. Other concerns were controling for the various physiological conditions of wild-caught mosquitoes, and selecting the ideal range of concentrations for chemical compounds. In addition, no single or set of statistical methods for analysis of data has been fully accepted nor has any test system been specifically designed to truely discriminate between contact excitation and noncontact repellency responses (Roberts et al., 1997). An improved excito-repellency test device that was able to clearly differentiate between excitation and spatial repellency was developed and initially tested against several field populations of *Anopheles albimanus* from Central America (Roberts et al., 1997; Chareonviriyaphap et al., 1997). Unfortunately, this fixed prototype was cumbersome to handle and required considerable time for attaching the chemcialtreated test papers. Several years on, a more field-friendly test system was designed that was both collapsible and easily transportable (Chareonviriyaphap & Aum-Aong, 2000; Chareonviriyaphap et al., 2002) and was used to investigate the behavioral responses of various mosquito species and geographical populations from Thailand and a few populations from elsewhere in Asia (Chareonviriyaphap et al., 2001; Sungvornyothin et al., 2001; Kongmee et al., 2004; Pothikasikorn et al., 2005). Recently, a novel modular assay system was developed for mass screening of chemical actions; including contact irritancy, spatial repellency, or toxicity responses, on adult mosquitoes (Grieco et al., 2005). This

behavioral responses and outcomes as a result of chemical exposure (Roberts, 1993; Roberts et al., 1997; Chareonviriyaphap et al., 1997). Studies have shown that most chemical compounds influence insect locomotor (movement) behavior, often resulting in profound excitation and pre-mature movement away from treated surfaces or areas, one or more kinetic mechanisms resulting in so-called "avoidance behavior" (Kongmee et al., 2004; Miller et al., 2009). The term "excito-repellency" is often used to describe behavior that is stimulated by direct contact with a chemical that results in abnormal excitation (sometimes termed 'irritancy') and spatial repellency that results without the insect making physical contact with the chemical (Roberts et al., 1997). This chapter describes the various behavioral responses to insecticides in some important mosquito vectors of malaria (*Anopheles* species) (Table 1) and dengue (*Aedes aegypti*) (Table 2) and the tropical house mosquito, *Culex quinquefasciatus* (Table 3) using two types of laboratory systems and field assays using

Behavioral responses of mosquitoes to chemical compounds can be directly demonstrated by using various laboratory devices and field assay systems. For laboratory assays, many of the variations have been reviewed (Roberts et al., 1997). The WHO developed the first test box using plywoood in its construction attempting to access the excitation ("irritability") of exposed mosquitoes following physical contact with insecticides (WHO, 1970). This system was subsequently referred to as an "excito-repellency" test box (Rachou et al., 1963). Subsequently, the test system was further modified by other investigators interested in behavioral avoidance responses exposed to DDT and some of the early synthetic pyrethroids (Charlwood & Paraluppi, 1978; Roberts et al., 1984; Bondareva et al., 1986; Rozendaal et al., 1989; Quinones & Suarez, 1989; Ree & Loong, 1989). Years later, a lightproof test chamber was designed to study the irritant response of *Anopheles gambiae*, an importent malaria vector in Africa, to several chemical compounds (Evans, 1993). One key concern with all these test systems was associated with the technical difficulties of the test boxes for introducing and removing test specimens. Other concerns were controling for the various physiological conditions of wild-caught mosquitoes, and selecting the ideal range of concentrations for chemical compounds. In addition, no single or set of statistical methods for analysis of data has been fully accepted nor has any test system been specifically designed to truely discriminate between contact excitation and noncontact repellency responses (Roberts et al., 1997). An improved excito-repellency test device that was able to clearly differentiate between excitation and spatial repellency was developed and initially tested against several field populations of *Anopheles albimanus* from Central America (Roberts et al., 1997; Chareonviriyaphap et al., 1997). Unfortunately, this fixed prototype was cumbersome to handle and required considerable time for attaching the chemcialtreated test papers. Several years on, a more field-friendly test system was designed that was both collapsible and easily transportable (Chareonviriyaphap & Aum-Aong, 2000; Chareonviriyaphap et al., 2002) and was used to investigate the behavioral responses of various mosquito species and geographical populations from Thailand and a few populations from elsewhere in Asia (Chareonviriyaphap et al., 2001; Sungvornyothin et al., 2001; Kongmee et al., 2004; Pothikasikorn et al., 2005). Recently, a novel modular assay system was developed for mass screening of chemical actions; including contact irritancy, spatial repellency, or toxicity responses, on adult mosquitoes (Grieco et al., 2005). This

**2. History of test systems to study mosquito behaviors** 

experimental huts.

modular system is substantially reduced in size compared to the previous excito-repellency box and minimizes the treated surface area and therefore the amount of chemical required for testing. In field observations, numerous attempts have been made to determine behavioal responses of mosquitoes using specially constructed experimental huts (Smith, 1965; Roberts et al., 1984, 1987; Roberts & Alecrim, 1991; Rozendaal et al., 1989; Bangs, 1999; Grieco et al., 2000; Grieco et al., 2007; Polsomboon et al., 2008; Malaithong et al., 2010). Most experimental hut studies have been conducted to observe the behavior of *Anopheles* mosquitoes with far fewer studies on other genera. Grieco et al., (2007) successfully demonstrated all 3 chemical actions using *Ae. aegypti* as a model system. The results obtained from both laboratory and field studies can help faciliate the choice of the most effective chemical measures to control house-frequenting adult mosquitoes.

Two standard systems used in the laboratory, the excito-repellency box (ERB) and the high throughput screening system (HITSS), and the experimental hut in the field to help characterize the behavioral responses of mosquitoes to chemical compounds are discussed herein.

### **2.1 Excito-Repellency Box (ERB) test system**

Given the complexities of insect behavioral research, the excito-repellency testing design and testing methodology along with methods of analyzing and interpreting test data, have yet to be universally accepted. However, tremendous improvement has occurred in recent years that effectively addressed a number of the previous drawbacks, opening up significance progress in behavioral studies involving innate responses to insecticides. The ERB test system (Fig.1) was developed to distinguish irritancy (Fig. 2) and repellency (Fig. 3) (Roberts, et al., 1997) and was first used to study the avoidance behavior of *Anopheles albimanus* to DDT and synthetic pyrethroids in Belize, Central America in which consistent and reliable results were generated measuring the escape behavior of female mosquitoes (Chareonviriyaphap et al., 1997). Over time, the initial system was modified into a collapsible test chamber with identical dimensions and operational attributes to help alleviate some of the previous handicaps (Chareonviriyaphap et al., 2002). This improved version has been used extensively in the evaluation of behavioral responses by laboratory and field mosquito populations in Thailand.

In 2006, a more field compatible device was designed with a substantial reduction in chamber size to minimize the amount of chemical and treated paper required, provide greater ease of test preparation and allow use of a small number of test specimens (15 vs. 25 females) compared to the larger version (Tanasinchayakul et al., 2006). Using this system, consistent and reliable results have been produced (Muenworn et al., 2006; Polsomboon et al., 2008; Thanispong et al., 2009; Mongkalagoon et al., 2009).

The system comprises of 4 outer stainless steel sheet metal walls (Fig. 1). Each wall is constructed with an aluminum sliding rib on each end and socket providing a surface for the test paper holder in the middle. The test paper holder consists of 2 sides: a sheet of fine mesh iron screen attached on one side and a panel to hold the test papers in place on top on the opposite side. There is a 0.8 cm gap between the test paper and screen barrier to prevent mosquitoes from making physical contact ('noncontact') on the treated paper surface during the repellency assay. The paper holder simply has to be inverted to provide the proper conditions to expose the paper in the contact test. On one end of the chamber a portal is made of overlapping sheets of dental dam and is used for placing mosquitoes inside the chamber and later for removing them after the exposure test period. A Plexiglas door serves

Behavioral Responses of Mosquitoes to Insecticides 205

Fig. 3. Excito-repellency test chamber: Repellency test design (Roberts et al., 1997)

Even though an ERB test system has been found valuable to evaluate the innate behavioral responses of mosquito to chemical compounds, this system is relatively resource intensive requiring a comparatively large amount of chemical to be used for treating papers and having to use a large number of test mosquitoes. Moreover, a current ERB design is not conducive for mass screening the candidate chemical compounds. With the development of the HITSS assay (Grieco et al., 2005), a smaller amount of chemical and a lower number of mosquitoes is required per test. In addition, the HITSS can also be configured to test each of the three actions of insecticide compounds, namely contact irritancy assay (CIA) (Fig. 4), spatial repellency assay (SRA) (Fig. 5) and the toxicity assay (TOX) (Fig. 6). This modular system is made from a variety of durable materials including a thick, clear plastic cylinder, metal chambers, hard plastic end caps and a butterfly valve to control the opening and closing of the door. For the CIA, a single clear plastic cylinder is connected to a metal chamber lined with either treated or untreated netting material using a butterfly value placed in the open position (Fig. 4). For the SRA, an assay comprises a single clear plastic cylinder and 2 metal chambers on either end containing either treated or untreated (control) netting. The plastic chamber is positioned between the 2 metal chambers using a butterfly value as a linking system (Fig. 5). For the TOX assay, a single metal chamber is equipped with plastic end caps (solid cap on one side and tunnel cap on the other end). Netting material treated with either chemical active ingredient (treatment) or acetone carrier only (control) lines the inner chamber (Fig. 6). The HITSS has been standardized and used to evaluate the 3 behavioral responses of *Ae. aegypti* against DEET, Bayrepel®, and SS220 (Grieco et al., 2005, 2007). Recently, HITSS has been used to define the behavioral responses among six field populations of *Ae. aegypti* from Thailand against three synthetic pyrethroids (Thanispong et al., 2010). From this study, it was clearly shown that the HITSS assay is an effective and easy to use tool for distinguishing the three actions of chemicals and screening

**2.2 High Throughput Screening System (HITSS)** 

Treated paper

Screen inner wall

new compounds.

to seal the chamber and allow the investigator to view the exposure chamber before and after the actual test period. A stainless steel outer rear door cover is used to shut off all external light inside the chamber when the experiment is being performed.

Each tests consists of enclosing 15 female mosquitoes in each of 4 chambers lined with either insecticide-treated or untreated (control) papers. On the opposite side of the Plexiglas portal is a single exit portal for mosquitoes to escape to a receiving cage. At the beginning of a test, a 3-min rest period allows mosquitoes to adjust to test chamber conditions before the exit portal is opened to initiate the observation period (30 or 60 min depending on experiment). The numbers of mosquitoes escaping from the chamber into receiving cage are recorded at 1 min intervals until test completion.

Fig. 1. Excito-repellency test chamber showing side of exit portal (Roberts et al., 1997).

Fig. 2. Excito-repellency test chamber: Irritability test design (Roberts et al., 1997).

to seal the chamber and allow the investigator to view the exposure chamber before and after the actual test period. A stainless steel outer rear door cover is used to shut off all

Each tests consists of enclosing 15 female mosquitoes in each of 4 chambers lined with either insecticide-treated or untreated (control) papers. On the opposite side of the Plexiglas portal is a single exit portal for mosquitoes to escape to a receiving cage. At the beginning of a test, a 3-min rest period allows mosquitoes to adjust to test chamber conditions before the exit portal is opened to initiate the observation period (30 or 60 min depending on experiment). The numbers of mosquitoes escaping from the chamber into receiving cage are recorded at 1

Fig. 1. Excito-repellency test chamber showing side of exit portal (Roberts et al., 1997).

Fig. 2. Excito-repellency test chamber: Irritability test design (Roberts et al., 1997).

external light inside the chamber when the experiment is being performed.

min intervals until test completion.

Treated paper

Screen inner wall

Fig. 3. Excito-repellency test chamber: Repellency test design (Roberts et al., 1997)

## **2.2 High Throughput Screening System (HITSS)**

Even though an ERB test system has been found valuable to evaluate the innate behavioral responses of mosquito to chemical compounds, this system is relatively resource intensive requiring a comparatively large amount of chemical to be used for treating papers and having to use a large number of test mosquitoes. Moreover, a current ERB design is not conducive for mass screening the candidate chemical compounds. With the development of the HITSS assay (Grieco et al., 2005), a smaller amount of chemical and a lower number of mosquitoes is required per test. In addition, the HITSS can also be configured to test each of the three actions of insecticide compounds, namely contact irritancy assay (CIA) (Fig. 4), spatial repellency assay (SRA) (Fig. 5) and the toxicity assay (TOX) (Fig. 6). This modular system is made from a variety of durable materials including a thick, clear plastic cylinder, metal chambers, hard plastic end caps and a butterfly valve to control the opening and closing of the door. For the CIA, a single clear plastic cylinder is connected to a metal chamber lined with either treated or untreated netting material using a butterfly value placed in the open position (Fig. 4). For the SRA, an assay comprises a single clear plastic cylinder and 2 metal chambers on either end containing either treated or untreated (control) netting. The plastic chamber is positioned between the 2 metal chambers using a butterfly value as a linking system (Fig. 5). For the TOX assay, a single metal chamber is equipped with plastic end caps (solid cap on one side and tunnel cap on the other end). Netting material treated with either chemical active ingredient (treatment) or acetone carrier only (control) lines the inner chamber (Fig. 6). The HITSS has been standardized and used to evaluate the 3 behavioral responses of *Ae. aegypti* against DEET, Bayrepel®, and SS220 (Grieco et al., 2005, 2007). Recently, HITSS has been used to define the behavioral responses among six field populations of *Ae. aegypti* from Thailand against three synthetic pyrethroids (Thanispong et al., 2010). From this study, it was clearly shown that the HITSS assay is an effective and easy to use tool for distinguishing the three actions of chemicals and screening new compounds.

Behavioral Responses of Mosquitoes to Insecticides 207

To better understand the behavior of a mosquito exposed to a residual avoidance of under more natural, realistic conditions, field studies should be performed using experimental huts. So-called hut studies provide valuable information that can facilitate vector control operations by helping select the most appropraite and effective tool in combating disease vectors. Since the 1940s, most attention has been directed to the study of *Anopheles* mosquitoes, yet few investigations have been carried out to describe the innate behaviors of mosquitoes entering, resting, biting (blood feeding) and exiting human dwellings (Gahan & Lindquist, 1945; Giglioli, 1948; Smith, 1965; Roberts et al., 1984; Bangs, 1999; Grieco et al., 2000; Pates & Curtis, 2005; Roberts et al., 1997). Another important vector species, *Ae. aegypti*, has received even less attention using of experimental hut assays (Suwannachote et al., 2009). The discovery of using natural pyrethrum extract to prevent human-vector contact inside homes was well known prior to 1945 (Muirhead-Thomson, 1951) and the first report of behavioral responses of malaria vectors to DDT was documented in 1947 (Kennedy, 1947, Brown, 1983). Strong behavioral responses to DDT were progressively reported up until 1975 (Muirhead-Thomson, 1960; Elliott & de Zulueta, 1975; Brown, 1983). For example, de Zulueta and Cullen (1963) observed significant reductions in the numbers of biting mosqutioes on human indoors and resting on walls in houses sprayed with DDT. However, there was no clear explanation on how (mechanism) DDT functioned by either repelling or preventing mosquitoes from locating host stimuli inside sprayed houses. A significant scientific observation on behavioral avoidance of *Anopheles ganbiae* was carried out in the mid 1960s (Smith & Webley, 1968) wherein 2 huts, 1 control and one treated with DDT were equipped with verandah traps and gas chromatography was used to evaluate the subsequent response of mosquitoes. A hut treated with DDT was observed to prevent between 60 and 70% of *An. gambiae* from normally entering indoors as an outflow of DDT was continuously decreasing. Additionally, those females that did enter the sprayed hut became stimulated (irritated) and escaped the hut much quicker than those mosqutioes present in the unsprayed hut. Another study comparing biting patterns of *Anopheles minimus* in Thailand showed a 71.5% decline in attempted blood feeding inside the DDT treated hut and a 42.8% reduction in blood feeding success in a deltamethrin treated hut (Polsomboon et al., 2008). Using the same huts, observing human landing patterns of *Anopheles dirus* complex found that the relative risk (odds) of female mosquitoes entering and attempting to feed were half the number when exposed to DDT compared with the

Of the possible responses a mosquto exposed to a chemcial can preform, spatial repellency is prehaps the most interesting and important. Observing true repellency presents problems of accurate messurement in the natural field situation. One of classic methods is to use the experimental huts wherein 2 experimental huts, 1 untreated control and 1 treated with an active ingredient is fitted with entrance (measure of repellency) and exit (irritancy) traps as illustrated in (Figs. 7-9). A landmark study was conducted by Grieco et al., (2007) on the movement patterns of *Ae. aegpyti* into and out of the experimental huts alternatively equipped with entrance (Fig. 8) and exit traps (Fig. 9) to describe the 3 chemical actions and mosquito responses as previously proposed in the mathematical framework for understanding the impact and relevance of repellency, irritability and toxicity on mosquito populations and disease transmission (Roberts et al. 2000). This study clearly showed that the 3 actions described and help to validate the model of actions. It was concluded that contact irritancy is the predominant action of synthetic pyrethroids, whereas spatial

**2.3 Field-based assay: experimental huts** 

deltamethrin treated hut (Malaithong et al., 2010).

Fig. 4 High throughput screening system (HITSS): (CIA) Contact Irritant Assay (Grieco et al., 2005)

Fig. 5. High throughput screening system (HITSS): (SRA) Spatial Repellency Asay (Grieco et al., 2005)

Fig. 6 High throughput screening system (HITSS): (TOX) Toxicity Assay (Grieco et al., 2005)

#### **2.3 Field-based assay: experimental huts**

206 Insecticides – Pest Engineering

Fig. 4 High throughput screening system (HITSS): (CIA) Contact Irritant Assay (Grieco et

Fig. 5. High throughput screening system (HITSS): (SRA) Spatial Repellency Asay (Grieco et

Fig. 6 High throughput screening system (HITSS): (TOX) Toxicity Assay (Grieco et al., 2005)

al., 2005)

al., 2005)

To better understand the behavior of a mosquito exposed to a residual avoidance of under more natural, realistic conditions, field studies should be performed using experimental huts. So-called hut studies provide valuable information that can facilitate vector control operations by helping select the most appropraite and effective tool in combating disease vectors. Since the 1940s, most attention has been directed to the study of *Anopheles* mosquitoes, yet few investigations have been carried out to describe the innate behaviors of mosquitoes entering, resting, biting (blood feeding) and exiting human dwellings (Gahan & Lindquist, 1945; Giglioli, 1948; Smith, 1965; Roberts et al., 1984; Bangs, 1999; Grieco et al., 2000; Pates & Curtis, 2005; Roberts et al., 1997). Another important vector species, *Ae. aegypti*, has received even less attention using of experimental hut assays (Suwannachote et al., 2009). The discovery of using natural pyrethrum extract to prevent human-vector contact inside homes was well known prior to 1945 (Muirhead-Thomson, 1951) and the first report of behavioral responses of malaria vectors to DDT was documented in 1947 (Kennedy, 1947, Brown, 1983). Strong behavioral responses to DDT were progressively reported up until 1975 (Muirhead-Thomson, 1960; Elliott & de Zulueta, 1975; Brown, 1983). For example, de Zulueta and Cullen (1963) observed significant reductions in the numbers of biting mosqutioes on human indoors and resting on walls in houses sprayed with DDT. However, there was no clear explanation on how (mechanism) DDT functioned by either repelling or preventing mosquitoes from locating host stimuli inside sprayed houses. A significant scientific observation on behavioral avoidance of *Anopheles ganbiae* was carried out in the mid 1960s (Smith & Webley, 1968) wherein 2 huts, 1 control and one treated with DDT were equipped with verandah traps and gas chromatography was used to evaluate the subsequent response of mosquitoes. A hut treated with DDT was observed to prevent between 60 and 70% of *An. gambiae* from normally entering indoors as an outflow of DDT was continuously decreasing. Additionally, those females that did enter the sprayed hut became stimulated (irritated) and escaped the hut much quicker than those mosqutioes present in the unsprayed hut. Another study comparing biting patterns of *Anopheles minimus* in Thailand showed a 71.5% decline in attempted blood feeding inside the DDT treated hut and a 42.8% reduction in blood feeding success in a deltamethrin treated hut (Polsomboon et al., 2008). Using the same huts, observing human landing patterns of *Anopheles dirus* complex found that the relative risk (odds) of female mosquitoes entering and attempting to feed were half the number when exposed to DDT compared with the deltamethrin treated hut (Malaithong et al., 2010).

Of the possible responses a mosquto exposed to a chemcial can preform, spatial repellency is prehaps the most interesting and important. Observing true repellency presents problems of accurate messurement in the natural field situation. One of classic methods is to use the experimental huts wherein 2 experimental huts, 1 untreated control and 1 treated with an active ingredient is fitted with entrance (measure of repellency) and exit (irritancy) traps as illustrated in (Figs. 7-9). A landmark study was conducted by Grieco et al., (2007) on the movement patterns of *Ae. aegpyti* into and out of the experimental huts alternatively equipped with entrance (Fig. 8) and exit traps (Fig. 9) to describe the 3 chemical actions and mosquito responses as previously proposed in the mathematical framework for understanding the impact and relevance of repellency, irritability and toxicity on mosquito populations and disease transmission (Roberts et al. 2000). This study clearly showed that the 3 actions described and help to validate the model of actions. It was concluded that contact irritancy is the predominant action of synthetic pyrethroids, whereas spatial

Behavioral Responses of Mosquitoes to Insecticides 209

To facilitate the study of behavioral responses of mosquitoes to chemical compounds, several test systems have been developed (Roberts et al., 1997; Chareonviriyaphap et al., 2001). The following is a brief review of important historical findings and more recent work from studies in Thialand on behavioral responses of mosquitoes to DDT and various

Some agricultural and medically important insects, including vectors of malaria, demonstrate what has been termed "behavioral resistance" to DDT (Lockwood et al., 1984). However, the term "behavioral avoidance" as an innate response rather than a permanent genetic shift in behavior is preferred since the development of behavioral changes due to insecticide selective pressure in nature has not been adequately documented (Muirhead-Thomson, 1960). The behavioral responses of mosquitoes have been investigated using either specially constructed experimental huts and/or ERB test system. The first study on the irritant effect of DDT residual deposits was conducted on *Anopheles quadrimaculatus* where females were found to be irritated after short contact with treated surfaces with many quickly escaping the DDT treated house without taking a blood meal (Gahan & Lindquist, 1945). Subsequent observations found that *An. quadrimaculatus* had received a lethal dose and perished within 24 hours (Metcalf et al., 1945). Unfortunately, these studies made the observations without having control (untreated houses) for comparison. Moreover, the high mortality seen with *An. quadrimaculatus* may have been caused by further contacts with toxic active ingredients while attempting to leave a treated house through a small outlet (Muirhead-Thomson, 1960). In the studies with *Anopheles albimanus* in Panama, Trapido (1954) concluded that wild-caught mosquitoes lacking re-exposure to DDT for a long period of time, showed the same susceptibility levels to DDT as those from a laboratory colony with no a history of previous exposure. Malaria vectors in some countries (e.g., Brazil, Thailand) have never developed resistance to DDT (Roberts et al., 1984; Chareonviriyaphap et al., 2001), suggesting that the particular mosquito population possibly avoids making direct physical contact with the chemical, thereby precluding any selection for resistance. Table 1 lists *Anopheles* species tested and levels of behavioral responses to DDT and synthetic pyrethroids. DDT was found to be a strong contact irritant and more moderately as a repellent among 3 test populations of *An. albimanus* and that most specimens that quickly escaped DDT exposure survived (Chareonviriyaphap et al., 1997). This finding is in agreement with the results of the field studies by Roberts and Alecrim (1991) who reported a strong repellent action of DDT with *Anopheles darlingi* in a sprayed house. In a similar study, both irritancy and repellency escape responses were observed in 2 populations of *Anopheles minimus* with a major action of DDT being contact excitation (Chareonviriyaphap et al., 2001). Another study was made on the 2 complex species within the Minimus Subgroup; *An. minimus* (species A) and *Anopheles harrisoni* (species C). DDT produced a rapid and striking irritancy response in both species. Additionally, repellency was more pronounced with DDT on *An. minimus* but was seen to be much weaker with *An. harrisoni* (Pothikasikorn et al. 2005). With *Ae. aegypti*, results demonstrated that the higher the degree of physiological resistance to DDT, the greater the apparent suppression of both contact irritant and noncontact repellency responses, yet avoidance behavior was still a significant event

**3. Behavioral responses** 

**3.1 Behaviroral responses to DDT**

(Thanispong et al., 2009).

synthetic pyrethroids commonly used in vector control.

repellency is the primary action of DDT. Dieldrin, a once commonly used cyclodiene compound, exhibited primarily a toxic action only.

Fig. 7 Experimental hut with traps attached to exterior walls.

Fig. 8 Experimental hut with entrance traps attached to interior walls.

Fig. 9. Experimental hut with exit traps attached to exterior walls.

## **3. Behavioral responses**

208 Insecticides – Pest Engineering

repellency is the primary action of DDT. Dieldrin, a once commonly used cyclodiene

Inside

compound, exhibited primarily a toxic action only.

Fig. 7 Experimental hut with traps attached to exterior walls.

Outside

Fig. 8 Experimental hut with entrance traps attached to interior walls.

Fig. 9. Experimental hut with exit traps attached to exterior walls.

To facilitate the study of behavioral responses of mosquitoes to chemical compounds, several test systems have been developed (Roberts et al., 1997; Chareonviriyaphap et al., 2001). The following is a brief review of important historical findings and more recent work from studies in Thialand on behavioral responses of mosquitoes to DDT and various synthetic pyrethroids commonly used in vector control.

### **3.1 Behaviroral responses to DDT**

Some agricultural and medically important insects, including vectors of malaria, demonstrate what has been termed "behavioral resistance" to DDT (Lockwood et al., 1984). However, the term "behavioral avoidance" as an innate response rather than a permanent genetic shift in behavior is preferred since the development of behavioral changes due to insecticide selective pressure in nature has not been adequately documented (Muirhead-Thomson, 1960). The behavioral responses of mosquitoes have been investigated using either specially constructed experimental huts and/or ERB test system. The first study on the irritant effect of DDT residual deposits was conducted on *Anopheles quadrimaculatus* where females were found to be irritated after short contact with treated surfaces with many quickly escaping the DDT treated house without taking a blood meal (Gahan & Lindquist, 1945). Subsequent observations found that *An. quadrimaculatus* had received a lethal dose and perished within 24 hours (Metcalf et al., 1945). Unfortunately, these studies made the observations without having control (untreated houses) for comparison. Moreover, the high mortality seen with *An. quadrimaculatus* may have been caused by further contacts with toxic active ingredients while attempting to leave a treated house through a small outlet (Muirhead-Thomson, 1960). In the studies with *Anopheles albimanus* in Panama, Trapido (1954) concluded that wild-caught mosquitoes lacking re-exposure to DDT for a long period of time, showed the same susceptibility levels to DDT as those from a laboratory colony with no a history of previous exposure. Malaria vectors in some countries (e.g., Brazil, Thailand) have never developed resistance to DDT (Roberts et al., 1984; Chareonviriyaphap et al., 2001), suggesting that the particular mosquito population possibly avoids making direct physical contact with the chemical, thereby precluding any selection for resistance. Table 1 lists *Anopheles* species tested and levels of behavioral responses to DDT and synthetic pyrethroids. DDT was found to be a strong contact irritant and more moderately as a repellent among 3 test populations of *An. albimanus* and that most specimens that quickly escaped DDT exposure survived (Chareonviriyaphap et al., 1997). This finding is in agreement with the results of the field studies by Roberts and Alecrim (1991) who reported a strong repellent action of DDT with *Anopheles darlingi* in a sprayed house. In a similar study, both irritancy and repellency escape responses were observed in 2 populations of *Anopheles minimus* with a major action of DDT being contact excitation (Chareonviriyaphap et al., 2001). Another study was made on the 2 complex species within the Minimus Subgroup; *An. minimus* (species A) and *Anopheles harrisoni* (species C). DDT produced a rapid and striking irritancy response in both species. Additionally, repellency was more pronounced with DDT on *An. minimus* but was seen to be much weaker with *An. harrisoni* (Pothikasikorn et al. 2005). With *Ae. aegypti*, results demonstrated that the higher the degree of physiological resistance to DDT, the greater the apparent suppression of both contact irritant and noncontact repellency responses, yet avoidance behavior was still a significant event (Thanispong et al., 2009).


from the matched control

Table 1. Degree of behavioral responses of female *Anopheles* species to synthetic pyrethroids and DDT at an operational field dose (mg/cm2).

Behavioral Responses of Mosquitoes to Insecticides 211

A number of synthetic pyrethroids, i.e. allethrin, deltamethrin, permethrin, cypermethrin, alpha-cypermethrin, cyfluthrin among others, are commonly used by home owners, private business and government sectors to control both household mosquitoes and those as important vectors. These pyrethroids have found to exhibit a moderate to strong repellent effect for many agricultural and medically important insects (Lockwood et al., 1984) and were observed to cause mosquitoes to move away ('avoidance') from sprayed areas (Miller, 1990; Lindsay et al., 1991). The extensive and continuing use of pyrethroids should be a major stimulus to intensify observations on the significance of pyrethroid-induced avoidance behavior in mosquito vectors and other arthropods. Given the role of indoor residual spraying of homes as a means of controlling malaria transmission, the precise role of excitation and repellent actions of pyrethriods should be well defined for specific malaria vectors prior to beginning any large scale control program. Following the refinement of the ERB test system allowing separation of the 2 types of primary behavioral actions (Roberts et al., 1997), a series of important findings on excito-repellency behavior in *Anopheles*  mosquitoes have been subsequently reported (Chareonviriyaphap et al., 1997, 2001, 2004; Pothikasikorn et al., 2005; Muenworn et al., 2006; Pothikasikorn et al., 2007). In general, synthetic pyrethroids produce much stronger irritant responses in *Anopheles* compared to repellency action (Table 1). For example, lambda-cyhalothrin and deltamethrin act as strong irritants on test populations of *An. minimus* complex mosquitoes while showing relatively weak repellency action (Chareonviriyaphap et al., 2001). Pothikasikorn et al. (2005) confirmed that Minimus complex species, *An. minimus* and *An. harrisoni* show a rapid irritancy to lambda-cyhalothrin and deltamethrin. Chareonviriyaphap et al., (2004) produced an extensive study to define the excito-repellency action of deltamethrin on 4 *Anopheles* species, all representing important malaria vectors in Thailand. Again, the findings demonstrated that deltamethrin produced a pronounced irritancy action compared to a much weaker repellency effect. Although repellency was less profound than contact excitation, the escape responses were statistically significant compared to the matched controls. A number of *Ae. aegypti* populations have been tested against a series of synthetic pyrethroids (deltamethrin, permethrin, alphacypermethrin, cyphenothrin, d-tetramethrin and tetramethrin) (Table 2). In general, all test populations of *Ae. aegypti* populations exhibit moderate to strong irritancy as compared with repellency (Grieco et al., 2005; Chareonviriyaphap et al., 2006; Paeporn et al., 2007; Thanispong et al., 2009, 2010). In addition, a few populations of *Culex quinqaefacsciatus* have been tested against the 3 principal classes of insecticides used in vector control; pyrethroids (deltamethrin), organophosphates (fenitrothion) and carbamates (propoxur) (Table 3). Striking differences in behavioral responses were seen between populations and active ingredients. Greater contact escape action was observed in a long-established colony exposed to deltamethrin and fenitrothion compared to two recent field populations (Sathantriphop et al., 2006). To summarize, the behavioral responses to insecticides by mosquitoes are important components of a chemical's overall effectiveness in reducing human-vector contact and transmision of disease. To date, there is no convincing example of behavioral resistance in mosquito species to insecticides, rather all evidence indicates actions on the part of exposed mosquitoes are part of an innate behaviral repertoire. Behavioral response can be split into 2 distinct categories, stimulus-dependent and stimulus-independent actions (Georghiou, 1972). A stimulus-dependent response requires sensory stimulation of the insect in order for an avoidance action to proceed. In general, this form of avoidance enables the insect to

**3.2 Behavioral response to synthetic pyrethroids** 

Table 1. Degree of behavioral responses of female *Anopheles* species to synthetic pyrethroids

*harrisoni*

from the matched control

and DDT at an operational field dose (mg/cm2).

#### **3.2 Behavioral response to synthetic pyrethroids**

A number of synthetic pyrethroids, i.e. allethrin, deltamethrin, permethrin, cypermethrin, alpha-cypermethrin, cyfluthrin among others, are commonly used by home owners, private business and government sectors to control both household mosquitoes and those as important vectors. These pyrethroids have found to exhibit a moderate to strong repellent effect for many agricultural and medically important insects (Lockwood et al., 1984) and were observed to cause mosquitoes to move away ('avoidance') from sprayed areas (Miller, 1990; Lindsay et al., 1991). The extensive and continuing use of pyrethroids should be a major stimulus to intensify observations on the significance of pyrethroid-induced avoidance behavior in mosquito vectors and other arthropods. Given the role of indoor residual spraying of homes as a means of controlling malaria transmission, the precise role of excitation and repellent actions of pyrethriods should be well defined for specific malaria vectors prior to beginning any large scale control program. Following the refinement of the ERB test system allowing separation of the 2 types of primary behavioral actions (Roberts et al., 1997), a series of important findings on excito-repellency behavior in *Anopheles*  mosquitoes have been subsequently reported (Chareonviriyaphap et al., 1997, 2001, 2004; Pothikasikorn et al., 2005; Muenworn et al., 2006; Pothikasikorn et al., 2007). In general, synthetic pyrethroids produce much stronger irritant responses in *Anopheles* compared to repellency action (Table 1). For example, lambda-cyhalothrin and deltamethrin act as strong irritants on test populations of *An. minimus* complex mosquitoes while showing relatively weak repellency action (Chareonviriyaphap et al., 2001). Pothikasikorn et al. (2005) confirmed that Minimus complex species, *An. minimus* and *An. harrisoni* show a rapid irritancy to lambda-cyhalothrin and deltamethrin. Chareonviriyaphap et al., (2004) produced an extensive study to define the excito-repellency action of deltamethrin on 4 *Anopheles* species, all representing important malaria vectors in Thailand. Again, the findings demonstrated that deltamethrin produced a pronounced irritancy action compared to a much weaker repellency effect. Although repellency was less profound than contact excitation, the escape responses were statistically significant compared to the matched controls. A number of *Ae. aegypti* populations have been tested against a series of synthetic pyrethroids (deltamethrin, permethrin, alphacypermethrin, cyphenothrin, d-tetramethrin and tetramethrin) (Table 2). In general, all test populations of *Ae. aegypti* populations exhibit moderate to strong irritancy as compared with repellency (Grieco et al., 2005; Chareonviriyaphap et al., 2006; Paeporn et al., 2007; Thanispong et al., 2009, 2010). In addition, a few populations of *Culex quinqaefacsciatus* have been tested against the 3 principal classes of insecticides used in vector control; pyrethroids (deltamethrin), organophosphates (fenitrothion) and carbamates (propoxur) (Table 3). Striking differences in behavioral responses were seen between populations and active ingredients. Greater contact escape action was observed in a long-established colony exposed to deltamethrin and fenitrothion compared to two recent field populations (Sathantriphop et al., 2006).

To summarize, the behavioral responses to insecticides by mosquitoes are important components of a chemical's overall effectiveness in reducing human-vector contact and transmision of disease. To date, there is no convincing example of behavioral resistance in mosquito species to insecticides, rather all evidence indicates actions on the part of exposed mosquitoes are part of an innate behaviral repertoire. Behavioral response can be split into 2 distinct categories, stimulus-dependent and stimulus-independent actions (Georghiou, 1972). A stimulus-dependent response requires sensory stimulation of the insect in order for an avoidance action to proceed. In general, this form of avoidance enables the insect to


Table 2. Degree of behavioral responses of female *Aedes aegypti* to synthetic pyrethroids and DDT at an operational field dose (mg/cm2).

Behavioral Responses of Mosquitoes to Insecticides 213

detect a chemical on direct contact or spatially before acquiring a lethal dose (Muirhead-Thomson, 1960). On the other hand, a stimulus-independent response does not require direct sensory stimulation of insect for avoidance to occur but rather involves other natural behavioral components such as exophily (outside resting) or zoophily (non-human blood preference) in which an insect avoids exposure to a chemical by preferentially utilizing habitats without active ingredients present (Byford & Sparks, 1987). This type of response has also been included in so-called "phenotypic and genotypic behaviors" (WHO, 1986). Stimulus-dependent behavioral responses include the avoidance behaviors discussed in detail in this chapter. The term *avoidance behavior* is generally used to describe actions that are stimulated by some combination of excitation (irritancy) and repellency, the former taking place following physical contact while spatial repellency results without physical

Populations Deltamethrin Fenitrothion Propoxur References

Bangkok\* ++ - ++ - + - Sathantriphop et al., 2006 Nontaburi\* + - + - + - Sathantriphop et al., 2006 Mae Sot\* + - + - + - Sathantriphop et al., 2006


Any compound used to control (eliminate, reduce or otherwise prevent harm) insect populations have been termed an "insecticide". An insecticide can also be defined as any compound that is used solely to kill insects. This single term of reference involving only one type of action (knockdown/death) is completely inadequate to describe the more meaningful and complete action of many insecticide compounds used against insect populations. Insects can respond to insecticides in at least 2 different ways; behavioral action, namely avoidance and toxicity. In the past, the prevailing practice has been to classify chemicals simply as toxicants for killing insects. As seen in this chapter, we introduce the term "chemical" in place of "insecticide" as it is more appropriate for

Chemicals protect humans from the bites of mosquitoes through 3 different actions: excitation, repellency and toxcity (Roberts et al., 2000 & Grieco et al., 2007). Historically the vast majority of chemical studies have focused on the direct toxicological responses (susceptibility and resistance) of chemicals on mosquito populations whereas very little emphasis has been placed on the vector's behavior in response to sub-lethal exposure. Knowledge of the mosquito's behavioral responses to particular chemicals is highly significant in the prioritization and design of appropriate vector prevention and control

recognizing the 2 other primary actions on mosquitoes vice toxicity alone.

 *I R I R I R* 

contact with an insecticide (Roberts et al., 1987).

\*Highly resistance to deltamethrin

++++: 81-100% escaped from treated chamber +++: 61-79% escaped from treated chamber ++: 41-59% escaped from treated chamber +: 21-40% escaped from treated chamber

and fenitrothion at an operational field dose (mg/cm2).

I: Irritancy, R: Repellency

**4. Conclusion** 

Table 2. Degree of behavioral responses of female *Aedes aegypti* to synthetic pyrethroids and

DDT at an operational field dose (mg/cm2).

detect a chemical on direct contact or spatially before acquiring a lethal dose (Muirhead-Thomson, 1960). On the other hand, a stimulus-independent response does not require direct sensory stimulation of insect for avoidance to occur but rather involves other natural behavioral components such as exophily (outside resting) or zoophily (non-human blood preference) in which an insect avoids exposure to a chemical by preferentially utilizing habitats without active ingredients present (Byford & Sparks, 1987). This type of response has also been included in so-called "phenotypic and genotypic behaviors" (WHO, 1986). Stimulus-dependent behavioral responses include the avoidance behaviors discussed in detail in this chapter. The term *avoidance behavior* is generally used to describe actions that are stimulated by some combination of excitation (irritancy) and repellency, the former taking place following physical contact while spatial repellency results without physical contact with an insecticide (Roberts et al., 1987).


\*Highly resistance to deltamethrin

I: Irritancy, R: Repellency

++++: 81-100% escaped from treated chamber

+++: 61-79% escaped from treated chamber

++: 41-59% escaped from treated chamber

+: 21-40% escaped from treated chamber


Table 3. Degree of behavioral responses of *Culex quinquefasciatus* to deltamethrin, propoxur and fenitrothion at an operational field dose (mg/cm2).

## **4. Conclusion**

Any compound used to control (eliminate, reduce or otherwise prevent harm) insect populations have been termed an "insecticide". An insecticide can also be defined as any compound that is used solely to kill insects. This single term of reference involving only one type of action (knockdown/death) is completely inadequate to describe the more meaningful and complete action of many insecticide compounds used against insect populations. Insects can respond to insecticides in at least 2 different ways; behavioral action, namely avoidance and toxicity. In the past, the prevailing practice has been to classify chemicals simply as toxicants for killing insects. As seen in this chapter, we introduce the term "chemical" in place of "insecticide" as it is more appropriate for recognizing the 2 other primary actions on mosquitoes vice toxicity alone.

Chemicals protect humans from the bites of mosquitoes through 3 different actions: excitation, repellency and toxcity (Roberts et al., 2000 & Grieco et al., 2007). Historically the vast majority of chemical studies have focused on the direct toxicological responses (susceptibility and resistance) of chemicals on mosquito populations whereas very little emphasis has been placed on the vector's behavior in response to sub-lethal exposure. Knowledge of the mosquito's behavioral responses to particular chemicals is highly significant in the prioritization and design of appropriate vector prevention and control

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strategies. Today, the development of insecticide resistance in insect pests and disease vectors occurs worldwide and on a increasing scale. However, resistance has still remained limited in many areas in spite of the long-term use of chemicals for control. This phenomenon suggests that behavioral responses likely play a significant role in how certain chemcials preform to interrupt human-vector contact while also reducing the selection pressure on a target insects for developing resistance (Roberts et al., 2000).

As discussed, at least 2 different types of mosqutio behavioral response outcomes to chemicals are recognized; excitation and repellency. Whether acting from direct contact or from a distance (spatially) both response activities are based on stimulus-response actions that result in clear movement away from an area with the chemical present. There have been numerous attempts to accurately measure the behavioral responses of mosquitoes to chemicals using various types of excito-repellency test systems (e.g., ERB and HITSS). However, no single system has yet been completely accepted as a standardized method of testing or analyzing behavioral responses. Currently, no test system as recommended by the WHO can discriminate between contact irritancy and noncontact repellency. The WHO tests are based on the concept that mosquitoes respond to chemicals only after physical contact and that spatial repellency plays little or no meaningful role in disease control, or may actually be a determental attribute of a chemical. The test systems describe in this chapter have the capacity to differentiate both key behavioral responses and thus assigning each with relative importance in the potential prevention of disease transmission. Both the ERB and HITSS systems have been used to study behavioral responses of several important mosquito vectors and pests (*Anopheles, Aedes,* and *Culex*) to various test chemicals currently used in vector control programs while providing valuable and highly reproducible results. This knowledge will allow better decision making on chemical selection, application method and future product development. From the detailed investigations previously mentioned and elsewhere, we conclude that the behavioral responses of mosquitoes to chemicals are an important, if not critical, components of disease control operations.

#### **5. Acknowledgments**

The author would like to thank Dr. Michael J. Bangs for the critical review of this chapter. I am especially grateful to the Thailand Research Fund (TRF) and the Kasetsart University Research and Development Institute (KURDI) for providing the financial support over the many years.

## **6. References**


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As discussed, at least 2 different types of mosqutio behavioral response outcomes to chemicals are recognized; excitation and repellency. Whether acting from direct contact or from a distance (spatially) both response activities are based on stimulus-response actions that result in clear movement away from an area with the chemical present. There have been numerous attempts to accurately measure the behavioral responses of mosquitoes to chemicals using various types of excito-repellency test systems (e.g., ERB and HITSS). However, no single system has yet been completely accepted as a standardized method of testing or analyzing behavioral responses. Currently, no test system as recommended by the WHO can discriminate between contact irritancy and noncontact repellency. The WHO tests are based on the concept that mosquitoes respond to chemicals only after physical contact and that spatial repellency plays little or no meaningful role in disease control, or may actually be a determental attribute of a chemical. The test systems describe in this chapter have the capacity to differentiate both key behavioral responses and thus assigning each with relative importance in the potential prevention of disease transmission. Both the ERB and HITSS systems have been used to study behavioral responses of several important mosquito vectors and pests (*Anopheles, Aedes,* and *Culex*) to various test chemicals currently used in vector control programs while providing valuable and highly reproducible results. This knowledge will allow better decision making on chemical selection, application method and future product development. From the detailed investigations previously mentioned and elsewhere, we conclude that the behavioral responses of mosquitoes to chemicals are an

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**10** 

*Cuba* 

**Essential Plant Oils and Insecticidal Activity** 

Plants have great importance for man because they are one of their sources of food, they provide us through the process of photosynthesis the oxygen we breathe and are essential to

Essential oils are volatile, usually distillable liquid fractions responsible for the aroma of the plant. The vast majority of them are pleasant smell and its metabolic and evolutionary significance lies in the role they play as attractor of pollinating agents (for its pleasant aroma), constitute elements of defense against the attack of parasites, herbivorous animals and insects, allow the adaptation of the plant when water is scarce and are part of the substances in reserve as the giver of H+ in the processes of electron. The organoleptic characteristics of the essential oils may be given by major components, although in other cases they are substances present in tiny quantities (traces) which define the taste, smell, or

These natural substances are known as secondary metabolites, name which refers to substances that are not involved in the basic mechanisms of life of the plant but that comply with specific functions. (Corbino, 2000). They tend to accumulate in large amounts without negative effects and represent a problem in the cell or on the plant. These metabolites have the property to form glycosides and are found soluble in the plant. For many years these metabolites were regarded as final products of metabolic processes without specific function

The study of these substances was initiated by organic chemists of the 19th century and early 20th century who were interested in these substances because of its importance in the medical industry, the manufacture of flavoring, etc. In fact, the study of the secondary metabolites stimulated the development of separating techniques, and spectroscopy for determining their structure and synthesis which constituted the basis of the contemporary

They can be found in different parts of the plant: leaves (wormwood, basil, buchú, cidrón, eucalyptus, mint, lemongrass, marjoram, mint, patchouli, quenopod, rosemary, Sage, lemon balm, etc.), in the roots (angelic, asaro, saffron, calamus, turmeric, galanga, ginger, sandalwood, Sasafras, Valerian, vetiver, etc.), in the pericarp of the fruit (lemon, Tangerine, Orange, etc.), seeds (anise, cardamom, dill, fennel, cumin, etc.), in the stem (cinnamon, caparrapí, etc.), flowers (arnica, lavender, chamomile, pyrethrum, thyme, clove scent, rose,

**1. Introduction** 

maintain the ecological balance (Corbino, 2000).

therapeutic properties (Scholes, 1995; Worwood, 1992).

or directly as a waste of plant products (Lopez, 2008).

organic chemistry (Lopez, 2008).

**in** *Culex quinquefasciatus*

*Institute Tropical "Pedro Kouri"* 

Maureen Leyva, Olinka Tiomno, Juan E. Tacoronte, Maria del Carmen Marquetti and Domingo Montada

World Health Organization. (1999). Prevention and Control and Dengue and Dengue Haemorrhagic Fever: Comprehensive Guidelines. *World Health Organization* Regional Publication, SEARO No. 29, New Delhi., pp. 134, ISSN 0378-2255.

## **Essential Plant Oils and Insecticidal Activity in** *Culex quinquefasciatus*

Maureen Leyva, Olinka Tiomno, Juan E. Tacoronte, Maria del Carmen Marquetti and Domingo Montada *Institute Tropical "Pedro Kouri" Cuba* 

## **1. Introduction**

220 Insecticides – Pest Engineering

World Health Organization. (1999). Prevention and Control and Dengue and Dengue

Regional Publication, SEARO No. 29, New Delhi., pp. 134, ISSN 0378-2255.

Haemorrhagic Fever: Comprehensive Guidelines. *World Health Organization*

Plants have great importance for man because they are one of their sources of food, they provide us through the process of photosynthesis the oxygen we breathe and are essential to maintain the ecological balance (Corbino, 2000).

Essential oils are volatile, usually distillable liquid fractions responsible for the aroma of the plant. The vast majority of them are pleasant smell and its metabolic and evolutionary significance lies in the role they play as attractor of pollinating agents (for its pleasant aroma), constitute elements of defense against the attack of parasites, herbivorous animals and insects, allow the adaptation of the plant when water is scarce and are part of the substances in reserve as the giver of H+ in the processes of electron. The organoleptic characteristics of the essential oils may be given by major components, although in other cases they are substances present in tiny quantities (traces) which define the taste, smell, or therapeutic properties (Scholes, 1995; Worwood, 1992).

These natural substances are known as secondary metabolites, name which refers to substances that are not involved in the basic mechanisms of life of the plant but that comply with specific functions. (Corbino, 2000). They tend to accumulate in large amounts without negative effects and represent a problem in the cell or on the plant. These metabolites have the property to form glycosides and are found soluble in the plant. For many years these metabolites were regarded as final products of metabolic processes without specific function or directly as a waste of plant products (Lopez, 2008).

The study of these substances was initiated by organic chemists of the 19th century and early 20th century who were interested in these substances because of its importance in the medical industry, the manufacture of flavoring, etc. In fact, the study of the secondary metabolites stimulated the development of separating techniques, and spectroscopy for determining their structure and synthesis which constituted the basis of the contemporary organic chemistry (Lopez, 2008).

They can be found in different parts of the plant: leaves (wormwood, basil, buchú, cidrón, eucalyptus, mint, lemongrass, marjoram, mint, patchouli, quenopod, rosemary, Sage, lemon balm, etc.), in the roots (angelic, asaro, saffron, calamus, turmeric, galanga, ginger, sandalwood, Sasafras, Valerian, vetiver, etc.), in the pericarp of the fruit (lemon, Tangerine, Orange, etc.), seeds (anise, cardamom, dill, fennel, cumin, etc.), in the stem (cinnamon, caparrapí, etc.), flowers (arnica, lavender, chamomile, pyrethrum, thyme, clove scent, rose,

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 223

Fig. 1. General pathways of plant secondary metabolism.

Fig. 2. Natural Monoterpenes.

etc.) and in the fruit (caraway, coriander, bay leaf, nutmeg, parsley, pepper, etc.). (www.buscasalud.com)

## **2. Essential oils**

Essential oils are complex mixtures of up to over 100 secondary metabolites, which can be classified in terpenoid compounds, phenolic and alkaloids based on their biosynthetic origins. Terpenes containing a single unit of isoprene are called monoterpenes, which contain three units named sesquiterpenes (Ikan, 1991; Silva, 2002).

Monoterpenes and sesquiterpenes are terpenes from 10 to 15 atoms of carbons biosynthetically derived from geranilpyrophosfates (SPG) and farnesilpyrophosfate (FPP) respectively. Monoterpenes and in general all the natural terpenoid compounds are synthetized by the route of the acetilCoA through a common intermediate which is the mevalonic acid. However, it has been proposed that some terpenoids are not originated by this route, and instead use an alternative route that may involve pyruvate, glyceraldehyde-3-phosphate (Fig 1) (Adams et al, 1998; Sponsel, 1995).

Essential oils are widely distributed in some 60 families of plants including the Compositae, Labiadas, Lauraceae, Myrtaceae, Rosaceae, Rutaceae, Umbelliferae, etc. Monoterpenoides are mainly found in plants in the family of Ranunculales, Violales, and Primulales, while they are rare in Rutales, Cornales, Lamiales, and Asterales. Moreover, the sesquiterpenoides are commonly found in Rutales, Cornales, Magnoliales and Asterales. Although in both essential oils: mono - sesquiterpenes and phenylpropan are found in free-form, those which are linked to carbohydrates, have been more recently investigated, because it is considered to be the immediate precursor of the essential oil.

From the chemical point of view, despite its complex composition with different types of substances, essential oils can be classified according to the type of substances of which they are the major components. According to this fact, the essential oils rich in monoterpenes are called essential oils sesquiterpenoids, those rich in phenylpropan are the essential oils phenylpropanoids. Although this classification is very general there are more complex classifications that take into account other chemical aspects (Fig 2,3,4) (González, 1984; Ikan, 1991; Judd et al, 2002; Stachenko, 1996).

#### **2.1 Methods of extraction**

According to www.chemkeys.com 2000; Gascon et al. 2002; www.herbotecnia. com.ar, 2002; www.losandes.com.ar 2002. essential oils can be extracted from plant samples through various methods samples such as: expression, distillation with steam, extraction with volatile solvents, enfleurage, extraction with supercritical fluids and hydrodistillation.

#### **2.2 Method of isolation**

From such oils, it is possible to its isolation through the use of one or several chromatographic methods such as thin layer chromatography and HPLC.

For the chromatographic column and thin layer methods the silica gel are widely used as stationary phase. Polar pure or mixed solvents such as: Toluene-acetate of ethyl benzene, chloroform, dichloromethane, ethyl, chloroform-benzene, chloroform-ethanol - chloroformbenzene acetic acid benzene-acetate are used as mobile phase.

etc.) and in the fruit (caraway, coriander, bay leaf, nutmeg, parsley, pepper, etc.).

Essential oils are complex mixtures of up to over 100 secondary metabolites, which can be classified in terpenoid compounds, phenolic and alkaloids based on their biosynthetic origins. Terpenes containing a single unit of isoprene are called monoterpenes, which

Monoterpenes and sesquiterpenes are terpenes from 10 to 15 atoms of carbons biosynthetically derived from geranilpyrophosfates (SPG) and farnesilpyrophosfate (FPP) respectively. Monoterpenes and in general all the natural terpenoid compounds are synthetized by the route of the acetilCoA through a common intermediate which is the mevalonic acid. However, it has been proposed that some terpenoids are not originated by this route, and instead use an alternative route that may involve pyruvate, glyceraldehyde-

Essential oils are widely distributed in some 60 families of plants including the Compositae, Labiadas, Lauraceae, Myrtaceae, Rosaceae, Rutaceae, Umbelliferae, etc. Monoterpenoides are mainly found in plants in the family of Ranunculales, Violales, and Primulales, while they are rare in Rutales, Cornales, Lamiales, and Asterales. Moreover, the sesquiterpenoides are commonly found in Rutales, Cornales, Magnoliales and Asterales. Although in both essential oils: mono - sesquiterpenes and phenylpropan are found in free-form, those which are linked to carbohydrates, have been more recently investigated, because it is considered

From the chemical point of view, despite its complex composition with different types of substances, essential oils can be classified according to the type of substances of which they are the major components. According to this fact, the essential oils rich in monoterpenes are called essential oils sesquiterpenoids, those rich in phenylpropan are the essential oils phenylpropanoids. Although this classification is very general there are more complex classifications that take into account other chemical aspects (Fig 2,3,4) (González, 1984; Ikan,

According to www.chemkeys.com 2000; Gascon et al. 2002; www.herbotecnia. com.ar, 2002; www.losandes.com.ar 2002. essential oils can be extracted from plant samples through various methods samples such as: expression, distillation with steam, extraction with volatile solvents, enfleurage, extraction with supercritical fluids and hydrodistillation.

From such oils, it is possible to its isolation through the use of one or several

For the chromatographic column and thin layer methods the silica gel are widely used as stationary phase. Polar pure or mixed solvents such as: Toluene-acetate of ethyl benzene, chloroform, dichloromethane, ethyl, chloroform-benzene, chloroform-ethanol - chloroform-

chromatographic methods such as thin layer chromatography and HPLC.

benzene acetic acid benzene-acetate are used as mobile phase.

contain three units named sesquiterpenes (Ikan, 1991; Silva, 2002).

3-phosphate (Fig 1) (Adams et al, 1998; Sponsel, 1995).

to be the immediate precursor of the essential oil.

1991; Judd et al, 2002; Stachenko, 1996).

**2.1 Methods of extraction** 

**2.2 Method of isolation** 

(www.buscasalud.com)

**2. Essential oils** 

Fig. 1. General pathways of plant secondary metabolism.

Fig. 2. Natural Monoterpenes.

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 225

of optically active components have been developed, and have developed methods for combined analysis HPLC HPLC-NMR of mixtures of sesquiterpenes and mass

**2.3 Test of recognition of monoterpenes and sesquiterpenes (Pino, 1999; Stam, 1970)**  Due to the diversity of functional groups that can be present in the components of mono and sesquiterpenics of an essential oil, there is a specific test for their recognition. However there are few experimental procedures that allow to recognize some of them by their coloration with different reagents, its absorption of 254 nm UV light and its Rf in thin layer chromatography. For example, limonene is recognized in the plates of TLC because it does not absorb UV 254 nm light, adds bromine, does not form a derivative 2, 4-

There is a procedure which combines the TLC with micro reactions (oxidation, reduction, dehydration, hydrolysis, etc.) described in the classic book already today of Ikan. Other reagents useful for revealing monoterpenes and sesquiterpenes are anisaldehid-sulfuric

Monoterpenes and sesquiterpenes, in a number, can be characterized chemically from the gas chromatography and mass spectra data as noted above, but when there are doubts of such characterization some spectral methods such as infrared ultraviolet and magnetic

Infrared Spectroscopy allows detection of the presence of groups hydroxyl, Carbonyl, aromatic rings, bond double C = C cis and trans, etc. To determine the spectrum just put a drop of the component in a cell of NaCl. For example, in the infrared spectrum of the 3-pmenten-7 present in the oil of cumin, the intense band in 1725 cm - 1 indicates a group not conjugated Carbonyl. The peak at 2710 cm - 1 is assigned to a proton aldehydic C-H. The bond around on 1375 cm - 1 indicates a group isopropyl, and the band of 817 cm- 1 average

UV spectrum ultraviolet of monoterpenes and sesquiterpenes allows the recognition of functional groups and chromophoric. E.g. limonene presents an absorption maximum at 262

Nuclear magnetic resonance (1 H - 13 C); due to the developments of the NMR has database of Spectra, especially of 13C -NMR for the monoterpenes and more distributed sesquiterpenes. The NMR spectrometry-13 C has the additional advantage that the carbons of terpenoid chemical displacement (and other natural and synthetic substances) can be calculated by computer programs available in the market as Chemwind and ACD-Lab. In addition, the recent development of two-dimensional methods homo - and heteronuclear, has allowed the fine structural determination of terpenoids and other natural substances,

There are databases that contain information and spectra of masses of the components of mono - and sesquiterpenoids of essential oils. These databases are now available in many

dinitrophenylhydrazone and produces Brown with sulfuric acid.

acid, sulfuric acid -vanillin and phosphomolibdic acid.

**2.3.1 Spectral characterization** 

resonance spectrometry can be used.

intensity indicates a double bond tri-substituted.

eliminating the ambiguity in the allocation of the observed signals.

**2.3.1.1 Infrared spectroscopy** 

**2.3.1.2 Ultraviolet spectroscopy** 

**2.3.1.3 Mass Spectrometry** 

nm ( = 6400).

spectrometry.

Fig. 3. Natural Sesquiterpenes.

However, currently more efficient and faster techniques of separation are used such as HPLC high efficiency liquid chromatography and gas (CG) chromatography, as well as also combinations "ON-LINE" HPLC- CG EM. These same methods are used for the analysis of the flower essences. The last technique, thanks to the recent development of capillary columns for high resolution, allows analyzing complex mixtures present in essential oils, and identify the components from the retention times through the so-called Kovats retention indexes (Ik). These values are characteristic for each component and there are databases with indexes of many components of essential oils. Ik values are determined in two chromatographic columns, one polar (e.g. CARBOWAX 20 M) and a non-polar (e.g. OV - 101 also called DB-1).

In addition, the docked technique chromatography of gases - mass spectrometry, (GC-MS) allows obtaining the spectrum of mass of each component which builds the molecular weight and structural information. Likewise there are databases with spectra of masses of many components, something similar goes for the index Kovats (determined in two columns of different polarity) and the spectra of mass are criteria for chemical allocation of many components of essential oils not just monoterpenes but also other types of substances characteristic of such oils. More recently the column chromatographic Chiral for separation of optically active components have been developed, and have developed methods for combined analysis HPLC HPLC-NMR of mixtures of sesquiterpenes and mass spectrometry.

## **2.3 Test of recognition of monoterpenes and sesquiterpenes (Pino, 1999; Stam, 1970)**

Due to the diversity of functional groups that can be present in the components of mono and sesquiterpenics of an essential oil, there is a specific test for their recognition. However there are few experimental procedures that allow to recognize some of them by their coloration with different reagents, its absorption of 254 nm UV light and its Rf in thin layer chromatography. For example, limonene is recognized in the plates of TLC because it does not absorb UV 254 nm light, adds bromine, does not form a derivative 2, 4 dinitrophenylhydrazone and produces Brown with sulfuric acid.

There is a procedure which combines the TLC with micro reactions (oxidation, reduction, dehydration, hydrolysis, etc.) described in the classic book already today of Ikan. Other reagents useful for revealing monoterpenes and sesquiterpenes are anisaldehid-sulfuric acid, sulfuric acid -vanillin and phosphomolibdic acid.

## **2.3.1 Spectral characterization**

224 Insecticides – Pest Engineering

However, currently more efficient and faster techniques of separation are used such as HPLC high efficiency liquid chromatography and gas (CG) chromatography, as well as also combinations "ON-LINE" HPLC- CG EM. These same methods are used for the analysis of the flower essences. The last technique, thanks to the recent development of capillary columns for high resolution, allows analyzing complex mixtures present in essential oils, and identify the components from the retention times through the so-called Kovats retention indexes (Ik). These values are characteristic for each component and there are databases with indexes of many components of essential oils. Ik values are determined in two chromatographic columns, one polar (e.g. CARBOWAX 20 M) and a non-polar (e.g. OV -

In addition, the docked technique chromatography of gases - mass spectrometry, (GC-MS) allows obtaining the spectrum of mass of each component which builds the molecular weight and structural information. Likewise there are databases with spectra of masses of many components, something similar goes for the index Kovats (determined in two columns of different polarity) and the spectra of mass are criteria for chemical allocation of many components of essential oils not just monoterpenes but also other types of substances characteristic of such oils. More recently the column chromatographic Chiral for separation

Fig. 3. Natural Sesquiterpenes.

Fig. 4. Naturals phenylpropanoids.

101 also called DB-1).

Monoterpenes and sesquiterpenes, in a number, can be characterized chemically from the gas chromatography and mass spectra data as noted above, but when there are doubts of such characterization some spectral methods such as infrared ultraviolet and magnetic resonance spectrometry can be used.

### **2.3.1.1 Infrared spectroscopy**

Infrared Spectroscopy allows detection of the presence of groups hydroxyl, Carbonyl, aromatic rings, bond double C = C cis and trans, etc. To determine the spectrum just put a drop of the component in a cell of NaCl. For example, in the infrared spectrum of the 3-pmenten-7 present in the oil of cumin, the intense band in 1725 cm - 1 indicates a group not conjugated Carbonyl. The peak at 2710 cm - 1 is assigned to a proton aldehydic C-H. The bond around on 1375 cm - 1 indicates a group isopropyl, and the band of 817 cm- 1 average intensity indicates a double bond tri-substituted.

#### **2.3.1.2 Ultraviolet spectroscopy**

UV spectrum ultraviolet of monoterpenes and sesquiterpenes allows the recognition of functional groups and chromophoric. E.g. limonene presents an absorption maximum at 262 nm ( = 6400).

Nuclear magnetic resonance (1 H - 13 C); due to the developments of the NMR has database of Spectra, especially of 13C -NMR for the monoterpenes and more distributed sesquiterpenes. The NMR spectrometry-13 C has the additional advantage that the carbons of terpenoid chemical displacement (and other natural and synthetic substances) can be calculated by computer programs available in the market as Chemwind and ACD-Lab.

In addition, the recent development of two-dimensional methods homo - and heteronuclear, has allowed the fine structural determination of terpenoids and other natural substances, eliminating the ambiguity in the allocation of the observed signals.

#### **2.3.1.3 Mass Spectrometry**

There are databases that contain information and spectra of masses of the components of mono - and sesquiterpenoids of essential oils. These databases are now available in many

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 227

Studies carried out from different concentrations of extract of Meliaceas show that this extract inhibits the feeding and negatively affects the development and survival of different species of insects. The anti-alimentary activity of this compound shows that at doses ranging from 5.5 to 27. 6 µg/cm2 caused an inhibitory activity of more than 75% the insects. The way of action of these compounds extracted from various species of Meliaceas are taken from a combination between an anti feeding effect and post-digestive toxicity.

The use of plants as repellents is an ancient knowledge. This practice is basically done with compounds that have odor or irritant effects such as chili and garlic. There are homemade recipes that describe the use of fennel (*Foniculum vulgare*), rude (*Ruta graveolens*) and eucalyptus (*Eucalyptus globules*) among other aromatic plants to repel moths of clothing.

Treatments with natural compounds such as essential oils or pure compounds (Awde & Ryan, 1992; Keane & Ryan, 1999;Ryan & Byrne, 1988) may cause symptoms that indicate neurotoxic activity including hyperactivity, seizures and tremors followed by paralysis (knock down), which are very similar to those produced by the insecticides pyrethroids

Acetyl cholinesterase enzyme catalyzes the hydrolysis of the neurotransmitter excess acetylcholine in the synaptic space between choline and acetic acid. It has been recognized

Many hydrophobic compounds incite the deactivation of protein and enzyme inhibition; the acetilcolinesterase (AchE) is an enzyme that is particularly susceptible to the hydrophobic

Is described in this context essential oil interfering with AchE are acting as potent of the central nervous system where all synapses cholinergic are virtually located (Bloomquist, 1999) of the cholinesterase inhibitors are known as anticolinesterasic, the chemical products

The majority of plant insecticide are extracts made by a group of active compounds of diverse chemical nature, which is hardly found in the same concentrations so the pressure of selection on the plague will not be always the same, i.e. in general, insects take more to develop resistance to a blend of compounds than any of its components separately (Silva,

With the beginning of the new millennium, assessment of a large number of essential oils from native or typical flora has increased in many countries of the Americas and Africa due

There have been studies with oils in laboratory conditions reporting larvicide action in species of the genus *Culex, Anopheles*, *Aedes* (Ansari, 2005; Assarn, 2003; Albuquerque et al, 2004; Amer et al, 2006a ; Bassole et al., 2003; Carvalho et to 2003; Cavalcanti et al, 2004; Cetin et al, 2006; Cheng et to 2004; Chung et 2010 b Dharmagda et al, 2005; of Mendonca et al., 2005; Faley et al., 2005; Hafeez et 2011; Khandagle et al, 2001; Mathew & Thoppil 2011;Morais et al, 2006; Prajapati et al., 2005; Pushpanathan et al, 2006; Phasomkusolsil & Soowera 2010; Pitarokili et al, 2011; Raví et al, 2006; Tare et al, 2004; Tomas et al, 2004). In the same way it has been evaluated the repellent capacity of some oils in the same species of mosquitoes; Amer et al, 2006b;Barnard et al, 2004; Byeoung-Soo, 2005; Caballero-

that interfere with the action of this enzyme are potent neurotoxins (López, 2008).

(www.cannabiscultura.com)

(Kostyukovsky et al., 2002).

inhibition (Ryan et al. 1992).

**2.6 Future prospects** 

2003).

**2.5.1 Mode of action of the natural insecticide** 

that essential oils could their effect through ACE inhibition.

to its potential use as an alternative method of control.

commercial instruments of analysis as a chromatograph of gases coupled with mass spectrometers.

## **2.4 Phenylpropan**

Phenylpropanes are secondary metabolites derived from phenylalanine, which is first converted to cinnamic acid and after a series of hidroxilations lead to acid cumaric, caffeic acid, pherulic acid etc. The conversion of these acids to their corresponding esters produces some of the volatile components responsible for the fragrance of herbs and flowers (Lopez, 2008).

Phenylpropanes of essential oils are extracted with the same methodology described above for mono - and sesquiterpenes. However, due to its aromatic ring they have advantages in detection by TLC and HPLC because absorb ultraviolet light (254 nm) and do not require to be disclosed with chemical agents, or need to be derivative, and therefore can be isolated and analyzed more easily.

## **2.4.1 Recognition of phenylpropan**

There are test assay for recognition to the aromatic ring as a reaction with formaldehyde and sulfuric acid. Likewise in the case of phenylpropan with phenolic hydroxyl these can be recognized by the test assay of ferric chloride, which produces green and blue coloring with phenolic substances in general.

Substances with aromatic ring, their infrared spectra show signs characteristic of these compounds and give information about the type of replacement of the aromatic ring in addition to the functional groups present in the molecule. For example, the spectrum of eugenol shows, among other, bands in 3500 (wide) due to the hydroxyl group, 1510 characteristic of aromatic, and three bands in 990, 920 and 938 cm-1 characteristics of a vinyl mono substituted group. The IR from the cinamaldehyde spectrum displays bands in (weak) 3330, 3050, 2820, 2750, 1660 (intense, due to the carbonyl group), 975, 740 and 695 cm-1 among others.

Unlike the majority of mono - and sesquiterpenes, the phenylpropanes absorb UV light with a maximum around 254 nm depending on the groups present in the molecule chromophors. For example, the isoeugenol shows maxima at 260 (15850) and 305 (7000), safrole in 286 nm, the myristicine at 276 nm, the isosafrole in 264 nm, trans- cinnamic acid in 273 nm and ciscinnamic acid in 264 nm.

Phenylpropan 1H-NMR spectra show signs of aromatic protons around 6-8 ppm whose multiplicities and coupling constants allow a clear structural assignment even with low resolution spectra. Trans-anethole there is a double signal around 1.9 ppm due to the protons from the methyl group, a singlet in 3.9 due to the protons of the methoxy group, a complex signal around 6.1 ppm due to the two protons olefinics provision trans each other, and a double around 6.9 ppm characteristic of 4 protons of an aromatic ring p-based.

#### **2.5 Natural insecticides way-of-action (Silva 2002)**

As growth regulators, this effect can manifest itself in several ways. The first is related to the molecules that inhibit the metamorphosis. Other compounds make the insect to have an earlier metamorphosis, occurring at a time which is not favorable. It has been observed that certain molecules can alter the function of the hormones that regulate these mechanisms so that there are insects with malformations, dead or sterile (Gunderson, 1985; Sangykurn, 1999).

commercial instruments of analysis as a chromatograph of gases coupled with mass

Phenylpropanes are secondary metabolites derived from phenylalanine, which is first converted to cinnamic acid and after a series of hidroxilations lead to acid cumaric, caffeic acid, pherulic acid etc. The conversion of these acids to their corresponding esters produces some of the volatile components responsible for the fragrance of herbs and flowers (Lopez,

Phenylpropanes of essential oils are extracted with the same methodology described above for mono - and sesquiterpenes. However, due to its aromatic ring they have advantages in detection by TLC and HPLC because absorb ultraviolet light (254 nm) and do not require to be disclosed with chemical agents, or need to be derivative, and therefore can be isolated

There are test assay for recognition to the aromatic ring as a reaction with formaldehyde and sulfuric acid. Likewise in the case of phenylpropan with phenolic hydroxyl these can be recognized by the test assay of ferric chloride, which produces green and blue coloring with

Substances with aromatic ring, their infrared spectra show signs characteristic of these compounds and give information about the type of replacement of the aromatic ring in addition to the functional groups present in the molecule. For example, the spectrum of eugenol shows, among other, bands in 3500 (wide) due to the hydroxyl group, 1510 characteristic of aromatic, and three bands in 990, 920 and 938 cm-1 characteristics of a vinyl mono substituted group. The IR from the cinamaldehyde spectrum displays bands in (weak) 3330, 3050, 2820, 2750, 1660 (intense, due to the carbonyl group), 975, 740 and 695 cm-1

Unlike the majority of mono - and sesquiterpenes, the phenylpropanes absorb UV light with a maximum around 254 nm depending on the groups present in the molecule chromophors. For example, the isoeugenol shows maxima at 260 (15850) and 305 (7000), safrole in 286 nm, the myristicine at 276 nm, the isosafrole in 264 nm, trans- cinnamic acid in 273 nm and cis-

Phenylpropan 1H-NMR spectra show signs of aromatic protons around 6-8 ppm whose multiplicities and coupling constants allow a clear structural assignment even with low resolution spectra. Trans-anethole there is a double signal around 1.9 ppm due to the protons from the methyl group, a singlet in 3.9 due to the protons of the methoxy group, a complex signal around 6.1 ppm due to the two protons olefinics provision trans each other,

As growth regulators, this effect can manifest itself in several ways. The first is related to the molecules that inhibit the metamorphosis. Other compounds make the insect to have an earlier metamorphosis, occurring at a time which is not favorable. It has been observed that certain molecules can alter the function of the hormones that regulate these mechanisms so that there are insects with malformations, dead or sterile (Gunderson, 1985; Sangykurn,

and a double around 6.9 ppm characteristic of 4 protons of an aromatic ring p-based.

**2.5 Natural insecticides way-of-action (Silva 2002)** 

spectrometers.

2008).

**2.4 Phenylpropan** 

and analyzed more easily.

**2.4.1 Recognition of phenylpropan** 

phenolic substances in general.

among others.

1999).

cinnamic acid in 264 nm.

Studies carried out from different concentrations of extract of Meliaceas show that this extract inhibits the feeding and negatively affects the development and survival of different species of insects. The anti-alimentary activity of this compound shows that at doses ranging from 5.5 to 27. 6 µg/cm2 caused an inhibitory activity of more than 75% the insects. The way of action of these compounds extracted from various species of Meliaceas are taken from a combination between an anti feeding effect and post-digestive toxicity. (www.cannabiscultura.com)

The use of plants as repellents is an ancient knowledge. This practice is basically done with compounds that have odor or irritant effects such as chili and garlic. There are homemade recipes that describe the use of fennel (*Foniculum vulgare*), rude (*Ruta graveolens*) and eucalyptus (*Eucalyptus globules*) among other aromatic plants to repel moths of clothing.

## **2.5.1 Mode of action of the natural insecticide**

Treatments with natural compounds such as essential oils or pure compounds (Awde & Ryan, 1992; Keane & Ryan, 1999;Ryan & Byrne, 1988) may cause symptoms that indicate neurotoxic activity including hyperactivity, seizures and tremors followed by paralysis (knock down), which are very similar to those produced by the insecticides pyrethroids (Kostyukovsky et al., 2002).

Acetyl cholinesterase enzyme catalyzes the hydrolysis of the neurotransmitter excess acetylcholine in the synaptic space between choline and acetic acid. It has been recognized that essential oils could their effect through ACE inhibition.

Many hydrophobic compounds incite the deactivation of protein and enzyme inhibition; the acetilcolinesterase (AchE) is an enzyme that is particularly susceptible to the hydrophobic inhibition (Ryan et al. 1992).

Is described in this context essential oil interfering with AchE are acting as potent of the central nervous system where all synapses cholinergic are virtually located (Bloomquist, 1999) of the cholinesterase inhibitors are known as anticolinesterasic, the chemical products that interfere with the action of this enzyme are potent neurotoxins (López, 2008).

The majority of plant insecticide are extracts made by a group of active compounds of diverse chemical nature, which is hardly found in the same concentrations so the pressure of selection on the plague will not be always the same, i.e. in general, insects take more to develop resistance to a blend of compounds than any of its components separately (Silva, 2003).

#### **2.6 Future prospects**

With the beginning of the new millennium, assessment of a large number of essential oils from native or typical flora has increased in many countries of the Americas and Africa due to its potential use as an alternative method of control.

There have been studies with oils in laboratory conditions reporting larvicide action in species of the genus *Culex, Anopheles*, *Aedes* (Ansari, 2005; Assarn, 2003; Albuquerque et al, 2004; Amer et al, 2006a ; Bassole et al., 2003; Carvalho et to 2003; Cavalcanti et al, 2004; Cetin et al, 2006; Cheng et to 2004; Chung et 2010 b Dharmagda et al, 2005; of Mendonca et al., 2005; Faley et al., 2005; Hafeez et 2011; Khandagle et al, 2001; Mathew & Thoppil 2011;Morais et al, 2006; Prajapati et al., 2005; Pushpanathan et al, 2006; Phasomkusolsil & Soowera 2010; Pitarokili et al, 2011; Raví et al, 2006; Tare et al, 2004; Tomas et al, 2004).

In the same way it has been evaluated the repellent capacity of some oils in the same species of mosquitoes; Amer et al, 2006b;Barnard et al, 2004; Byeoung-Soo, 2005; Caballero-

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 229

The percentage of hatching inhibition was 36.47% of the total number of larvae. The larvae completing their development to adults corresponded to 60.54% of surviving larvae. As a figure of interest the greatest number of semi emergency in adults occurred at 72 hours after exposure to the oil of turpentine (with a predominance of males). At 96 hour, the largest proportion was in favor of females, who became non-existent for this sex at 120 and 168

More recently, oil of turpentine and the bicyclic α-pinene obtained by fractional distillation of oil of turpentine have been evaluated in *Culex quinquefasciatus*, important vector of West

The purification of the α-pinene was made using column chromatography in system of variable stationary phase (SiO2, Al2O3, and their mixtures in dependence of the polarities of the mobile phases for elution and separation).) Molecular characterization was developed, using techniques of FTIR and NMR, as well as methodologies for its extension to field conditions type TLC (chromatography on thin layer in the presence of plates of glass with silica gel 200-G254 activated with AgNO3 / acetonitril via wet impregnation with methanol). The plates are eluted with mixtures of solvents of media polarity (ethyl-acetone alcohol isopropyl acetate). Figure 5 shows the spectroscopic characterization of the α pinene. In table 1 and 2 is shown the concentrations finally used, the obtained mortalities and lethal concentrations that causes 50% to 95% percent of mortality, the slope of the regression line

The lowest lethal concentrations were obtained with oils of turpentine and turpentine modified followed by *Curcuma longa, Psidium rotundatum* and *Chenopodium ambrosoide*. By applying a χ2-square test (p < 0.05) to the results of the bioassays, the results showed that the mortalities are associated with the used dose. The higher slopes of the lines were equal with the oil of turpentine, *Eugenia melanadenia, Psidium rotundatum* and *Chenopodium ambrosiode*, corresponding to the lower value of the slope to the terpene α-pinene. To the slope values obtained were applied a χ2 test finding significant difference between them

for p < 0.0001, indicating that these oils have a different response for the same strain.

which it has active defense before any insecticide action.

In our assessed results we find the insecticidal action of oil in *Cx quinquefasciatus* and is important to note that lethal concentrations obtained are higher than concentrations found in *Aedes aegypti* to these same oils (Leyva,2008a; 2009a,b). This response could be that this species has developed physiological resistance and some tolerance to insecticides with

Oil of *Curcuma longa* presents in its composition more than 50% of sesquiterpenes, apart from the monoterpenes and α -pinene to which can be attributed the insecticide action. If we compare studies conducted with *Curcuma aromatica* (Choochate et al., 2005) and *Curcuma zedoaria* in *Aedes aegypti* these oils show lower values of LC50 and CL99 (Champakaew et al,

Within the family Piperaceae, notably members of the genus Piper have noted insecticidal action, molluscicide (Chansang et al., 2005; Parmar et 1997) this is due to the presence of the secondary metabolites as alkaloids, phenylpropanoids, lignans, neolignanos, terpenes, flavononas, among others (Pino et al, 2004; Smith & Kassim 1979). *Piper aduncum* specifically shows in its composition a high percent of dilapiol, α-pinene and 1.8- cineole (Bottia et 2007). In Mexico studies were not mortalities obtained with a kind of Piper in *Culex quinquefasciatus* in aqueous extracts to 5 and 15% where used in the aerial part of plants

hours (5-7 d)

Nile virus and encephalitis

and diagnostic dose.

2007).

(Perez-Pacheco et al 2004).

Gallardo, et-2011; Chang et al, 2006; Jaenson et al, 2006; Das et al., 2003; Kim et al, 2004; Oyedale et al, 2002; Odalo et al., 2005; Pohilt et 2011; Phasomkusolsil & Soowera 2010; Tawatsin et al, 2006; Trongtokit et al. 2005a; 2005b; Yang et al., 2005; Zhu et al, 2006; Less has been evaluated adulticide effect found only two reports described by Chaiyasit et al 2006 and Miot et al 2004.

## **3. Studies developing in Cuba with essential oils for controlling of public health pests**

In Cuba there is a large plant biodiversity with a lot of endemic species. Myrtaceas, Piperaceas, Zingiberaceae, Pinaceae families possess insecticidal action potential based to their essential oils. To this day, there have been verified in public health pests such as; *Blatella germanica*, *Musca domestica* and *Aedes aegypti* (Aguilera et al 2003; 2004;Leyva et al, 2007 a, b, 2008a, b; 2009a, b; 2010) and more recently in *Culex quinquefasciatus.* 

Evaluated essential oils are essentially from *Pimenta racemosa, Eugenia melanadenia, Psidium rotundatum* and *Melaleuca leucadendron* of the botanical family Myrtaceae, *Piper auritum, Piper aduncum* of the family Piperacea, *Curcuma longa* in the family Zingiberaceae, *Artemisia abrotamum* in the family Asteraceae, *Pinus tropicalis* and *Pinus caribbaea* of the Pinacea family. A derivative of essential oil of pinaceas has also been evaluated; turpentine oil obtained by hidrodestylation of the resin of endemic pine trees, mostly composed of terpenes (α-and βpinene and a modified turpentine where a part of the original turpentine oil was modified by photoizomerization of the and β-pinene to compound of the type verbenone and pulegone.)

In all tested oils, there has been found necessary dosages that elicited from 5% to 95% of mortality. After testing CHI2 they have a probability greater than 0.05 by which can raise all the mortalities occurring in each are associated with the used dose.

 Studies with these oils in larvae of *Aedes aegypti* show high insecticidal action by the low lethal concentrations obtained and the high slopes of the regression lines; however, in *Blatella germanica* and *Musca domestica* the bioassays were conducted in adults, showing a very low activity given the high lethal concentrations obtained.

Natural essences of plants owe its insecticide and repellent action to the presence in its composition of derivative monoterpenic as d-limonene, α -terpineol, β - myrceno, linolool, 1.8-cineole, 4-terpineol, thymol, carvacrol, α-pinene and β-pinene.) Studied oils possess structures lactones as the 1.8 cineole, 4-terpinol, α and β - pinene, cineole, safrole, turmenon, chavicol, eugenol, beta-phellandrene, longifoleno among others, which account for a 18-68% of its total membership, and are that they can be attributed the high insecticidal action in *Aedes aegypti*.

One aspect assessed for reasons of eco-sustainability was ovicidal and inhibitory action of the development by the oil of turpentine in larvae of *Ae aegypti* 

The largest ovicidal effect was shown with the dose diagnosis of oil of turpentine with photochemical treatment. In a test of hypothesis of different proportions for each dose of oil, the diagnostic doses were compared with every CL95 finding differences between the doses tested with modified oil of turpentine for p=0.05 while for oil not modified there was only significant difference comparing the CL95 and the diagnostic doses. (p=0.05). By comparing the CL95 and the diagnostic dose, he found significant difference for a p = 0.05. If we compare both types of essences we may conclude that although modified turpentine oil presents a CL95 dose higher than oil without changing this it has a greater effect ovicidal and therefore more protector.

Gallardo, et-2011; Chang et al, 2006; Jaenson et al, 2006; Das et al., 2003; Kim et al, 2004; Oyedale et al, 2002; Odalo et al., 2005; Pohilt et 2011; Phasomkusolsil & Soowera 2010;

Less has been evaluated adulticide effect found only two reports described by Chaiyasit et al

In Cuba there is a large plant biodiversity with a lot of endemic species. Myrtaceas, Piperaceas, Zingiberaceae, Pinaceae families possess insecticidal action potential based to their essential oils. To this day, there have been verified in public health pests such as; *Blatella germanica*, *Musca domestica* and *Aedes aegypti* (Aguilera et al 2003; 2004;Leyva et al,

Evaluated essential oils are essentially from *Pimenta racemosa, Eugenia melanadenia, Psidium rotundatum* and *Melaleuca leucadendron* of the botanical family Myrtaceae, *Piper auritum, Piper aduncum* of the family Piperacea, *Curcuma longa* in the family Zingiberaceae, *Artemisia abrotamum* in the family Asteraceae, *Pinus tropicalis* and *Pinus caribbaea* of the Pinacea family. A derivative of essential oil of pinaceas has also been evaluated; turpentine oil obtained by hidrodestylation of the resin of endemic pine trees, mostly composed of terpenes (α-and βpinene and a modified turpentine where a part of the original turpentine oil was modified by photoizomerization of the and β-pinene to compound of the type verbenone and pulegone.) In all tested oils, there has been found necessary dosages that elicited from 5% to 95% of mortality. After testing CHI2 they have a probability greater than 0.05 by which can raise all

 Studies with these oils in larvae of *Aedes aegypti* show high insecticidal action by the low lethal concentrations obtained and the high slopes of the regression lines; however, in *Blatella germanica* and *Musca domestica* the bioassays were conducted in adults, showing a

Natural essences of plants owe its insecticide and repellent action to the presence in its composition of derivative monoterpenic as d-limonene, α -terpineol, β - myrceno, linolool, 1.8-cineole, 4-terpineol, thymol, carvacrol, α-pinene and β-pinene.) Studied oils possess structures lactones as the 1.8 cineole, 4-terpinol, α and β - pinene, cineole, safrole, turmenon, chavicol, eugenol, beta-phellandrene, longifoleno among others, which account for a 18-68% of its total membership, and are that they can be attributed the high insecticidal action in

One aspect assessed for reasons of eco-sustainability was ovicidal and inhibitory action of

The largest ovicidal effect was shown with the dose diagnosis of oil of turpentine with photochemical treatment. In a test of hypothesis of different proportions for each dose of oil, the diagnostic doses were compared with every CL95 finding differences between the doses tested with modified oil of turpentine for p=0.05 while for oil not modified there was only significant difference comparing the CL95 and the diagnostic doses. (p=0.05). By comparing the CL95 and the diagnostic dose, he found significant difference for a p = 0.05. If we compare both types of essences we may conclude that although modified turpentine oil presents a CL95 dose higher than oil without changing this it has a greater effect ovicidal and

Tawatsin et al, 2006; Trongtokit et al. 2005a; 2005b; Yang et al., 2005; Zhu et al, 2006;

**3. Studies developing in Cuba with essential oils for controlling of public** 

2007 a, b, 2008a, b; 2009a, b; 2010) and more recently in *Culex quinquefasciatus.* 

the mortalities occurring in each are associated with the used dose.

very low activity given the high lethal concentrations obtained.

the development by the oil of turpentine in larvae of *Ae aegypti* 

2006 and Miot et al 2004.

**health pests** 

*Aedes aegypti*.

therefore more protector.

The percentage of hatching inhibition was 36.47% of the total number of larvae. The larvae completing their development to adults corresponded to 60.54% of surviving larvae. As a figure of interest the greatest number of semi emergency in adults occurred at 72 hours after exposure to the oil of turpentine (with a predominance of males). At 96 hour, the largest proportion was in favor of females, who became non-existent for this sex at 120 and 168 hours (5-7 d)

More recently, oil of turpentine and the bicyclic α-pinene obtained by fractional distillation of oil of turpentine have been evaluated in *Culex quinquefasciatus*, important vector of West Nile virus and encephalitis

The purification of the α-pinene was made using column chromatography in system of variable stationary phase (SiO2, Al2O3, and their mixtures in dependence of the polarities of the mobile phases for elution and separation).) Molecular characterization was developed, using techniques of FTIR and NMR, as well as methodologies for its extension to field conditions type TLC (chromatography on thin layer in the presence of plates of glass with silica gel 200-G254 activated with AgNO3 / acetonitril via wet impregnation with methanol). The plates are eluted with mixtures of solvents of media polarity (ethyl-acetone alcohol isopropyl acetate). Figure 5 shows the spectroscopic characterization of the α pinene. In table 1 and 2 is shown the concentrations finally used, the obtained mortalities and lethal concentrations that causes 50% to 95% percent of mortality, the slope of the regression line and diagnostic dose.

The lowest lethal concentrations were obtained with oils of turpentine and turpentine modified followed by *Curcuma longa, Psidium rotundatum* and *Chenopodium ambrosoide*. By applying a χ2-square test (p < 0.05) to the results of the bioassays, the results showed that the mortalities are associated with the used dose. The higher slopes of the lines were equal with the oil of turpentine, *Eugenia melanadenia, Psidium rotundatum* and *Chenopodium ambrosiode*, corresponding to the lower value of the slope to the terpene α-pinene. To the slope values obtained were applied a χ2 test finding significant difference between them for p < 0.0001, indicating that these oils have a different response for the same strain.

In our assessed results we find the insecticidal action of oil in *Cx quinquefasciatus* and is important to note that lethal concentrations obtained are higher than concentrations found in *Aedes aegypti* to these same oils (Leyva,2008a; 2009a,b). This response could be that this species has developed physiological resistance and some tolerance to insecticides with which it has active defense before any insecticide action.

Oil of *Curcuma longa* presents in its composition more than 50% of sesquiterpenes, apart from the monoterpenes and α -pinene to which can be attributed the insecticide action. If we compare studies conducted with *Curcuma aromatica* (Choochate et al., 2005) and *Curcuma zedoaria* in *Aedes aegypti* these oils show lower values of LC50 and CL99 (Champakaew et al, 2007).

Within the family Piperaceae, notably members of the genus Piper have noted insecticidal action, molluscicide (Chansang et al., 2005; Parmar et 1997) this is due to the presence of the secondary metabolites as alkaloids, phenylpropanoids, lignans, neolignanos, terpenes, flavononas, among others (Pino et al, 2004; Smith & Kassim 1979). *Piper aduncum* specifically shows in its composition a high percent of dilapiol, α-pinene and 1.8- cineole (Bottia et 2007). In Mexico studies were not mortalities obtained with a kind of Piper in *Culex quinquefasciatus* in aqueous extracts to 5 and 15% where used in the aerial part of plants (Perez-Pacheco et al 2004).

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 231


Table 1. Concentrations used and rates obtained with derivatives of Pinaceae in *Cx* 

Conc (%) Mortality (%) Lethal Conc Slope of the Line *Chenopodium ambrosoide* 0.0040 10 11.8

*Curcuma longa* 0.0040 46 5.22

*Psidium rotundatum* 0.0040 28 8.16

*Piper aduncum* 0.0100 36 6.72

*Pimenta racemosa* 0.0040 10 5.23

*Eugenia melanadenia* 0.0240 14 18.5

Diagnostic Dose 0.044 (440 mg/L)

> Diagnostic Dose : 0.0162 (162 mg/L)

> Diagnostic Dose : 0.0232 (232 mg/L)

> Diagnostic Dose 0.018 (180 mg/L)

> Diagnostic Dose 0.050 (500 mg/L)

> Diagnostic Dose 0.0380 (380 mg/L)

> Diagnostic Dose 0.0718 (718 mg/L)

0.0040 35 CL50 0.0052 3.7

0.0050 42 CL50 0.0051

0.0070 95 CL95 0.0071

0.0050 66 CL50 0.0041

0.0070 86 CL95 0.0086

0.0050 58 CL50 0.0047

0.0070 92 CL95 0.007

0.0120 53 CL50 0.0110

0.0160 85 CL95 0.0200

0.0050 20 CL50 0.0070

0.0070 50 CL95 0.0140

0.0260 41 CL50 0.026

0.0300 78 CL95 0.033

Table 2. Concentrations used and rates obtained with essential oils tested in *Cx* 

0.0050 46

0.0070 67 0.0080 78

*quinquefasciatus* larvae.

0.0060 56 CL95 0.014

0.0060 80

0.0080 98

0.0060 80

0.0080 94

0.0060 80

0.0080 97

0.0140 73

0.0180 90 0.0200 95

0.0060 44

0.0080 62 0.0100 79

0.0280 70

0.0320 90

*quinquefasciatus* larvae.

The larvicidal activity of *Chenopodium ambrosioide* was evaluated in *Ae aegypti*, with extracts metanolics to different concentrations resulting most important inhibition of pupal development and an increase in the time of larval development (Suparvarn et al 1974) has also been evaluated in mosquito repellent activity (Gillij et al 2008). *Ch ambrosiodes* has as one of its components majority the carvacrol reported in the literature as insecticide terpene (Silva et al 2008; Kordali et al 2008). *Pimenta racemosa, Eugenia melanadenia* and *Psidium rotundatum* presented in its composition more than 50% of monoterpenes and sesquiterpenes standing out in his majority composition 1,8 cineole, 4-terpineole and αpinene terpenes with high insecticidal action (Pino et al, 2004,2005).

Oil of turpentine in *Aedes aegypti* obtained strong insecticide and inhibitory action of development in previous studies (Lucia et al, 2007; Leyva et al, 2009b 2010). In this study isolated terpene α -pinene (one of the major components of turpentine) was higher than the own turpentine lethal concentrations, this may be due to the synergistic effect with several monoterpens (α and β -pinene) at the same time and not isolated and evaluated by themselves in insects.

Fig. 5. Spectroscopic characterization of the α pinene.


The larvicidal activity of *Chenopodium ambrosioide* was evaluated in *Ae aegypti*, with extracts metanolics to different concentrations resulting most important inhibition of pupal development and an increase in the time of larval development (Suparvarn et al 1974) has also been evaluated in mosquito repellent activity (Gillij et al 2008). *Ch ambrosiodes* has as one of its components majority the carvacrol reported in the literature as insecticide terpene (Silva et al 2008; Kordali et al 2008). *Pimenta racemosa, Eugenia melanadenia* and *Psidium rotundatum* presented in its composition more than 50% of monoterpenes and sesquiterpenes standing out in his majority composition 1,8 cineole, 4-terpineole and α-

Oil of turpentine in *Aedes aegypti* obtained strong insecticide and inhibitory action of development in previous studies (Lucia et al, 2007; Leyva et al, 2009b 2010). In this study isolated terpene α -pinene (one of the major components of turpentine) was higher than the own turpentine lethal concentrations, this may be due to the synergistic effect with several monoterpens (α and β -pinene) at the same time and not isolated and evaluated by

Turpentine

Modified Turpentine

Slope of the Line

Diagnostic Dose 0.0108 (108 mg/L)

Diagnostic Dose 0.0112 (112 mg/L)

pinene terpenes with high insecticidal action (Pino et al, 2004,2005).

Fig. 5. Spectroscopic characterization of the α pinene.

0.0035 5 CL50 0.0042 20.9

0.0030 5 CL50 0.0038 14.4

Lethal Conc

themselves in insects.

Conc (%)

0.0040 31

0.0060 100

0.0040 60

0.0060 100

Mortality (%)

0.0050 94 CL95 0.0055

0.0050 93 CL95 0.0050


Table 1. Concentrations used and rates obtained with derivatives of Pinaceae in *Cx quinquefasciatus* larvae.


Table 2. Concentrations used and rates obtained with essential oils tested in *Cx quinquefasciatus* larvae.

Essential Plant Oils and Insecticidal Activity in *Culex quinquefasciatus* 233

Alburquerque, MR; Silveira,ER; De Ochoa, DE; Lemus, TL; Souza, EB; Santiago, GM; Pessoa,

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## **4. Conclusions**

An interest in natural products from plants has been increased due to the development of resistance to synthetic insecticides, which are applied in order to reduce the populations of insects.

The biological activity of natural compounds is based on its structure and the doses used for such purposes. Vegetable insecticides have the great advantage of being compatible with other acceptable low-risk options from the ecological point of view on the control of insects. The secondary metabolites produced by plants against the attacks of predators and insects make them natural candidates in the control of species of insects, both vector of diseases and pests of agriculture. It is not logical to come to jump to the idea that they will completely replace the synthetic insecticides. Logical thinking is to have in them a complementary use to optimize and increase the sustainability of current integrated pest control strategies.

There are many publications of lists of plants with insecticidal properties. To use such plants, it is not enough to be regarded as promising or proven insecticidal properties. Analysis of risks to the environment and health should also be made. It is not to recommend the use of plants that are endangered, with limited biomass or that their use involves major alterations to the density in which they are in the nature. An ideal insecticide plant must be perennial, be widely distributed and in large amounts in nature or that can be cultivated, using renewable plant bodies such as leaves, flowers or fruits, not be destroyed every time you need to collect material to (avoid the use of roots and bark), agro-technitian minimum requirements and be eco-sustainability, have additional uses (such as medicines), not having a high economic value, be effective at low doses, possess potential scaling biotechnology (Silva;2002).

Insecticide plants have the advantage of having other uses as medicinal, a rapid degradation which decreases the risk of residues in food and therefore can be more specific for pest insect and less aggressive with natural enemies. They also develop resistance more slowly in comparison with synthetic insecticides. By the other hand, the disadvantages include that they can be degraded more quickly by ultraviolet rays so its residual effect is low, however not all insecticides from plants are less toxic than synthetic and residual is not established.

We can conclude that studies in our country, essential oils and their derivatives from the different families of evaluated plants have a high insecticide activity on larvae of *Aedes aegypti* and *Culex quinquefasciatus* and derived specifically from pinaceas, they have ovicide and inhibitory action of development in *Aedes aegypti*, which are potential candidates for control alternatives in these species of insects.

## **5. References**


An interest in natural products from plants has been increased due to the development of resistance to synthetic insecticides, which are applied in order to reduce the populations of

The biological activity of natural compounds is based on its structure and the doses used for such purposes. Vegetable insecticides have the great advantage of being compatible with other acceptable low-risk options from the ecological point of view on the control of insects. The secondary metabolites produced by plants against the attacks of predators and insects make them natural candidates in the control of species of insects, both vector of diseases and pests of agriculture. It is not logical to come to jump to the idea that they will completely replace the synthetic insecticides. Logical thinking is to have in them a complementary use to optimize and increase the sustainability of current integrated pest control strategies. There are many publications of lists of plants with insecticidal properties. To use such plants, it is not enough to be regarded as promising or proven insecticidal properties. Analysis of risks to the environment and health should also be made. It is not to recommend the use of plants that are endangered, with limited biomass or that their use involves major alterations to the density in which they are in the nature. An ideal insecticide plant must be perennial, be widely distributed and in large amounts in nature or that can be cultivated, using renewable plant bodies such as leaves, flowers or fruits, not be destroyed every time you need to collect material to (avoid the use of roots and bark), agro-technitian minimum requirements and be eco-sustainability, have additional uses (such as medicines), not having a high economic value, be effective at low doses, possess potential scaling biotechnology

Insecticide plants have the advantage of having other uses as medicinal, a rapid degradation which decreases the risk of residues in food and therefore can be more specific for pest insect and less aggressive with natural enemies. They also develop resistance more slowly in comparison with synthetic insecticides. By the other hand, the disadvantages include that they can be degraded more quickly by ultraviolet rays so its residual effect is low, however not all insecticides from plants are less toxic than synthetic and residual is not established. We can conclude that studies in our country, essential oils and their derivatives from the different families of evaluated plants have a high insecticide activity on larvae of *Aedes aegypti* and *Culex quinquefasciatus* and derived specifically from pinaceas, they have ovicide and inhibitory action of development in *Aedes aegypti*, which are potential candidates for

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**4. Conclusions** 

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**11** 

*1Mexico 2France* 

**Biological Control of Mosquito Larvae by**

*Bacillus thuringiensis* **subsp.** *israelensis*

Chemical insecticides provide many benefits to food production and human health and has proven very effective at increasing agriculture and forestry productivities. However, they also pose some hazards as contamination of water and food sources, poisoning of non-target fauna and flora, concentration in the food chain and selection of insect pest populations resistent to the chemical insecticides (Wojciech & Korsten 2002). It is well documented that chemical pesticides reduced natural-enemy populations and chemical applications can disrupt biological control and may cause outbreaks of secundary pests previously suppressed by natural enemies (Bartlett, 1964) and pest species develop pesticide resistance

The use of synthetic organic pesticides has had serious economic, social and environmental ramifications. Economically, the rapidy increasing cost for development and production of petrochemically derived insecticides, together with the declining effectiveness due to widespread insect resistance. As a result the chemical pesticide industry continues to develop new more expensive compounds and increasing pesticide prices. Socially and ecologically they have caused death and disease in human and damaged the environment. It is estimated that only a minute fraction of the insecticides applied is required for suppression of the target pest. The remainder, more than 99.9%, enters the environment

Alternative methods of insect management offer adequate levels of pest control and pose fewer hazards. One such alternative is the use of microbial insecticides, that contain microorganisms or their by-products. Microbial insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low. Compared to other commonly used insecticides, they are safe for both, the pesticide user and consumers of treated crops. Microbial insecticides also are known as biological pathogens, and biological control agents. Chemical insecticides are far more commonly used in the world than microbial control, however some microbial control agents, at least in part, can be used to replace some hazardous chemical pest control agents. A number of biological control agents formulated with bacteria, fungi, virus, pheromones, and plant extracts have been in use mainly for the control of insects responsible for the destruction of forests and agriculture

but natural enemies not (Johnson & Tabashnick, 1999).

through soil, water and food cycles (Metcalf, 1986).

crops (McDonald & Linde, 2002).

**1. Introduction** 

Mario Ramírez-Lepe1 and Montserrat Ramírez-Suero2

*1Instituto Tecnológico de Veracruz, UNIDA, Veracruz, 2Université de Haufe-Alsace, LVBE EA-3991, Colmar,* 


## **Biological Control of Mosquito Larvae by** *Bacillus thuringiensis* **subsp.** *israelensis*

Mario Ramírez-Lepe1 and Montserrat Ramírez-Suero2

*1Instituto Tecnológico de Veracruz, UNIDA, Veracruz, 2Université de Haufe-Alsace, LVBE EA-3991, Colmar, 1Mexico 2France* 

## **1. Introduction**

238 Insecticides – Pest Engineering

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29-53.

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caged and free flying *An stephensi* mosquitoes Southeast Asian. *J Trop Med. Public* 

Chemical insecticides provide many benefits to food production and human health and has proven very effective at increasing agriculture and forestry productivities. However, they also pose some hazards as contamination of water and food sources, poisoning of non-target fauna and flora, concentration in the food chain and selection of insect pest populations resistent to the chemical insecticides (Wojciech & Korsten 2002). It is well documented that chemical pesticides reduced natural-enemy populations and chemical applications can disrupt biological control and may cause outbreaks of secundary pests previously suppressed by natural enemies (Bartlett, 1964) and pest species develop pesticide resistance but natural enemies not (Johnson & Tabashnick, 1999).

The use of synthetic organic pesticides has had serious economic, social and environmental ramifications. Economically, the rapidy increasing cost for development and production of petrochemically derived insecticides, together with the declining effectiveness due to widespread insect resistance. As a result the chemical pesticide industry continues to develop new more expensive compounds and increasing pesticide prices. Socially and ecologically they have caused death and disease in human and damaged the environment. It is estimated that only a minute fraction of the insecticides applied is required for suppression of the target pest. The remainder, more than 99.9%, enters the environment through soil, water and food cycles (Metcalf, 1986).

Alternative methods of insect management offer adequate levels of pest control and pose fewer hazards. One such alternative is the use of microbial insecticides, that contain microorganisms or their by-products. Microbial insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low. Compared to other commonly used insecticides, they are safe for both, the pesticide user and consumers of treated crops. Microbial insecticides also are known as biological pathogens, and biological control agents. Chemical insecticides are far more commonly used in the world than microbial control, however some microbial control agents, at least in part, can be used to replace some hazardous chemical pest control agents. A number of biological control agents formulated with bacteria, fungi, virus, pheromones, and plant extracts have been in use mainly for the control of insects responsible for the destruction of forests and agriculture crops (McDonald & Linde, 2002).

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 241

According to Federici (1999) the main reasons for the success of *Bt* include (i) the high efficacy of its insecticidal proteins (ii) the existence of a diversity of proteins that are effective against a range of important pests (iii) its relatively safety to nontarget insect predators and parasites (iv) its easy to mass production at a relatively low cost, and (v) its

Advantages and disadvantages of *Bt* with chemical insecticides were summarized by Rowe

High specificity, i.e. no mammalian or nontarget effects; use permitted up to date of

 Adaptable to many types of formulations; potential to incorporate feeding stimulants or baits to increase the attractiveness of the formulations to the insects and thereby

Probability of producing more potent formulations and reducing production costs

 High probability that strain selection and/or genetic engineering will lead to better control of pest insects by newly found or created strains of Bt having novel host spectra

Proper timing of application require due to slower effect than chemical insecticides

adaptability to conventional formulations and application technology.

No broad insect resistance observed or expected to develop.

through improved-fermentation technology.

Lack of patent protection on new strains

Fig. 1. *Bacillus thuringiensis* subsp. *israelensis.*

**2.3 Advantages and disadvantages of** *Bt* 

and Margaritis (1987). Advantages of *Bt:* 

increase their efficacy.

or increased activity

Disadvantages of *Bt:*  Narrow host spectrum

harvest.

The microbial insecticides most widely used in the world are preparations of *Bacillus thuringiensis* (*Bt*). The insecticidal activity of *Bt* is due to the proteic parasporal inclusions that are produced during sporulation. Insecticides based on the proteinaceous -endotoxin of *Bt* constitute part of a more ecologically rational pest control strategy. *Bt* strains have been isolated world wide from many habitats, including soil, insects, stored-product dust, and deciduous and coniferous leaves, all of which have a limited host range, however together span a wide range of insects orders which include: Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera, Orthoptera, Acari, and Mallophaga and other organisms such as nematodes, mites, and protozoa (Federici, 1999). *Bacillus thuringiensis* subsp. *israelensis* (*Bti*) or serotype H-14, exhibit acute toxicity towards dipteran insects such as larval mosquitoes and black flies (de Barjac 1978) and is currently used in mosquito control programs world wide (Priest, 1992). The World Health Organization's (WHO) Onchocerciasis Control Program (OCP) in West Africa using *Bti* toxins has been one of the success stories of international co-operation in the control of infectious diseases program (Webb 1992; Drobniewski 1993). Due to the importance of *Bti* to control several tropical diseases such malaria and dengue, our purpose in this chapter is to provide an overview for non-*Bt* specialists of the basic knowledge of *Bti.* 

## **2.** *Bacillus thuringiensis* **strains**

#### **2.1 Origin of some strains of** *B. thuringiensis*

A large number of strain of *Bt* have been isolated from which to date. *Bt* as currently recognized is actually a complex of subspecies. They have grouped in 79 serotypes (Zeigler, 1999). The first *Bt* strain was isolated from diseased larvae of the silkworm, *Bombi mori*, in Japan by Ishiwata (1901). Iwabushi (1908) describe the bacillus as *Bacillus sotto.* Aoki & Chigasaki (1915) and Mitani & Wataral (1916) purified a highly toxic substance from sporulated *Bt* cultures. It was not officially described, however, until it was reisolated by Berliner in 1915 from diseased larvae of the Mediterranean flour moth, *Anagasta kuehniella*, in Thuringia, Germany, hence the derivation of species name thuringiensis (Federici, 1999). A *Bt* strain was isolated again by Mattes (1927) and described briefly the inclusion rhomboidal body. The activity of the *Bt* strains against lepidopteran larvae was described by Metalnikov (1930) and by Husz (1931). The association of the inclusion bodies of *B. thuringiensis* with toxicity against insects was established by Steinhaus. In 1951 published a paper which described the morphology of *Bt* and its possible use in the biological control against the alfalfa caterpillar. In France, a product named "Sporeine" was developed and used against *Ephestia kuhniella* (Lepidoptera) (Jacobs, 1950). Hannay, (1953) described a parasporal body in bipyramidal shape produced by the bacterium during sporulation and suggested that the crystal was involved in the toxic activity. The protein nature of the crystals was determined by Hanna and Fitz-James (1955).

#### **2.2 Features of** *B. thuringiensis* **strains**

*Bt* is a facultative anaerobic, gram-positive bacterium that forms characteristic protein inclusions adjacent to the endospore (Fig.1). The crystalline inclusion are composed of proteins known as ICPs crystal proteins. Cry proteins, or -endotoxins is the basis for commercial insecticidal formulations of *Bt.* Insecticides containing *Bt* in pest control programs is now considered as a viable strategy, which has proven to be both safe and reliable over the last 45 years (Chungjatupornchai et al*.* 1988).

Fig. 1. *Bacillus thuringiensis* subsp. *israelensis.*

## **2.3 Advantages and disadvantages of** *Bt*

According to Federici (1999) the main reasons for the success of *Bt* include (i) the high efficacy of its insecticidal proteins (ii) the existence of a diversity of proteins that are effective against a range of important pests (iii) its relatively safety to nontarget insect predators and parasites (iv) its easy to mass production at a relatively low cost, and (v) its adaptability to conventional formulations and application technology.

Advantages and disadvantages of *Bt* with chemical insecticides were summarized by Rowe and Margaritis (1987).

Advantages of *Bt:* 

240 Insecticides – Pest Engineering

The microbial insecticides most widely used in the world are preparations of *Bacillus thuringiensis* (*Bt*). The insecticidal activity of *Bt* is due to the proteic parasporal inclusions that are produced during sporulation. Insecticides based on the proteinaceous -endotoxin of *Bt* constitute part of a more ecologically rational pest control strategy. *Bt* strains have been isolated world wide from many habitats, including soil, insects, stored-product dust, and deciduous and coniferous leaves, all of which have a limited host range, however together span a wide range of insects orders which include: Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera, Orthoptera, Acari, and Mallophaga and other organisms such as nematodes, mites, and protozoa (Federici, 1999). *Bacillus thuringiensis* subsp. *israelensis* (*Bti*) or serotype H-14, exhibit acute toxicity towards dipteran insects such as larval mosquitoes and black flies (de Barjac 1978) and is currently used in mosquito control programs world wide (Priest, 1992). The World Health Organization's (WHO) Onchocerciasis Control Program (OCP) in West Africa using *Bti* toxins has been one of the success stories of international co-operation in the control of infectious diseases program (Webb 1992; Drobniewski 1993). Due to the importance of *Bti* to control several tropical diseases such malaria and dengue, our purpose in this chapter is to provide an

A large number of strain of *Bt* have been isolated from which to date. *Bt* as currently recognized is actually a complex of subspecies. They have grouped in 79 serotypes (Zeigler, 1999). The first *Bt* strain was isolated from diseased larvae of the silkworm, *Bombi mori*, in Japan by Ishiwata (1901). Iwabushi (1908) describe the bacillus as *Bacillus sotto.* Aoki & Chigasaki (1915) and Mitani & Wataral (1916) purified a highly toxic substance from sporulated *Bt* cultures. It was not officially described, however, until it was reisolated by Berliner in 1915 from diseased larvae of the Mediterranean flour moth, *Anagasta kuehniella*, in Thuringia, Germany, hence the derivation of species name thuringiensis (Federici, 1999). A *Bt* strain was isolated again by Mattes (1927) and described briefly the inclusion rhomboidal body. The activity of the *Bt* strains against lepidopteran larvae was described by Metalnikov (1930) and by Husz (1931). The association of the inclusion bodies of *B. thuringiensis* with toxicity against insects was established by Steinhaus. In 1951 published a paper which described the morphology of *Bt* and its possible use in the biological control against the alfalfa caterpillar. In France, a product named "Sporeine" was developed and used against *Ephestia kuhniella* (Lepidoptera) (Jacobs, 1950). Hannay, (1953) described a parasporal body in bipyramidal shape produced by the bacterium during sporulation and suggested that the crystal was involved in the toxic activity. The protein nature of the

*Bt* is a facultative anaerobic, gram-positive bacterium that forms characteristic protein inclusions adjacent to the endospore (Fig.1). The crystalline inclusion are composed of proteins known as ICPs crystal proteins. Cry proteins, or -endotoxins is the basis for commercial insecticidal formulations of *Bt.* Insecticides containing *Bt* in pest control programs is now considered as a viable strategy, which has proven to be both safe and

overview for non-*Bt* specialists of the basic knowledge of *Bti.* 

crystals was determined by Hanna and Fitz-James (1955).

reliable over the last 45 years (Chungjatupornchai et al*.* 1988).

**2.2 Features of** *B. thuringiensis* **strains** 

**2.** *Bacillus thuringiensis* **strains** 

**2.1 Origin of some strains of** *B. thuringiensis* 


Disadvantages of *Bt:* 


Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 243

Serotype Serovar Serotype Serovar 1 thuringiensis 28a, 28c jeghatensan 2 finitimus 29 amagiensis 3a, 3b, 3c kurstaki 31 toguchini 3a, 3c alesti 32 cameroun 3a, 3d sumiyoshiensi*s* 33 leesis 3a, 3d, 3e fukuokaensis 34 konkukian 4a, 4b soto/dendrolimus 35 seoulensis 4a, 4c kenyae 36 malayensis 5a, 5b galleriae 37 andralousiensis 5a,5c canadensis 38 owaldocruzi 6 entomocidus/subtoxicus 39 brasilensis 7 aizawai/pacificus 40 huazhongensis 8a.8b morrisoni 41 sooncheon 8a, 8c ostriniae 42 jinghongiensis 8b, 8d nigeriensis 43 guiyangiensis 9 tolworthi 44 higo 10a, 10b darmstadiensis 45 roskildensis 10a, 10c londrina 46 chanpaisis 11a, 11b toumanoffi 47 wratislaviensis 11a, 11c kyushuensis 48 balearica 12 thompsoni 49 muju 13 pakistani 50 navarrensis 14 israelensis 51 xiaguangiensis 15 dakota 52 kim 16 indiana 53 asturiensis 17 tohokuensis 54 poloniensis 18a, 18b kumamtoensis 55 palmanyolensis 18a, 18c yosoo 56 rongseni 19 tochigiensis 57 pirenaica 20a, 20b yunnanensis 58 argentinensis 20a, 20c pondicheriensis 59 iberica 21 colmeri 60 pingluonsis 22 shanongiensis 61 sylvestriensis 23 japonensis 62 zhaodongensis 24a, 24b neolonensis 64 azorensis 24a, 24c novosibirsk 65 pulsiensis 25 coreanensis 66 graciosensis 26 silo 67 vazensis 27 mexicanensis none wuhanensis

28a, 28b monterrey

Table 1. Classification of *B.thuringiensis* strains by serotype (modified after Zeigler, 1999).

The *cryI* genes can be distinguished from the other *cry* genes simply by sequence homology (>50% aminoacid identy) and encode 130 to 140 kDa proteins which accumulate in


## **2.4 Clasification of strains of** *Bacillus thuringiensis*

*Bt* is a crystalliferous spore-forming bacterium close genetic relationship with *B. cereus, B. anthracis* and *B. mycoides* (Höffe & Whiteley, 1989). The classification of *Bt* is difficult because DNA sequencing studies of conserved gene regions of theses species have suggested that they belong to a single specie. *Bt* strains are distinguished from *B. cereus, B. anthracis* and *B. Mycoides* by the ability to produce parasporal crystalline inclusions during sporulation (de Barjac, 1978). Crystal formation is the criterion for distinguishing between *B. cereus* and *Bt*, otherwise they could be considered as the same specie. Research based on a comparative study of 16s rRNA sequences, *Bt* and *B. cereus* var. *mycoides* differed from each other and from *B. anthracis* and *cereus* by less than nine nucleotides (Ash and Collins 1992). Chen & Tsen (2002) amplified 16S rDNA and *gyrB* gene by PCR and they found that the discrimination between *B. cereus* and *Bt* strains, when a large number of Bacillus strains were tested was difcult. They proposed, to distinguish *Bt* from *B. cereus*, a single feature, such as the presence of a parasporal crystal protein or cry gene is reliable.

Several attemps were made to classify *Bt* strains. de Barjac and Frachon (1990) didn´t find correlation between biochemical reactions and 27 H serotypes using 1,600 *B. thuringiensis* isolates. They demonstrated that the current biochemical tests have no value as the sole criteria for differentiating *Bt* strains. Another approach ot these authors for classification was the use of susceptibility to certain bacterial viruses called phages. There are 14 bacteriophages that have been used for *Bt* but phage typing is inconsistent with serotyping and does not permit classification. They found frequent cross-reactions.

One widely clasification system for *Bt* strains is based in the determination of the H-flagellar antigen technique described by Barjac & Bonnefoi (1962). This technique needs very motile bacterial cultures to prepare flagellar suspensions. These suspensions are titrated against antisera directed against *B. thuringiensis* strains of each serotype. Presently *B. thuringiensis* strains are classified within 79 serotypes. Table 1 shows the classification of *Bt* strains by serotypes.

*Bt* strains that had discovered previously to 1977 were pathogens towards lepidopteran larvae. However, in 1975 was discovered *B. thuringiensis* serovar *israelensis* toxic to mosquito larvae (Goldberg & Margarit 1977) and in 1983 a strain from *Bt* serovar *morrisoni* was found to be pathogenic to Coleoptera larvae (Krieg et al. 1983). According to these findings, serological clasification, although still in use as a basic method to clasify *Bt* strains could not be related with pathogenicity. Subsequently studies showed that, within a serotype, different activity, spectra can be found. For example, some strains of *B. thuringiensis* serotype *morrisoni* in their parasporal inclusión bodies contain different proteins and have activity against Diptera, Coleoptera or Lepidoptera.

With the knowledge of sequence of the genes that encode the proteins cry it was proposed a classification based on the cry toxin genes. Höfte and Whiteley (1989) proposed a nomenclature clasification scheme for *Bt* crystal proteins based in their structural aminoacid sequence, deduced from the DNA and host range. They named *cry* (crystal protein) genes and their related proteins, "Cry proteins". They clasified 42 *Bt* crystal protein genes into 14 distinct genes grouped into 4 major clases. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera-and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Dipter-specific).

Activity dependent of ingestion, and feeding activity depends on environmental

*Bt* is a crystalliferous spore-forming bacterium close genetic relationship with *B. cereus, B. anthracis* and *B. mycoides* (Höffe & Whiteley, 1989). The classification of *Bt* is difficult because DNA sequencing studies of conserved gene regions of theses species have suggested that they belong to a single specie. *Bt* strains are distinguished from *B. cereus, B. anthracis* and *B. Mycoides* by the ability to produce parasporal crystalline inclusions during sporulation (de Barjac, 1978). Crystal formation is the criterion for distinguishing between *B. cereus* and *Bt*, otherwise they could be considered as the same specie. Research based on a comparative study of 16s rRNA sequences, *Bt* and *B. cereus* var. *mycoides* differed from each other and from *B. anthracis* and *cereus* by less than nine nucleotides (Ash and Collins 1992). Chen & Tsen (2002) amplified 16S rDNA and *gyrB* gene by PCR and they found that the discrimination between *B. cereus* and *Bt* strains, when a large number of Bacillus strains were tested was difcult. They proposed, to distinguish *Bt* from *B. cereus*, a single feature,

Several attemps were made to classify *Bt* strains. de Barjac and Frachon (1990) didn´t find correlation between biochemical reactions and 27 H serotypes using 1,600 *B. thuringiensis* isolates. They demonstrated that the current biochemical tests have no value as the sole criteria for differentiating *Bt* strains. Another approach ot these authors for classification was the use of susceptibility to certain bacterial viruses called phages. There are 14 bacteriophages that have been used for *Bt* but phage typing is inconsistent with serotyping

One widely clasification system for *Bt* strains is based in the determination of the H-flagellar antigen technique described by Barjac & Bonnefoi (1962). This technique needs very motile bacterial cultures to prepare flagellar suspensions. These suspensions are titrated against antisera directed against *B. thuringiensis* strains of each serotype. Presently *B. thuringiensis* strains are

*Bt* strains that had discovered previously to 1977 were pathogens towards lepidopteran larvae. However, in 1975 was discovered *B. thuringiensis* serovar *israelensis* toxic to mosquito larvae (Goldberg & Margarit 1977) and in 1983 a strain from *Bt* serovar *morrisoni* was found to be pathogenic to Coleoptera larvae (Krieg et al. 1983). According to these findings, serological clasification, although still in use as a basic method to clasify *Bt* strains could not be related with pathogenicity. Subsequently studies showed that, within a serotype, different activity, spectra can be found. For example, some strains of *B. thuringiensis* serotype *morrisoni* in their parasporal inclusión bodies contain different proteins and have

With the knowledge of sequence of the genes that encode the proteins cry it was proposed a classification based on the cry toxin genes. Höfte and Whiteley (1989) proposed a nomenclature clasification scheme for *Bt* crystal proteins based in their structural aminoacid sequence, deduced from the DNA and host range. They named *cry* (crystal protein) genes and their related proteins, "Cry proteins". They clasified 42 *Bt* crystal protein genes into 14 distinct genes grouped into 4 major clases. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera-and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Dipter-specific).

classified within 79 serotypes. Table 1 shows the classification of *Bt* strains by serotypes.

Relatively higher cost compared to chemical insecticides

such as the presence of a parasporal crystal protein or cry gene is reliable.

and does not permit classification. They found frequent cross-reactions.

activity against Diptera, Coleoptera or Lepidoptera.

**2.4 Clasification of strains of** *Bacillus thuringiensis* 

conditions


Table 1. Classification of *B.thuringiensis* strains by serotype (modified after Zeigler, 1999).

The *cryI* genes can be distinguished from the other *cry* genes simply by sequence homology (>50% aminoacid identy) and encode 130 to 140 kDa proteins which accumulate in

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 245

*Bti* is highly pathogenic against *Culicidae* (mosquitoes ) and *Simuliidae* (blackflies), and has some virulence against certain others Diptera, especially Chironomidae (midges). Mosquito have four distinct stages in their life cycle: egg, larva, pupa and adult. Depending on the specie a female lays between 30 and 300 eggs at a time on the surface of the water, singly (*Anopheles*), in floating rafts (*Culex*) or just above the water line or on wet mud (*Aedes*). Once hatched the larvae grow in four different stages (instars). The first instar measures 1.5 mm in length, the fourth instar about 8-10 mm. The fully grown larvae then changes into a comma shaped pupa. When mature, the pupal skin splits at one end and a fully developed adult emerges. The entire period from egg to adult takes about 7-13 days under good conditions (Wada, 1989). First instar is more suceptible to *Bti* than fourth instar (Mulla *et al.* 1990). Pupa does not feed and therefore is not affected by *Bti*. For almost all species tested, increasing age of the larvae resulted in reduced susceptibility in mosquito (Chen et al. 1984: Mulla *et al.*

**3.3 Susceptibility of mosquito species to** *B. thuringiensis* **serotype** *israelensis* 

1985). *Bti* was found to be specific toxic to larvae of 109 mosquito species (Table 3).

Total 109

Mosquito genus Species *Aedes* 40 *Anopheles* 27 *Culex* 19 *Culiseta* 5 *Mansonia* 5 *Psorophora* 3 *Armigeres* 3 *Toxorhynchites* 2 *Limatus* 2 *Trichophospon* 1 *Uranotaenia* 1 *Tripteroides* 1

Table 3. Larvicidal activity of *Bti* on mosquito species (Glare & O´Callaghan 1998).

*aegypti* larvae (Goldberg & Margalit 1977; Nugud & White 1982)

Among mosquitoes, different preparations of *Bti* have shown different levels of toxicity to host species. Others factors influencing the susceptibility of mosquito larva to *Bti*. For example, the effect of a given dosage of toxin could produce different results depending on weather the lethal dose is administered all at once or in same doses over a long period (Aly et al. 1988). *Bti* has an LC50 in the range of 10–13 ng/ml against the fourth instar of many mosquito species (Federici *et al.* 2003). Generally, *Culex* and *Aedes* are highly susceptible while *Anopheles* are less suceptible, but can be killed with *Bti* (Balaraman *et al*. 1983). Much higher concentrations of *Bti* are required to induce mortality in anopheline larvae than in *Aedes* 

The exception is *A. franciscanus* that is as susceptible as other genera (García *et al.* 1980, Sun *et al.* 1980). Furhtermore, even within one genus, some species are more suceptible than others (Chui et al. 1993). Sun et al. (1980) suggested that a difference in feeding behavior might account for differences in susceptibilities. Filtering rates vary between genera and species. For example Aly (1988) showed that in the absence of *Bti*, larvae cleared the suspensions with constant relative filtration rates of 632 (*Ae. aegypti*), 515 (*Cx*. *quinquefasciatus*) or 83.9

bypiramidal crystalline inclusions during the sporulation of *Bt*. *cryII* genes encode 65-kDa proteins which form cuboidal inclusions in strains of several species. *cryIII* genes produces romboidal crystals containing one major protein, a 72kDa protein. cryIV class genes (*cryIVA*, cryIVB, cryIVC, and cryIVD) as well as *cytA* were all isolated from the same 72-Mda plasmid present in *Bt* subsp. *israelensis.* 

## **3.** *Bacillus thuringiensis* **subsp.** *israelensis* **as an important part of mosquito control**

## **3.1 Properties of** *Bti*

In 1975-76 under a World Health Organization sponsored project, a new *Bt* strain was discovered in the Negev desert in Israel by Goldberg and Margalit (1977). The strain was isolated from *Culex* sp. dead larvae mosquito. Later was identified as *Bt israelensis*, serotype H14 according to its fagellar antigenicity by de Barjac (1978).

*Bti* has all the features taxonomic, morphological, growth, sporulation, of isolation, cultivation of other varieties of *B. thuringiensis* (Fig.1)*.* The larvicidal activity of *Bt israelensis*  on mosquito transmitted diseases was the most important feature of the strain. The insecticidal properties of this bacteria are due primarily to insecticidal proteins produced during sporulation. The key proteins are Cyt1A(27.3·kDa), Cry4A (128·kDa), Cry4B (134·kDa) and Cry11A (72·kDa) and in three different inclusion types assembled into a spherical parasporal body held together by lamellar envelope (Ibarra & Federici, 1986). The inclusions are realtively small (0.1 to 0.5m) and there are usually two to four inclusions per cell which vary in shape from cuboidal to bipyramidal, ovoid or anamorph (Charles & de Barjac 1982; Mikola *et al.* 1982; Yamamoto *et al.* 1983)

## **3.2 Mosquitoes, important vectors of tropical diseases**

Mosquitoes are important vectors of several tropical diseases that suck blood from human and animals. They are vectors of multiple of diseases of man through transmision of pathogenic viruses, bacteria, protozoa and nematodes (Priest, 1992). From the medical point of view, mosquitoes are among the most important insects due their capacity to transmit human diseases such as malaria and dengue.


Table 2. Some diseases transmitted by mosquito (Rawlins, 1989; Walsh 1986).

There are about 3000 species of mosquito, of which about 100 are vectors of human diseases. Some of the more important of these diseases are listed in Table 2. It is estimated two billion people worldwide living in areas where these are endemic (World Health Organization, 1999). Thus, there is an urgent need for new agents and strategies to control these diseases.

bypiramidal crystalline inclusions during the sporulation of *Bt*. *cryII* genes encode 65-kDa proteins which form cuboidal inclusions in strains of several species. *cryIII* genes produces romboidal crystals containing one major protein, a 72kDa protein. cryIV class genes (*cryIVA*, cryIVB, cryIVC, and cryIVD) as well as *cytA* were all isolated from the same 72-Mda

**3.** *Bacillus thuringiensis* **subsp.** *israelensis* **as an important part of mosquito** 

In 1975-76 under a World Health Organization sponsored project, a new *Bt* strain was discovered in the Negev desert in Israel by Goldberg and Margalit (1977). The strain was isolated from *Culex* sp. dead larvae mosquito. Later was identified as *Bt israelensis*, serotype

*Bti* has all the features taxonomic, morphological, growth, sporulation, of isolation, cultivation of other varieties of *B. thuringiensis* (Fig.1)*.* The larvicidal activity of *Bt israelensis*  on mosquito transmitted diseases was the most important feature of the strain. The insecticidal properties of this bacteria are due primarily to insecticidal proteins produced during sporulation. The key proteins are Cyt1A(27.3·kDa), Cry4A (128·kDa), Cry4B (134·kDa) and Cry11A (72·kDa) and in three different inclusion types assembled into a spherical parasporal body held together by lamellar envelope (Ibarra & Federici, 1986). The inclusions are realtively small (0.1 to 0.5m) and there are usually two to four inclusions per cell which vary in shape from cuboidal to bipyramidal, ovoid or anamorph (Charles & de

Mosquitoes are important vectors of several tropical diseases that suck blood from human and animals. They are vectors of multiple of diseases of man through transmision of pathogenic viruses, bacteria, protozoa and nematodes (Priest, 1992). From the medical point of view, mosquitoes are among the most important insects due their capacity to transmit

*Culex* Lymphatic filariasis, Japanese encephalitis,

There are about 3000 species of mosquito, of which about 100 are vectors of human diseases. Some of the more important of these diseases are listed in Table 2. It is estimated two billion people worldwide living in areas where these are endemic (World Health Organization, 1999). Thus, there is an urgent need for new agents and strategies to control these diseases.

other viral diseases

filariasis

Yellow fever, dengue, dengue hemorrhagic fever, other viral diseases, lymphatic

plasmid present in *Bt* subsp. *israelensis.* 

H14 according to its fagellar antigenicity by de Barjac (1978).

Barjac 1982; Mikola *et al.* 1982; Yamamoto *et al.* 1983)

human diseases such as malaria and dengue.

**3.2 Mosquitoes, important vectors of tropical diseases** 

**Vector Disease** 

*Anopheles* Malaria, lymphatic filariasis

Table 2. Some diseases transmitted by mosquito (Rawlins, 1989; Walsh 1986).

*Mansonia* Lymphatic filariasis

**control** 

*Aedes* 

**3.1 Properties of** *Bti* 

## **3.3 Susceptibility of mosquito species to** *B. thuringiensis* **serotype** *israelensis*

*Bti* is highly pathogenic against *Culicidae* (mosquitoes ) and *Simuliidae* (blackflies), and has some virulence against certain others Diptera, especially Chironomidae (midges). Mosquito have four distinct stages in their life cycle: egg, larva, pupa and adult. Depending on the specie a female lays between 30 and 300 eggs at a time on the surface of the water, singly (*Anopheles*), in floating rafts (*Culex*) or just above the water line or on wet mud (*Aedes*). Once hatched the larvae grow in four different stages (instars). The first instar measures 1.5 mm in length, the fourth instar about 8-10 mm. The fully grown larvae then changes into a comma shaped pupa. When mature, the pupal skin splits at one end and a fully developed adult emerges. The entire period from egg to adult takes about 7-13 days under good conditions (Wada, 1989). First instar is more suceptible to *Bti* than fourth instar (Mulla *et al.* 1990). Pupa does not feed and therefore is not affected by *Bti*. For almost all species tested, increasing age of the larvae resulted in reduced susceptibility in mosquito (Chen et al. 1984: Mulla *et al.* 1985). *Bti* was found to be specific toxic to larvae of 109 mosquito species (Table 3).


Table 3. Larvicidal activity of *Bti* on mosquito species (Glare & O´Callaghan 1998).

Among mosquitoes, different preparations of *Bti* have shown different levels of toxicity to host species. Others factors influencing the susceptibility of mosquito larva to *Bti*. For example, the effect of a given dosage of toxin could produce different results depending on weather the lethal dose is administered all at once or in same doses over a long period (Aly et al. 1988).

*Bti* has an LC50 in the range of 10–13 ng/ml against the fourth instar of many mosquito species (Federici *et al.* 2003). Generally, *Culex* and *Aedes* are highly susceptible while *Anopheles* are less suceptible, but can be killed with *Bti* (Balaraman *et al*. 1983). Much higher concentrations of *Bti* are required to induce mortality in anopheline larvae than in *Aedes aegypti* larvae (Goldberg & Margalit 1977; Nugud & White 1982)

The exception is *A. franciscanus* that is as susceptible as other genera (García *et al.* 1980, Sun *et al.* 1980). Furhtermore, even within one genus, some species are more suceptible than others (Chui et al. 1993). Sun et al. (1980) suggested that a difference in feeding behavior might account for differences in susceptibilities. Filtering rates vary between genera and species. For example Aly (1988) showed that in the absence of *Bti*, larvae cleared the suspensions with constant relative filtration rates of 632 (*Ae. aegypti*), 515 (*Cx*. *quinquefasciatus*) or 83.9

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 247

One major problem with insects control via chemical insecticides is the evolution in insects of resistance to those insecticides. The use of *Bti* on biological control of mosquitoes has no resulted in the development of resistence in host populations. Laboratory attemps to induce resistance by continual exposure to *Bti* have generally failed to detect resistence (Lee &

The lack of resistance development to *Bti* could be due to its complex mode of action, involving synergistic interactions between up to four proteins (Becker & Maragrit 1993). Use of a single protein from *Bti* for mosquito control resulted in resistance after only a few generations in the laboratory (Becker and Margalit 1993). Georghiou & Wirth (1997) also showed that resistance could be raised in only a few generations when single *Bti* toxin was used (i.e. Cry 4Aa, 4Ba, 10Aa or 11Aa), and was progressively more difficult to raise in mosquitoes with combinations of two, and three toxins. When all four *Bti* toxins were used, resistance incidence was remarkably low. On the other hand Wirth et al. (2005) have shown that the lack of resistance in *Bti* is due to the presence of the Cyt1Aa protein in the crystal. For example, *Culex quinquefascitus* populations resistant to CryIVA, Cry4B and Cry11A have been obtained in the laboratory but not mosquito larvae resistant to Cry and Cyt1Aa

*Bti* produces four crystal proteins Cry (4Aa, 4Ba, 10Aa, and 11Aa) and two Cyt (1Aa and 2Ba) (Guerchicoff *et al.* 1997). No single crystal component is as toxic as the intact crystal complex (Chan *et al.* 1993; Wu *et al.* 1994; Chilcott & Ellar, 1998). One possible explanation for this is that two or more proteins act synergisticall, yielding a higher activity than would be expected on the basis of the specific toxicity of the individual protein (Finney, 1971). For example, the toxicity against mosquito larvae of Cyt1Aa is lower compared to each of the four Cry proteins (Crickmore et al. 1995). However, cytA can potentiate the activity of the toxins and synergistic interactions that seems to account for the high toxicity of the *Bti*

Tabashnick (1992) proposed a method to measure synergistic effect. Using the proposed method discuss the data reported by two authors: in bioassays with *A. aegypti* larvae, Wu and Chang (1985) found that mixtures of the 27- and 65-kDa proteins from *B. thuringiensis* subsp. *israelensis* were more toxic than expected on the basis of their individual toxicities, however, Chilcott and Ellar (1988) concluded from their own data that no synergism between these two proteins occurred. With this new interpretation of Tabashnick method, both studies support the same conclusions: positive synergism between the 27-kDa protein (CytA) and either of the CryIV proteins (65 and 130 kDa) and no such synergism between CryIV proteins (65 and 130 kDa). Other studies have been carried out with the aim of increasing the synergistic activity of *Bti.* Ramirez-Suero et al. (2011) evaluated the synergistic effect of *S. griseus* and *Bt aizawai* chitinases with *Bt israelensis* spore-toxin complex against *Aedes aegypti* larvae. The synergistic factor values according to Tabashnik

*Bti* has no direct effect on aquatic organisms other than mosquitoes, blackflies and chironomids. Other aquatic organisms, such as shrimps, mites and oysters are generally unaffected (Glare & O'Callaghan, 1998). This large safety margin of preparations of *Bti* for

**3.5 Resistance** 

Chong 1985; Georghiou & Wirth 1997).

proteins (Georghiou & Wirth 1997)

strains (Delecluse et al. 1993).

(1992) method were 2 and 1.4, respectively.

**3.7 Effect of** *Bti* **on no-target organisms** 

**3.6 Synergism** 

L/Larvae/h (*An. albimanus*). Another factor to be considered for the suceptibility to *Bti* is the behavior of the larvae. *Anopheles* larvae filter-feed on food particles present at the surface of water or a few centimeters below it, where as *Culex* and *Aedes* larvae not only feed faster but are capable of filter-feeding at much deeper water dephts (Aly et al. 1988).

Mosquito species which are not filter feeders do not seem suceptible. For example, against *Culicoides occidentalis* (Colwell 1982) and *Coquillettidia perturbans*, *Bti* larvicides had no effect (Walker et al 1985). Solubilization of the protein reduced the toxicity dramatically (50- to - 100 fold) this is attributed to reduced level of toxin ingestion by larvae owing to their filter feeding behaviour (Chungjatupornchai et al. 1988).

## **3.4 Susceptibility of mosquito species to purified** *Bt* **proteic crystals**

Several efforts have been made to purify the proteins Cry of *Bti* and other *Bt* subps. with the purpose of studying the chemistry of Cry proteins, the synergism between them, and the effect of each crystal on larvae of different mosquito species. It is somewhat difficult to separate the *Bt* spores and crystals because they are of similar size and surface characteristics. For that reason several methods have been used to purify *Bt* crystals proteins. Using NaBr gradients (Chang et al. 1992), sucrose gradients (Debro et al. 1986), renografin gradients (Aronson et al. 1991), and in a separatory funnels (Delafield et al. 1968). Bioassays of purified Cry proteins have been allowed to know that not only *Bti* Cry proteins have activity on mosquito larva. Other purified Cry proteins of other *Bt* strains have also activity on mosquito larvae. Table 4 shows the mosquito toxicity of purified crystals of some *Bt* strains.



## **3.5 Resistance**

246 Insecticides – Pest Engineering

L/Larvae/h (*An. albimanus*). Another factor to be considered for the suceptibility to *Bti* is the behavior of the larvae. *Anopheles* larvae filter-feed on food particles present at the surface of water or a few centimeters below it, where as *Culex* and *Aedes* larvae not only feed faster but

Mosquito species which are not filter feeders do not seem suceptible. For example, against *Culicoides occidentalis* (Colwell 1982) and *Coquillettidia perturbans*, *Bti* larvicides had no effect (Walker et al 1985). Solubilization of the protein reduced the toxicity dramatically (50- to - 100 fold) this is attributed to reduced level of toxin ingestion by larvae owing to their filter

Several efforts have been made to purify the proteins Cry of *Bti* and other *Bt* subps. with the purpose of studying the chemistry of Cry proteins, the synergism between them, and the effect of each crystal on larvae of different mosquito species. It is somewhat difficult to separate the *Bt* spores and crystals because they are of similar size and surface characteristics. For that reason several methods have been used to purify *Bt* crystals proteins. Using NaBr gradients (Chang et al. 1992), sucrose gradients (Debro et al. 1986), renografin gradients (Aronson et al. 1991), and in a separatory funnels (Delafield et al. 1968). Bioassays of purified Cry proteins have been allowed to know that not only *Bti* Cry proteins have activity on mosquito larva. Other purified Cry proteins of other *Bt* strains have also activity on mosquito

are capable of filter-feeding at much deeper water dephts (Aly et al. 1988).

**3.4 Susceptibility of mosquito species to purified** *Bt* **proteic crystals** 

larvae. Table 4 shows the mosquito toxicity of purified crystals of some *Bt* strains.

**Name Source Strain Mosquito toxicity**

Cry4Aa1 *B.t. israelensis* 4Q2-72 *Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Cry11Aa1 *B.t. israelensis* HD-567 *Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Cry11Ba1 *B.t. jegathensan* 367 *Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Cry11Bb1 *B.t. medellin Aedes agypti, Anopheles albimanus*, *Culex* 

Cry19Aa1 *B.t. jegethesan Anopheles stephensi*, *Culex pipiens* (Diptera:

Cyt1Ab1 *B.t. medellin* 163-131 *Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Cyt2Aa1 *B.t. kyushuensis Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Table 4. Mosquitocidal activity of Cry and Cyt proteins (Modified after "Zeigler, 1999").

Cry20Aa1 *B.t. fukuokaensis Aedes agypti,* (Diptera: Cuclidae) Cry21Aa1 *B.t. higo Culex pipiens molestus* (Diptera: Cuclidae) Cyt1Aa1 *B.t. israelensis* IPS82 *Aedes agypti, Anopheles stephensi*, *Culex pipiens* 

Cry4Ba1 *B.t. israelensis* 4Q2-72 *Aedes agypti* (Diptera: Cuclidae) Cry10Aa1 *B.t. israelensis* ONR60A *Aedes agypti,* (Diptera: Cuclidae)

(Diptera: Cuclidae)

(Diptera: Cuclidae)

(Diptera: Cuclidae)

*quinquefasciatus* (Diptera: Cuclidae)

*Aedes agypti, Anopheles stephensi*, *Culex pipiens*  (Diptera: Cuclidae)

Cuclidae)

(Diptera: Cuclidae)

(Diptera: Cuclidae)

(Diptera: Cuclidae)

feeding behaviour (Chungjatupornchai et al. 1988).

Cry16Aa1 *Clostridium bifermentans* 

*malasya* CH18

One major problem with insects control via chemical insecticides is the evolution in insects of resistance to those insecticides. The use of *Bti* on biological control of mosquitoes has no resulted in the development of resistence in host populations. Laboratory attemps to induce resistance by continual exposure to *Bti* have generally failed to detect resistence (Lee & Chong 1985; Georghiou & Wirth 1997).

The lack of resistance development to *Bti* could be due to its complex mode of action, involving synergistic interactions between up to four proteins (Becker & Maragrit 1993). Use of a single protein from *Bti* for mosquito control resulted in resistance after only a few generations in the laboratory (Becker and Margalit 1993). Georghiou & Wirth (1997) also showed that resistance could be raised in only a few generations when single *Bti* toxin was used (i.e. Cry 4Aa, 4Ba, 10Aa or 11Aa), and was progressively more difficult to raise in mosquitoes with combinations of two, and three toxins. When all four *Bti* toxins were used, resistance incidence was remarkably low. On the other hand Wirth et al. (2005) have shown that the lack of resistance in *Bti* is due to the presence of the Cyt1Aa protein in the crystal. For example, *Culex quinquefascitus* populations resistant to CryIVA, Cry4B and Cry11A have been obtained in the laboratory but not mosquito larvae resistant to Cry and Cyt1Aa proteins (Georghiou & Wirth 1997)

## **3.6 Synergism**

*Bti* produces four crystal proteins Cry (4Aa, 4Ba, 10Aa, and 11Aa) and two Cyt (1Aa and 2Ba) (Guerchicoff *et al.* 1997). No single crystal component is as toxic as the intact crystal complex (Chan *et al.* 1993; Wu *et al.* 1994; Chilcott & Ellar, 1998). One possible explanation for this is that two or more proteins act synergisticall, yielding a higher activity than would be expected on the basis of the specific toxicity of the individual protein (Finney, 1971). For example, the toxicity against mosquito larvae of Cyt1Aa is lower compared to each of the four Cry proteins (Crickmore et al. 1995). However, cytA can potentiate the activity of the toxins and synergistic interactions that seems to account for the high toxicity of the *Bti* strains (Delecluse et al. 1993).

Tabashnick (1992) proposed a method to measure synergistic effect. Using the proposed method discuss the data reported by two authors: in bioassays with *A. aegypti* larvae, Wu and Chang (1985) found that mixtures of the 27- and 65-kDa proteins from *B. thuringiensis* subsp. *israelensis* were more toxic than expected on the basis of their individual toxicities, however, Chilcott and Ellar (1988) concluded from their own data that no synergism between these two proteins occurred. With this new interpretation of Tabashnick method, both studies support the same conclusions: positive synergism between the 27-kDa protein (CytA) and either of the CryIV proteins (65 and 130 kDa) and no such synergism between CryIV proteins (65 and 130 kDa). Other studies have been carried out with the aim of increasing the synergistic activity of *Bti.* Ramirez-Suero et al. (2011) evaluated the synergistic effect of *S. griseus* and *Bt aizawai* chitinases with *Bt israelensis* spore-toxin complex against *Aedes aegypti* larvae. The synergistic factor values according to Tabashnik (1992) method were 2 and 1.4, respectively.

## **3.7 Effect of** *Bti* **on no-target organisms**

*Bti* has no direct effect on aquatic organisms other than mosquitoes, blackflies and chironomids. Other aquatic organisms, such as shrimps, mites and oysters are generally unaffected (Glare & O'Callaghan, 1998). This large safety margin of preparations of *Bti* for

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 249

gained or lost (Federici, 1999). The mere presence of plasmids in *Bt* does not prove that they are involved in crystal formation, many non-crystalliferous bacteria also contain plasmids The *cry* genes are located on large plasmids although some Cry genes have been reported on the chromosome (Baume & Malvar, 1995). As mentioned earlier, *Bti* produces four different Cry proteins: CryIVA, CryIVB, Cry11A, and the cytolytic CytA protein (Hoffe and Whiteley, 1989). The Cry proteins are codified by *cryIVA*, c*ryIVB*, *cry11A*, and *cytA* genes, respectively. These genes responsible for the toxicity of *Bti* have been sequenced by various researchers

**Coding Region Reference** 

(Table 5).

**Gene name GenBank** 

Table 5. *cry* and *cytA* genes DNA sequences of *Bti.* 

*Bti* (Modified after Ben-Dov *et al.* 1996).

**5.2** *Bti* **operon of** *cryIVD* **gene** 

**Accesion No.** 

*cryIVAa1* Y00423 1-3540 Ward &Ellar, 1987 *cry4Aa2* D00248, E01676 393-3935 Sen *et al*. 1988

*cry4Ba4* D00247, E01905 461-3865 Sen *et al*. 1988 *cry11Aa1* M31737 41-1969 Donovan *et al.* 1989 *cry11Aa2* M22860 1-235 Adams *et al.* 1989 *cyt1Aa1* X03182 140-886 Waalwijk *et al.* 1985 *cyt1Aa2* X04338 509-1255 Ward & Ellar, 1986 *cyt1Aa3* Y00135 36-782 Earp a& Ellar, 1987 *cyt1Aa4* M35968 67-813 Galjart *et al*. 1987

(Aronson 1993). Figure 2 shows the partial map of the *Bti* 125 kb plasmid.

*cry4Ba1* X07423, X05692 157-3564 Chungjatupornchai *et al*. 1988 *cry4Ba2* X07082 151-3558 Tungpradubkul *et al.* 1988 *cry4Ba3* M20242 526-3930 Yamamoto *et al.* 1988

In *Bti* the elements responsible of the toxicity against mosquito larvae are located in a large plasmid of 72 MDa (125 kb) and contribute to the formation of a complex parasporal body

> *Sac*I *Hind*III **~26 kb**

Fig. 2. Map of 26kb DNA fragment carrying the DNA genes responsibles of the toxicity of

*cryIVA cryIVB cryIVC orf2 p20 cryIVD p19* 

The DNA sequence indicated that *cryIVD* gene is the second gene of an operon which includes three genes. A gene that encodes a 19 kDa polypeptide, *cryIVD* gene and a gene that encodes a 20 kDa polypeptide (Dervyn et al. 1995). Transcription of *cryIVD* gene in *Bti* is induced 9 h after the beginning of the sporulation. DNA sequence analysis and potential promoters are recognized by the RNA polimerase associated with the 35 and 28 , specific sigma subunit of the RNA polymerase genes related with control of sporulation of *B.* 

non-target organisms indicate their suitability for mosquito control programs in areas where protection of the natural ecosystem is important (Sinegre et al. 1980)

Several authors have reviewed the non target effects of *Bti* (Becker & Margalit 1993; Lacey & Mulla 1990). Field applications have often been monitored for effects on non-target organisms but no significant non-target effects have been reported (Ali, 1981; Jackson et al. 1994; Hershey et al. 1995)

## **4. Production of** *Bti* **by fermentation**

#### **4.1 Culture medium for** *Bti* **production**

Commercial production of *Bti* is performed using culture media based on complex nutrients sources. The main purpose of the fermentation is to obtain high quantities of *Bti* crystals. The *Bti* parasporal crystal can account for up to 25% of the sporulated cell dry weight. To optimize the cry production it is necessary to have a suitable culture medium because the toxicity obtained at the end of the fermentation depend on the culture medium and operating conditions. The culture media that have been reported in the literature for high growth and sporulation can be used for any variety of *Bt*. Not always a high cell growth ensures an elevated Cry protein production or an increased insecticidal activity. Various culture mediums have been used for high growth and sporulation of *Bt* in the laboratory: 2XSG medium (Leighton & Doi, 1971), PA medium (Thorne, 1968), G-Tris medium (Aronson et al. 1971), CDGS medium (Nakata, 1964). Other media with inexpensive substrates have been reported by Pearson & Ward (1988), Smith (1982), Foda et al. (1985), Dulmage et al. (1970), Salama et al. (1983), Goldberg et al. (1980).

## **4.2 Factors affecting Cry production**

There are several factors that influence the production of crystals: (1) Carbon source. Glucose is the most appropiate carbon source either for high *Bt* growth and sporulation (Smith, 1982). When glucose has been exhausted in the fermentation, the abscence of this can trigger sporulation. The use of one or other carbon source affects the biological activity and the morphology of the crystals (Dulmage, 1970). (2) Nitrogen source. An appropiate source of aminoacids provides high growth rates and high sporulation of *Bt* strains. Its absence delays sporulation and low yield in Cry proteins (Goldberg et al. 1980) (3) Carbon:Nitrogen ratio. Higher C:N rates glucose do not deplete at the end of fermentation and biomass yield decrease. Several authors have recomended a carbon nitrogen ratio of 7.5:1. (Salama et al. 1983; Foda et al. 1985) (4) Oxygen. High aeration rates are important for high spore and toxin formation. As kLa, increase biomass and Cry protein formation are increased (Rowe & Margaritis, 1987)(5) pH. Optimum pH for *Bt* growth is 6.8-7.2. If pH rises to 9.0 Cry protein can be dissolved (6) Temperature. Optimum temperature of *Bt* is 28-32C. Higher temperatures favours plasmid losses or *Bt* mutants (Rowe & Margaritis, 1987).

## **5. Molecular biology of** *Bti*

#### **5.1** *Bti cry* **and** *cytA* **genes**

All *Bt* strains contain extrachromosomal DNA. *Bt* strains are well known for its numerous plasmids ranging in size from 1.5 MDa to 130 MDa. Plasmids have been found in each variety examined and the plasmid profiles appear to be strain specific. However these plasmid profiles depend on the media type and growth rate of the strain and can be readily

non-target organisms indicate their suitability for mosquito control programs in areas where

Several authors have reviewed the non target effects of *Bti* (Becker & Margalit 1993; Lacey & Mulla 1990). Field applications have often been monitored for effects on non-target organisms but no significant non-target effects have been reported (Ali, 1981; Jackson et al.

Commercial production of *Bti* is performed using culture media based on complex nutrients sources. The main purpose of the fermentation is to obtain high quantities of *Bti* crystals. The *Bti* parasporal crystal can account for up to 25% of the sporulated cell dry weight. To optimize the cry production it is necessary to have a suitable culture medium because the toxicity obtained at the end of the fermentation depend on the culture medium and operating conditions. The culture media that have been reported in the literature for high growth and sporulation can be used for any variety of *Bt*. Not always a high cell growth ensures an elevated Cry protein production or an increased insecticidal activity. Various culture mediums have been used for high growth and sporulation of *Bt* in the laboratory: 2XSG medium (Leighton & Doi, 1971), PA medium (Thorne, 1968), G-Tris medium (Aronson et al. 1971), CDGS medium (Nakata, 1964). Other media with inexpensive substrates have been reported by Pearson & Ward (1988), Smith (1982), Foda et al. (1985),

There are several factors that influence the production of crystals: (1) Carbon source. Glucose is the most appropiate carbon source either for high *Bt* growth and sporulation (Smith, 1982). When glucose has been exhausted in the fermentation, the abscence of this can trigger sporulation. The use of one or other carbon source affects the biological activity and the morphology of the crystals (Dulmage, 1970). (2) Nitrogen source. An appropiate source of aminoacids provides high growth rates and high sporulation of *Bt* strains. Its absence delays sporulation and low yield in Cry proteins (Goldberg et al. 1980) (3) Carbon:Nitrogen ratio. Higher C:N rates glucose do not deplete at the end of fermentation and biomass yield decrease. Several authors have recomended a carbon nitrogen ratio of 7.5:1. (Salama et al. 1983; Foda et al. 1985) (4) Oxygen. High aeration rates are important for high spore and toxin formation. As kLa, increase biomass and Cry protein formation are increased (Rowe & Margaritis, 1987)(5) pH. Optimum pH for *Bt* growth is 6.8-7.2. If pH rises to 9.0 Cry protein can be dissolved (6) Temperature. Optimum temperature of *Bt* is 28-32C. Higher

temperatures favours plasmid losses or *Bt* mutants (Rowe & Margaritis, 1987).

All *Bt* strains contain extrachromosomal DNA. *Bt* strains are well known for its numerous plasmids ranging in size from 1.5 MDa to 130 MDa. Plasmids have been found in each variety examined and the plasmid profiles appear to be strain specific. However these plasmid profiles depend on the media type and growth rate of the strain and can be readily

protection of the natural ecosystem is important (Sinegre et al. 1980)

Dulmage et al. (1970), Salama et al. (1983), Goldberg et al. (1980).

1994; Hershey et al. 1995)

**4. Production of** *Bti* **by fermentation 4.1 Culture medium for** *Bti* **production** 

**4.2 Factors affecting Cry production** 

**5. Molecular biology of** *Bti*  **5.1** *Bti cry* **and** *cytA* **genes** 

gained or lost (Federici, 1999). The mere presence of plasmids in *Bt* does not prove that they are involved in crystal formation, many non-crystalliferous bacteria also contain plasmids The *cry* genes are located on large plasmids although some Cry genes have been reported on the chromosome (Baume & Malvar, 1995). As mentioned earlier, *Bti* produces four different Cry proteins: CryIVA, CryIVB, Cry11A, and the cytolytic CytA protein (Hoffe and Whiteley, 1989). The Cry proteins are codified by *cryIVA*, c*ryIVB*, *cry11A*, and *cytA* genes, respectively. These genes responsible for the toxicity of *Bti* have been sequenced by various researchers (Table 5).


Table 5. *cry* and *cytA* genes DNA sequences of *Bti.* 

In *Bti* the elements responsible of the toxicity against mosquito larvae are located in a large plasmid of 72 MDa (125 kb) and contribute to the formation of a complex parasporal body (Aronson 1993). Figure 2 shows the partial map of the *Bti* 125 kb plasmid.

Fig. 2. Map of 26kb DNA fragment carrying the DNA genes responsibles of the toxicity of *Bti* (Modified after Ben-Dov *et al.* 1996).

## **5.2** *Bti* **operon of** *cryIVD* **gene**

The DNA sequence indicated that *cryIVD* gene is the second gene of an operon which includes three genes. A gene that encodes a 19 kDa polypeptide, *cryIVD* gene and a gene that encodes a 20 kDa polypeptide (Dervyn et al. 1995). Transcription of *cryIVD* gene in *Bti* is induced 9 h after the beginning of the sporulation. DNA sequence analysis and potential promoters are recognized by the RNA polimerase associated with the 35 and 28 , specific sigma subunit of the RNA polymerase genes related with control of sporulation of *B.* 

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 251

Expression of *Bti* genes either individually or in combination in crystal-negative *Bt* or other strains have being carried on by several researchers. The genes encoding these proteins have been expressed in *Caulobacter* (Thanabalu et al. 1992), cyanobacteria (Manasherob et al. 2002; Murphy & Stevens 1992), *Escherichia coli* (McLean & Whiteley 1987; Tanapongpipat et al. 2003), *Bacillus subtilis* (Ward et al. 1986), and *Bt* (Crickmore et al. 1990). However, *Bt* toxins have been expressed as active or inactive toxins, especially when expressed in *E. coli*

Quintano et al. (2005) reported the expression of *cry11A* from *Bti* in *S. cerevisiae*. The *cry11A* gene was expressed as fusion proteins with gluthathione *S*-transferase under the control of the *S. cerevisiae HXK1* promoter. The protein was purified by affinity chromatography using gluthathione *S*-transferase–Sepharose beads. Insecticidal activity against third-instar *Aedes aegypti* larvae of the recombinant *S. cerevisiae* cell extracts (LC50 = 4.10g protein/mL) and purified GST-cry11A fusion protein (LC50 = 4.10g protein/mL ) was detected in cells grown

Servant et al. (1999) constructed a recombinant *B. sphaericus* strain containing the *cry11A* gene from *Bti*. They found an LC50 for the cry11A protein of 1.175 g/mL against fourth-instar *A. aegypti* larvae. Poncet *et al.* (1997) constructed a recombinant *B. sphaericus* strain with *cry11A* and *p20* genes integrated into the chromosome. In this case, the LC50 value reported against third- instar *A. aegypti* larvae was 0.023 g/mL. Xu *et al.* (2001) studied the expression of cry11A and *cry11A*+*p20* gene cluster in recombinant *E. coli* and *Pseudomonas putida*. They found that both recombinant bacteria contained higher levels of Cry11A protein when the adjacent *p20* gene was present on the same DNA fragment. Yamagiwa et al. (2004) reported that the solubilized cry11A protein, obtained from a nonrecombinant *Bti*, was less toxic against *Culex pipiens* larvae than the crystal itself (LC50 of 0.267 and 0.008 g/mL, respectively). These authors obtained 2 GST fusion proteins of 36 and 32 kDa from cry11A. The LC50 against *C. pipiens* larvae obtained using both proteins were 0.818 g/mL. In other study with the purpose to preserve the toxicity of sunlight-sensitive Cry proteins, Manasherob et al. (2002) constructed a transgenic cyanobacterium *Anabaena* PCC 7120 to express the genes *cry4Aa*, *cry11Aa* and an accesory protein (*p20*) under control of two tandem strong promotors. Cyanobacterium *Anabaena* can multiply in breeding sites of mosquito larvae and serve as their food source.

Higher toxicity against *Aedes aegypti* larvae was obtained in this study.

**6. Mode of action of** *Bti* **Cry proteins against mosquito larvae** 

*Bt* Cry and Cyt toxins belong to a class of bacterial toxins known as pore-forming toxins (PFT) that are secreted as water-soluble proteins undergoing conformational changes in order to insert into, or to translocate across, cell membranes of their host (Bravo et al. 2007). In most cases, PFT are activated by host proteases after receptor binding inducing the formation of an oligomeric structure that is insertion competent. Finally membrane insertion is triggered, in most cases, by a decrease in pH that induces a molten globule state of the protein (Parker and Feil, 2005). Cry and Cyt proteins are PFT proteins. Both proteins are solubilized in the gut of suceptible diperans insects and proteolytic activated by midgut proteases. For the Cry 11Aa protoxin, proteolytic activation involves amino-terminal processing and intramolecular cleavage leading to two fragments of 36 and 32 kDa that

**5.4 Expression of** *Bti* **genes in other strains** 

(Ogunjimi et al. 2002).

**6.1 Pore-forming-toxins** 

remain associated and retain insect toxicity.

in ethanol.

*thuringiensis*. *cryIVA* and *cryIVB* promotors are activated in the mid-sporulation phase (Ben-Dov et al. 1996), *cryIVA* is regulated by the 35 RNA polymerase gene and *cryIVB* is under control of 35 (Yoshisue et al. 1994). *cytA* gene is transcribed by two promotors, pBtI and pBtII, regulated by the RNA polymerase35 and 28, respectively (Brown & Whiteley, 1988, 1990). These results have demostrated that *cryIVD* transcription is subjected to <sup>35</sup> regulation.

## **5.3 Identication of** *Bacillus thuringiensis* **pesticidal crystal genes by PCR**

The polymerase chain reaction (PCR) is a molecular tool widely used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The identification of *Bt* toxin genes by PCR can partially predict the insecticidal activity of a given strain. Several studies have reported that the type of *cry* and *cyt* genes present in a *Bt* strain correlates to some extent with its insecticidal activity (Porcar & Juarez-Perez). Thus, the identification of the gene content in a *Bt* strain can be used to predict its insecticidal potential. The PCR-based identication of *B. thuringiensis* cry genes was first developed by Carozzi et al. (1991), who introduced this technique as a tool to predict insecticidal activity. They found correspondence with the toxicity predicted on the basis of the amplification product profiles. Carozzi et al. proposed PCR as an accurate, fast methodology for the identification of novel strains and the prediction of insecticidal activity of new isolates, and they also forecast the possible use of PCR for the discovery of previously unknown cry genes. highly conserved regions and recognizing entire cry gene subfamilies are often used in a preliminary screening prior to performing a second PCR with specific primers. The primers used to amplify *cry4A, cry4B, cry11A*, and *cytA* genes have been designed by various researchers and are shown in Table 6.


Table 6. PCR primers pairs and the cry or cyt genes they amplify of *Bti.* Sources: Ben-Dov *et al.* (1997)\*, Carozzi et al. (1991)\*\*, Bravo *et al*. (1998)\*\*\*

## **5.4 Expression of** *Bti* **genes in other strains**

250 Insecticides – Pest Engineering

*thuringiensis*. *cryIVA* and *cryIVB* promotors are activated in the mid-sporulation phase (Ben-Dov et al. 1996), *cryIVA* is regulated by the 35 RNA polymerase gene and *cryIVB* is under control of 35 (Yoshisue et al. 1994). *cytA* gene is transcribed by two promotors, pBtI and pBtII, regulated by the RNA polymerase35 and 28, respectively (Brown & Whiteley, 1988, 1990). These results have demostrated that *cryIVD* transcription is subjected to <sup>35</sup>

The polymerase chain reaction (PCR) is a molecular tool widely used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The identification of *Bt* toxin genes by PCR can partially predict the insecticidal activity of a given strain. Several studies have reported that the type of *cry* and *cyt* genes present in a *Bt* strain correlates to some extent with its insecticidal activity (Porcar & Juarez-Perez). Thus, the identification of the gene content in a *Bt* strain can be used to predict its insecticidal potential. The PCR-based identication of *B. thuringiensis* cry genes was first developed by Carozzi et al. (1991), who introduced this technique as a tool to predict insecticidal activity. They found correspondence with the toxicity predicted on the basis of the amplification product profiles. Carozzi et al. proposed PCR as an accurate, fast methodology for the identification of novel strains and the prediction of insecticidal activity of new isolates, and they also forecast the possible use of PCR for the discovery of previously unknown cry genes. highly conserved regions and recognizing entire cry gene subfamilies are often used in a preliminary screening prior to performing a second PCR with specific primers. The primers used to amplify *cry4A, cry4B, cry11A*, and *cytA* genes have been designed by various researchers and

**5.3 Identication of** *Bacillus thuringiensis* **pesticidal crystal genes by PCR** 

**Direct Sequences (5´3) Reverse Sequences** 

AACCCCACTT\*

TTTGGC\*\*

ATCAACCAACT\*

TGCGCCA\*

ACAGCAAGG\*\*\*

Table 6. PCR primers pairs and the cry or cyt genes they amplify of *Bti.* Sources: Ben-Dov *et* 

EE-4A(d) GGGTATGGCACTC

Dip2A(d) GGTGCTTCCTATTC

EE-4B(d) GAGAACACACCTA

EE-11A(d) CCGAACCTACTAT

gral cyt1(d) AACCCCTCAATCA

*al.* (1997)\*, Carozzi et al. (1991)\*\*, Bravo *et al*. (1998)\*\*\*

**(5´3)** 

Un4(r) GCGTGACATACCC ATTTCCAGGTCC\*

Dip2B TGACCAGGTCCCT TGATTAC\*\*

Un4(r) GCGTGACATACCC ATTTCCAGGTCC\*

EE-11A(r) CTCCCTGCTAGGA TTCCGTC\*

gral cyt(r) GGTACACAATACA TAACGCCACC\*\*\*

**Amplifies/Prod uct (bp)** 

cry4A/1529

*cry4A*/1290

*cry4B*/1951

*cry11A*/445

*cyt1*/522-525

regulation.

are shown in Table 6.

Expression of *Bti* genes either individually or in combination in crystal-negative *Bt* or other strains have being carried on by several researchers. The genes encoding these proteins have been expressed in *Caulobacter* (Thanabalu et al. 1992), cyanobacteria (Manasherob et al. 2002; Murphy & Stevens 1992), *Escherichia coli* (McLean & Whiteley 1987; Tanapongpipat et al. 2003), *Bacillus subtilis* (Ward et al. 1986), and *Bt* (Crickmore et al. 1990). However, *Bt* toxins have been expressed as active or inactive toxins, especially when expressed in *E. coli* (Ogunjimi et al. 2002).

Quintano et al. (2005) reported the expression of *cry11A* from *Bti* in *S. cerevisiae*. The *cry11A* gene was expressed as fusion proteins with gluthathione *S*-transferase under the control of the *S. cerevisiae HXK1* promoter. The protein was purified by affinity chromatography using gluthathione *S*-transferase–Sepharose beads. Insecticidal activity against third-instar *Aedes aegypti* larvae of the recombinant *S. cerevisiae* cell extracts (LC50 = 4.10g protein/mL) and purified GST-cry11A fusion protein (LC50 = 4.10g protein/mL ) was detected in cells grown in ethanol.

Servant et al. (1999) constructed a recombinant *B. sphaericus* strain containing the *cry11A* gene from *Bti*. They found an LC50 for the cry11A protein of 1.175 g/mL against fourth-instar *A. aegypti* larvae. Poncet *et al.* (1997) constructed a recombinant *B. sphaericus* strain with *cry11A* and *p20* genes integrated into the chromosome. In this case, the LC50 value reported against third- instar *A. aegypti* larvae was 0.023 g/mL. Xu *et al.* (2001) studied the expression of cry11A and *cry11A*+*p20* gene cluster in recombinant *E. coli* and *Pseudomonas putida*. They found that both recombinant bacteria contained higher levels of Cry11A protein when the adjacent *p20* gene was present on the same DNA fragment. Yamagiwa et al. (2004) reported that the solubilized cry11A protein, obtained from a nonrecombinant *Bti*, was less toxic against *Culex pipiens* larvae than the crystal itself (LC50 of 0.267 and 0.008 g/mL, respectively). These authors obtained 2 GST fusion proteins of 36 and 32 kDa from cry11A. The LC50 against *C. pipiens* larvae obtained using both proteins were 0.818 g/mL. In other study with the purpose to preserve the toxicity of sunlight-sensitive Cry proteins, Manasherob et al. (2002) constructed a transgenic cyanobacterium *Anabaena* PCC 7120 to express the genes *cry4Aa*, *cry11Aa* and an accesory protein (*p20*) under control of two tandem strong promotors. Cyanobacterium *Anabaena* can multiply in breeding sites of mosquito larvae and serve as their food source. Higher toxicity against *Aedes aegypti* larvae was obtained in this study.

## **6. Mode of action of** *Bti* **Cry proteins against mosquito larvae**

## **6.1 Pore-forming-toxins**

*Bt* Cry and Cyt toxins belong to a class of bacterial toxins known as pore-forming toxins (PFT) that are secreted as water-soluble proteins undergoing conformational changes in order to insert into, or to translocate across, cell membranes of their host (Bravo et al. 2007). In most cases, PFT are activated by host proteases after receptor binding inducing the formation of an oligomeric structure that is insertion competent. Finally membrane insertion is triggered, in most cases, by a decrease in pH that induces a molten globule state of the protein (Parker and Feil, 2005). Cry and Cyt proteins are PFT proteins. Both proteins are solubilized in the gut of suceptible diperans insects and proteolytic activated by midgut proteases. For the Cry 11Aa protoxin, proteolytic activation involves amino-terminal processing and intramolecular cleavage leading to two fragments of 36 and 32 kDa that remain associated and retain insect toxicity.

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 253

1985). The international standard recognized for *Bti* is IPS82. LC50 and LC90 are the dose require to kill 50 and 90 percent of the mosquito larvae of a tested population after 24h tested duration. Each sample is bioassayed at least 3 times on various days and the results are average values. LC50 and LC90 are measured in micrograms or milligrams of material per

For potency calculations, it is used the international recognized standard for mosquito assay, IPS82 (15 000 ITU/mg) provided by Institute Pasteur, Paris, France. Standard vials

Potency (A) = Potency (std) LC50 (std)/LC50 (A)

The size of the particle could be a factor that influences the potency of the toxin. A product with small particles is more homogenously distributed in the water than a product with larger particles and small particles which sink slower. Changes in LC50 are not necesary regarded to reflect changes in amount of toxin, but could be a function of particle size/distribution (Skovmand et al. 1997). Change of particle size also change LC50 . Decreasing particle increased LC50 and thus decrease the calculated potency. The slope measured between LC50 and LC90 values should have high value. Higher value will require a smaller quantity to kill a greater number of larvae. The slope of the dosage-mortality curve is in function of the heterogeneity of the product effect. If product availability is in function of particle sizes, particles with broad ranges of particle size distribution will also have low

A variety of *Bti* formulations have been studied for mosquito control under laboratory and field conditions. *Bti* fluid formulations are not stable in heat and high humidity and cannot be stored for months under tropical conditions (McGuirre et al. 1996) . In many cases, and especially in areas exposed to the sun, the residual effect is very short (Leong et al. 1980) and the product has to be reapplied. Photoinactivation seems to be one of the major environment factors affecting the stability of *Bti* delta-endotoxin (Morris, 1983). Yu-Tien et al. (1993) reported that *Bti* completely lost its toxicity to mosquito larvae when exposed to irradiation at 253nm. Poszgay et al. (1987) showed that exposure of *B. thuringiensis* toxin to 40 h of ultraviolet light irradiation resulted in lost activity. Cry proteins inactivation by the solar radiation is the result of the destruction of the tryphtophan (Pusztai et al. 1991). Research and development efforts are focusing on formulations to avoid the ultraviolet light effect. Ramirez-Lepe et al. (2003) encapsulated *Bti* spore-toxin within aluminum/ carboxymethylcellulose using green malachite, congo red or ponceau red as photoprotective agents against ultraviolet light in lab conditions. The encapsulated form of the *Bti* sporetoxin complex with photoprotectors avoided the limitation in controlling mosquito larvae caused by ultraviolet light. Yu-Tien et al. (1993) achieved photoprotection of the spore-toxin

Other *Bti* formulations have been developed. For example, Ramirez-Suero et al. (2005) evaluated maltodextrin, nixtamalized corn flour and corn starch for entraping active materials in *Bti* spore-toxin complexes dried by aspersion. Dried products had water activity values below 0.7 suggesting that the formulations are long shelf-life because keep

liter, or parts per million (ppm).

Product potency is calculated by the Abbott (1925) formula:

where (a) is the product and (std) is the standard.

are kept at -18C.

slopes.

**7.2** *Bti* **formulations** 

complex by addition of melanin.

## **6.2 Mechanism of action of** *Bti* **toxins**

An acepted model for Cry toxin action against mosquito larvae is that it is a multistage process. (i) Ingestion of Cry protein by the larvae (ii) Solubilization of the crystals in the alkaline midgut (iii) Proteolytic activation of the insecticidal solubilised protein (iv) Toxin binds to receptors located on the apical microvellus membrana of ephitelial midgut cell walls (v) Alter the toxin binds the receptor, it is though that there is a change in the toxin conformation allowing toxin insertion into the membrane (vi) Electrophysiological and biochemical evidence suggest that the toxins generate pores in the cell membrane, thus disturbing the osmotic balance, consecuently the cells swell and lyse (vii) The gut becomes paralyzed and the insect stops feeding. Most mosquito larvae die whitin few hours of ingestion, generally cease feeding within 1 hour, show reduced activity by two hours and die six hours after ingestion (Chicott et al. 1990; Marrone & Macintosh, 1993).

Several authors have studied the mechanisms of action of the -endotoxin of *Bti* on mosquito larvae. Thomas and Ellar (1983) found that -endotoxin active against mosquito larvae was inactivated by prior incubation with lipids extracted from ephitelial midgut *Aedes albopictus* cells. They reported that toxin binds to membrane lipids (phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine). According to their results, they proposed a mechanism in which the interaction of toxin with membrane lipids causes a detergent-like rearrangement of the lipids and as a consequence cytolisis. Others authors have corroborated these results: Cyt protein, unlike Cry toxins, do not recognize specific binding sites and do not bind to protein receptors, directly interact with membrane lipids inserting into the membrane and forming pores (Knowles *et al.* 1989; Promdonkoy & Ellar, 2000) or destroying the membrane by a detergent-like interaction (Butko, 2003).

The high efficacy of *Bti* is because of the production of multiple toxins with different modes of action. Perez *et al.* (2005) reported that Cyt1Aa protein functions as a receptor for the Cry11Aa toxin and suggest that this interaction explains the synergism between the Cyt1A and Cry11A proteins. Further, the Cyt proteins in *Bti* synergize the toxic effect of Cry11A and Cry4 toxins and, even more, suppresses the resistance to these Cry toxins (Wirth *et al.* 1997).

## **7. Formulation of** *Bti* **toxins**

## **7.1 Potency in** *Bti* **formulations**

Formulation is a preparation of an insecticide for a particular application method. Formulation plays an important rol in determining final virulence. The vast majority of the formulations of *Bt* have been developed to control agricultural and forest pests, mainly *Lepidoptera*. However, the feeding habits of *Lepidoptera* are different to feeding habits of *Diptera*. Mosquito larvae feed by filtering water and concentrate organic particles. Product formulations based on *Bti* should consider the mosquito larvae habits and the environmental conditions, promote that Cry proteins retain their toxic activity and promote that the larva have access to them. Products based on strains of *Bti* are given a potency based on bioassays on third or fourth instar mosquito larvae. Bioassays are conducted using 6 to 7 dilutions of the toxin by duplicate in 100 mL cups containing 20 third instar *Aedes aegypti* larvae. Duplicate cups with 20 mosquito larvae in 100 mL of deionized water without test material serves as a control (McLaughlin *et al*. 2004). Concentration-mortality data are obtained, transformed to a log-probit scale, and potency is obtained by comparing the estimated LC50 of a test substance with that of a standard with a known potency (de Barjac 1985). The international standard recognized for *Bti* is IPS82. LC50 and LC90 are the dose require to kill 50 and 90 percent of the mosquito larvae of a tested population after 24h tested duration. Each sample is bioassayed at least 3 times on various days and the results are average values. LC50 and LC90 are measured in micrograms or milligrams of material per liter, or parts per million (ppm).

For potency calculations, it is used the international recognized standard for mosquito assay, IPS82 (15 000 ITU/mg) provided by Institute Pasteur, Paris, France. Standard vials are kept at -18C.

Product potency is calculated by the Abbott (1925) formula:

Potency (A) = Potency (std) LC50 (std)/LC50 (A)

where (a) is the product and (std) is the standard.

The size of the particle could be a factor that influences the potency of the toxin. A product with small particles is more homogenously distributed in the water than a product with larger particles and small particles which sink slower. Changes in LC50 are not necesary regarded to reflect changes in amount of toxin, but could be a function of particle size/distribution (Skovmand et al. 1997). Change of particle size also change LC50 . Decreasing particle increased LC50 and thus decrease the calculated potency. The slope measured between LC50 and LC90 values should have high value. Higher value will require a smaller quantity to kill a greater number of larvae. The slope of the dosage-mortality curve is in function of the heterogeneity of the product effect. If product availability is in function of particle sizes, particles with broad ranges of particle size distribution will also have low slopes.

### **7.2** *Bti* **formulations**

252 Insecticides – Pest Engineering

An acepted model for Cry toxin action against mosquito larvae is that it is a multistage process. (i) Ingestion of Cry protein by the larvae (ii) Solubilization of the crystals in the alkaline midgut (iii) Proteolytic activation of the insecticidal solubilised protein (iv) Toxin binds to receptors located on the apical microvellus membrana of ephitelial midgut cell walls (v) Alter the toxin binds the receptor, it is though that there is a change in the toxin conformation allowing toxin insertion into the membrane (vi) Electrophysiological and biochemical evidence suggest that the toxins generate pores in the cell membrane, thus disturbing the osmotic balance, consecuently the cells swell and lyse (vii) The gut becomes paralyzed and the insect stops feeding. Most mosquito larvae die whitin few hours of ingestion, generally cease feeding within 1 hour, show reduced activity by two hours and

Several authors have studied the mechanisms of action of the -endotoxin of *Bti* on mosquito larvae. Thomas and Ellar (1983) found that -endotoxin active against mosquito larvae was inactivated by prior incubation with lipids extracted from ephitelial midgut *Aedes albopictus* cells. They reported that toxin binds to membrane lipids (phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine). According to their results, they proposed a mechanism in which the interaction of toxin with membrane lipids causes a detergent-like rearrangement of the lipids and as a consequence cytolisis. Others authors have corroborated these results: Cyt protein, unlike Cry toxins, do not recognize specific binding sites and do not bind to protein receptors, directly interact with membrane lipids inserting into the membrane and forming pores (Knowles *et al.* 1989; Promdonkoy & Ellar,

The high efficacy of *Bti* is because of the production of multiple toxins with different modes of action. Perez *et al.* (2005) reported that Cyt1Aa protein functions as a receptor for the Cry11Aa toxin and suggest that this interaction explains the synergism between the Cyt1A and Cry11A proteins. Further, the Cyt proteins in *Bti* synergize the toxic effect of Cry11A and Cry4 toxins and, even more, suppresses the resistance to these Cry toxins (Wirth *et al.*

Formulation is a preparation of an insecticide for a particular application method. Formulation plays an important rol in determining final virulence. The vast majority of the formulations of *Bt* have been developed to control agricultural and forest pests, mainly *Lepidoptera*. However, the feeding habits of *Lepidoptera* are different to feeding habits of *Diptera*. Mosquito larvae feed by filtering water and concentrate organic particles. Product formulations based on *Bti* should consider the mosquito larvae habits and the environmental conditions, promote that Cry proteins retain their toxic activity and promote that the larva have access to them. Products based on strains of *Bti* are given a potency based on bioassays on third or fourth instar mosquito larvae. Bioassays are conducted using 6 to 7 dilutions of the toxin by duplicate in 100 mL cups containing 20 third instar *Aedes aegypti* larvae. Duplicate cups with 20 mosquito larvae in 100 mL of deionized water without test material serves as a control (McLaughlin *et al*. 2004). Concentration-mortality data are obtained, transformed to a log-probit scale, and potency is obtained by comparing the estimated LC50 of a test substance with that of a standard with a known potency (de Barjac

die six hours after ingestion (Chicott et al. 1990; Marrone & Macintosh, 1993).

2000) or destroying the membrane by a detergent-like interaction (Butko, 2003).

**6.2 Mechanism of action of** *Bti* **toxins** 

1997).

**7. Formulation of** *Bti* **toxins 7.1 Potency in** *Bti* **formulations**  A variety of *Bti* formulations have been studied for mosquito control under laboratory and field conditions. *Bti* fluid formulations are not stable in heat and high humidity and cannot be stored for months under tropical conditions (McGuirre et al. 1996) . In many cases, and especially in areas exposed to the sun, the residual effect is very short (Leong et al. 1980) and the product has to be reapplied. Photoinactivation seems to be one of the major environment factors affecting the stability of *Bti* delta-endotoxin (Morris, 1983). Yu-Tien et al. (1993) reported that *Bti* completely lost its toxicity to mosquito larvae when exposed to irradiation at 253nm. Poszgay et al. (1987) showed that exposure of *B. thuringiensis* toxin to 40 h of ultraviolet light irradiation resulted in lost activity. Cry proteins inactivation by the solar radiation is the result of the destruction of the tryphtophan (Pusztai et al. 1991). Research and development efforts are focusing on formulations to avoid the ultraviolet light effect. Ramirez-Lepe et al. (2003) encapsulated *Bti* spore-toxin within aluminum/ carboxymethylcellulose using green malachite, congo red or ponceau red as photoprotective agents against ultraviolet light in lab conditions. The encapsulated form of the *Bti* sporetoxin complex with photoprotectors avoided the limitation in controlling mosquito larvae caused by ultraviolet light. Yu-Tien et al. (1993) achieved photoprotection of the spore-toxin complex by addition of melanin.

Other *Bti* formulations have been developed. For example, Ramirez-Suero et al. (2005) evaluated maltodextrin, nixtamalized corn flour and corn starch for entraping active materials in *Bti* spore-toxin complexes dried by aspersion. Dried products had water activity values below 0.7 suggesting that the formulations are long shelf-life because keep

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 255

Briquettes for mosquitoes with continual successive generations like *Culex* spp (Becker & Margalit 1993; Kase & Branton 1986). Briquetts or pellets, in particular seem to be useful for overcoming lack of persistence, which is one of main limitations of *Bti.* Granules using plant, such corn (maiz) grits or clay carriers are particular useful in aerial application to breeding sites with dense foliage as salt marshes or rice fields. Sustained release formulations such as floating briquettes or semi submersible pellets are designed to provide long-lasting larvicidal activity in containers or small ponds. Ingestion of the toxin depends on the rate of feeding, the rate at which the toxin falls to the bottom of the pool and becomes inaccesible, and competition to ingestion from other suspended organic materials. In turbid and polluted waters the rate of application needs to be at least two-fold greater than in clear water (Mulla et al*.* 1985). The feeding habits of mosquito larvae influence formulation design. *Anopheles* larvae are surface feeders and ingest particulate material from water surface such as yeast or flour and filter feed poorly. This has led to the development of formulations that present the toxin at, or just below, the water surface and such prepartions

One of the major drawbacks in the use of *Bti* is its rapid inactivation (24-48 h) in the environment (Mulla et al*.* 1993). Thus larvae populations of stagnant water mosquitoes recover within 5-7 days following treatment. Therefore the use of *Bti* is limited by the low efficacies of current preparations under field conditions (Tyanum & Mulla 1999). Since there is a little persistence of the toxin further applications are necessary to effect continuous control. Formulation and applications techniques can extend the persistence of activity for over one month in some situations, but activity remains sensitive to factors like UV degradation. In areas exposed to the sun, the residual effect is very short (Leong et al. 1980) and the product has to be reapplied. Other factors that affect the toxicity of *Bti* are particle sedimentation (Rushed & Mulla, 1989), protein adsorption onto silt particles, organic matter, elevated temperatures (Ohana et al. 1987), consumption by other organisms to which the toxin is not lethal (Blaustein and Margalit, 1991), dissolved tannins (Lord & Undeen, 1990) and inactivation by sunlight. Other factors that have been observed are that *Bti* does not recycle, under simulated field conditions, is unable to germinate and multiply in mud at the botton of pools although it did remain viable viable for up to 22 days, and higher water

Early reports showed that a primary powder formulations of *Bti* had virtually no residual effect against mosquito larvae beyond application (Aguilar-Meza et al. 2010), although the delta-endotoxin remained chemically stable in neutral and acid waters (Sinegre et al 1980). Extend persistence with *Bti* is possible through use of improved formulations. Gunasekaran *et al.* (2002) tested a floating sustained release formulation of *Bti* in polluted water habitats against *Culex quinquefasciatus* larvae. Briquetts may result in more prolonged control than liquid formulations as these products have greater persistence through slow release (Kase &

On the other hand, Aguilar-Meza et al. (2010) tested the residual insecticidal activity after field exposure of an aluminum-carboxymethylcellulose microencapsulated formulation of *Bti* spore-toxin complex with malachite green as photoprotective agent. The formulation improved the activity against *Aedes aegypti* larvae for 30 days and was comparable to that of

are particulary effective against certain *Anopheles* larvae (Aly et al. 1987)

depth where applied decrease its activity (Ohana et al. 1987)

**8. Field application** 

Branton 1986).

the chemical insecticide temephos.

Fig. 3. Scanning electron micrograph of spray/dried formulation of *Bti* spore/toxin complex with Grits a) 379X and b)1500X and Nixtamalized corn flour c) 370X and d) 1500X.

the products without microorganisms for longer periods and increases the larval feeding and as a consequence have higher activity against mosquito larvae. Fig. 3 shows the scanning electron microscope of spray/dried formulations with grits and nixtamalized corn flour. It shows smoth spherical grits and corn particles entrapping the spore/toxin ingredient. Manasherob et al. (1996) encapsulated *Bti* in the protozoan *Tetrahymena pyriformis* and the activity against *A. stephensi* was enhanced 8 times when exposed to protozoan cells filled with *Bti* compared to exposed to the same concentration of *Bti* alone. Elcin (1995) encapsulated *Bti* in alginate microcapsules and increase its stability and its effect against *Culex* sp larvae. Another alternative to protect *Bti* crystals from ultraviolet light is obtaining mutants that protectrs *Bti* crystal. Hoti & Balaraman (1993) obtained a mutant of *Bti* that produced a brown pigment during sporulation, the pigment was identified as melanin. Other *Bti* formulations have been developed. Culigel superabsorbent polymer controlled-release system for the slow release of *Bti* to mosquito larvae (Levy et al. 1990). Combination of chemical and biological agents susch as insect growth regulator smethoprene + *Bti* (Bassi et al. 1989). Sprayed-dried *Bti* powder as a fizzy tablet (Skovmand & Eriksen 1993). Floating bait formulations designed to improve the effect of bacterial toxins, especially against *Anopheles* spp (Aly et al. 1987).

Highly concentrated liquid formulations are available for control of floodwater mosquitoes while formulations which float for as long possible have been developed for use in fastflowing or turbulent waters. Formulations which settle and persist at the bottom are required for buttom feeders. Granules which float on the surface are the most effective against surface feeders such as *Anopheles* spp. (Mulla et al. 2004).

Briquettes for mosquitoes with continual successive generations like *Culex* spp (Becker & Margalit 1993; Kase & Branton 1986). Briquetts or pellets, in particular seem to be useful for overcoming lack of persistence, which is one of main limitations of *Bti.* Granules using plant, such corn (maiz) grits or clay carriers are particular useful in aerial application to breeding sites with dense foliage as salt marshes or rice fields. Sustained release formulations such as floating briquettes or semi submersible pellets are designed to provide long-lasting larvicidal activity in containers or small ponds. Ingestion of the toxin depends on the rate of feeding, the rate at which the toxin falls to the bottom of the pool and becomes inaccesible, and competition to ingestion from other suspended organic materials. In turbid and polluted waters the rate of application needs to be at least two-fold greater than in clear water (Mulla et al*.* 1985). The feeding habits of mosquito larvae influence formulation design. *Anopheles* larvae are surface feeders and ingest particulate material from water surface such as yeast or flour and filter feed poorly. This has led to the development of formulations that present the toxin at, or just below, the water surface and such prepartions are particulary effective against certain *Anopheles* larvae (Aly et al. 1987)

## **8. Field application**

254 Insecticides – Pest Engineering

Fig. 3. Scanning electron micrograph of spray/dried formulation of *Bti* spore/toxin complex

the products without microorganisms for longer periods and increases the larval feeding and as a consequence have higher activity against mosquito larvae. Fig. 3 shows the scanning electron microscope of spray/dried formulations with grits and nixtamalized corn flour. It shows smoth spherical grits and corn particles entrapping the spore/toxin ingredient. Manasherob et al. (1996) encapsulated *Bti* in the protozoan *Tetrahymena pyriformis* and the activity against *A. stephensi* was enhanced 8 times when exposed to protozoan cells filled with *Bti* compared to exposed to the same concentration of *Bti* alone. Elcin (1995) encapsulated *Bti* in alginate microcapsules and increase its stability and its effect against *Culex* sp larvae. Another alternative to protect *Bti* crystals from ultraviolet light is obtaining mutants that protectrs *Bti* crystal. Hoti & Balaraman (1993) obtained a mutant of *Bti* that produced a brown pigment during sporulation, the pigment was identified as melanin. Other *Bti* formulations have been developed. Culigel superabsorbent polymer controlled-release system for the slow release of *Bti* to mosquito larvae (Levy et al. 1990). Combination of chemical and biological agents susch as insect growth regulator smethoprene + *Bti* (Bassi et al. 1989). Sprayed-dried *Bti* powder as a fizzy tablet (Skovmand & Eriksen 1993). Floating bait formulations designed to improve the effect of bacterial

Highly concentrated liquid formulations are available for control of floodwater mosquitoes while formulations which float for as long possible have been developed for use in fastflowing or turbulent waters. Formulations which settle and persist at the bottom are required for buttom feeders. Granules which float on the surface are the most effective

with Grits a) 379X and b)1500X and Nixtamalized corn flour c) 370X and d) 1500X.

toxins, especially against *Anopheles* spp (Aly et al. 1987).

against surface feeders such as *Anopheles* spp. (Mulla et al. 2004).

One of the major drawbacks in the use of *Bti* is its rapid inactivation (24-48 h) in the environment (Mulla et al*.* 1993). Thus larvae populations of stagnant water mosquitoes recover within 5-7 days following treatment. Therefore the use of *Bti* is limited by the low efficacies of current preparations under field conditions (Tyanum & Mulla 1999). Since there is a little persistence of the toxin further applications are necessary to effect continuous control. Formulation and applications techniques can extend the persistence of activity for over one month in some situations, but activity remains sensitive to factors like UV degradation. In areas exposed to the sun, the residual effect is very short (Leong et al. 1980) and the product has to be reapplied. Other factors that affect the toxicity of *Bti* are particle sedimentation (Rushed & Mulla, 1989), protein adsorption onto silt particles, organic matter, elevated temperatures (Ohana et al. 1987), consumption by other organisms to which the toxin is not lethal (Blaustein and Margalit, 1991), dissolved tannins (Lord & Undeen, 1990) and inactivation by sunlight. Other factors that have been observed are that *Bti* does not recycle, under simulated field conditions, is unable to germinate and multiply in mud at the botton of pools although it did remain viable viable for up to 22 days, and higher water depth where applied decrease its activity (Ohana et al. 1987)

Early reports showed that a primary powder formulations of *Bti* had virtually no residual effect against mosquito larvae beyond application (Aguilar-Meza et al. 2010), although the delta-endotoxin remained chemically stable in neutral and acid waters (Sinegre et al 1980). Extend persistence with *Bti* is possible through use of improved formulations. Gunasekaran *et al.* (2002) tested a floating sustained release formulation of *Bti* in polluted water habitats against *Culex quinquefasciatus* larvae. Briquetts may result in more prolonged control than liquid formulations as these products have greater persistence through slow release (Kase & Branton 1986).

On the other hand, Aguilar-Meza et al. (2010) tested the residual insecticidal activity after field exposure of an aluminum-carboxymethylcellulose microencapsulated formulation of *Bti* spore-toxin complex with malachite green as photoprotective agent. The formulation improved the activity against *Aedes aegypti* larvae for 30 days and was comparable to that of the chemical insecticide temephos.

Biological Control of Mosquito Larvae by *Bacillus thuringiensis* subsp. *israelensis* 257

Baum, J. A. & T. Malvar, T. (1995). Regulation of insecticidal crystal protein production in

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## **9. Conclusions**

*Bti* is a bacterium that has been applied with success in biological control programs against mosquitoes and flies larvae all over the world. The study in each of its facets addressed in this review will open new perspectives to improve their effectiveness in biological control.

## **10. Acknowledgments**

We thank Dr. Vladimir Sanchez-López of the University of Papaloapan, Tuxtepec, Oaxaca and Tiburcio Laez Aponte of the INECOL, Xalapa, Veracruz for taking optical an electronic microscope microphotographs, respectively.

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**12** 

*Thailand* 

**Metabolism of Pyrethroids by Mosquito** 

**Cytochrome P450 Enzymes:** 

Pornpimol Rongnoparut1, Sirikun Pethuan1,

Songklod Sarapusit2 and Panida Lertkiatmongkol1

*1Department of Biochemistry, Faculty of Science, Mahidol University, 2Department of Biochemistry, Faculty of Science, Burapha University,* 

Cytochrome P450 enzymes (P450s) are heme-containing monooxygenases that catalyze metabolisms of various endogenous and exogenous compounds. These P450s constitute a superfamily of enzymes present in various organisms including mammals, plants, bacteria, and insects. P450 enzymes are diverse and metabolize a wide variety of substrates, but their structures are largely conserved. A universal nomenclature has been assigned to P450 superfamily based on their amino acid sequence homology (Nelson et al., 1996). In eukaryotes, P450 is membrane-bound and in general functions to insert one molecule of oxygen into its substrate, with its heme prosthetic group playing a role in substrate oxidation. This catalytic reaction requires a pair of electrons shuttled from NADPH via the NADPH-cytochrome P450 reductase (CYPOR) enzyme, a P450 redox partner, to target P450s (Ortiz de Montellano, 2005). In contrast in bacteria and mitochondria, ferredoxin reductase and iron-sulfur ferredoxin proteins act as a bridge to transfer reducing equivalent from NAD(P)H to target P450s. In insects, P450s are membrane-bound enzymes that play key roles in endogenous metabolisms (i.e. metabolisms of steroid molting and juvenile hormones, and pheromones) and xenobiotic metabolisms, as well as detoxification of insecticides (Feyereisen, 1999). It becomes evident that P450s are implicated in pyrethroid

Insecticides form a mainstay for vector control programs of vector-borne diseases. However intensive uses of insecticides have led to development of insecticide resistance in many insects thus compromising success of insect vector control. In particular pyrethroid resistance has been found widespread in many insects such as house flies, cockroaches, and mosquitoes (Acevedo et al., 2009; Awolola et al., 2002; Cochran, 1989; Hargreaves et al., 2000; Jirakanjanakit et al., 2007). Two major mechanisms have been recorded responsible for insecticide resistance, which are alteration of target sites and metabolic resistance (Hemingway et al., 2004). Metabolic resistance is conferred by increased activities of detoxification enzymes such as P450s, non-specific esterases (Hemingway et al., 2004; Price, 1991). Initial approaches to detect involvement of detoxification mechanisms in metabolic resistance are to compare activities of detoxification enzymes between resistant and

**1. Introduction** 

resistance in insects.

**Impact on Vector Control** 


## **Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control**

Pornpimol Rongnoparut1, Sirikun Pethuan1, Songklod Sarapusit2 and Panida Lertkiatmongkol1 *1Department of Biochemistry, Faculty of Science, Mahidol University, 2Department of Biochemistry, Faculty of Science, Burapha University, Thailand* 

## **1. Introduction**

264 Insecticides – Pest Engineering

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236

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from *Bacillus thuringiensis* subsp. *israelensis* crystal. FEBS Letters, Vol.190, pp.232-

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Protection from ultraviolet irradiation by melanin mosquitocidal activity of *Bacillus* 

Cytochrome P450 enzymes (P450s) are heme-containing monooxygenases that catalyze metabolisms of various endogenous and exogenous compounds. These P450s constitute a superfamily of enzymes present in various organisms including mammals, plants, bacteria, and insects. P450 enzymes are diverse and metabolize a wide variety of substrates, but their structures are largely conserved. A universal nomenclature has been assigned to P450 superfamily based on their amino acid sequence homology (Nelson et al., 1996). In eukaryotes, P450 is membrane-bound and in general functions to insert one molecule of oxygen into its substrate, with its heme prosthetic group playing a role in substrate oxidation. This catalytic reaction requires a pair of electrons shuttled from NADPH via the NADPH-cytochrome P450 reductase (CYPOR) enzyme, a P450 redox partner, to target P450s (Ortiz de Montellano, 2005). In contrast in bacteria and mitochondria, ferredoxin reductase and iron-sulfur ferredoxin proteins act as a bridge to transfer reducing equivalent from NAD(P)H to target P450s. In insects, P450s are membrane-bound enzymes that play key roles in endogenous metabolisms (i.e. metabolisms of steroid molting and juvenile hormones, and pheromones) and xenobiotic metabolisms, as well as detoxification of insecticides (Feyereisen, 1999). It becomes evident that P450s are implicated in pyrethroid resistance in insects.

Insecticides form a mainstay for vector control programs of vector-borne diseases. However intensive uses of insecticides have led to development of insecticide resistance in many insects thus compromising success of insect vector control. In particular pyrethroid resistance has been found widespread in many insects such as house flies, cockroaches, and mosquitoes (Acevedo et al., 2009; Awolola et al., 2002; Cochran, 1989; Hargreaves et al., 2000; Jirakanjanakit et al., 2007). Two major mechanisms have been recorded responsible for insecticide resistance, which are alteration of target sites and metabolic resistance (Hemingway et al., 2004). Metabolic resistance is conferred by increased activities of detoxification enzymes such as P450s, non-specific esterases (Hemingway et al., 2004; Price, 1991). Initial approaches to detect involvement of detoxification mechanisms in metabolic resistance are to compare activities of detoxification enzymes between resistant and

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 267

insight into pyrethroid metabolisms in this mosquito species and knowledge obtained could

*An. minimus* is one of malaria vectors in Southeast Asia, including Thailand, Loas, Cambodia and Vietnam. We previously established a deltamethrin-selected mosquito strain of *An. minimus* species A, by exposure of subsequent mosquito generations to LD50 and LT50 values of deltamethrin (Chareonviriyaphap et al., 2002). Biochemical assays suggested that deltamethrin-resistant *An. minimus* predominantly employ P450s to detoxify pyrethroids (Chareonviriyaphap et al., 2003). We next set out on isolation of P450 genes that have a causal linkage in conferring deltamethrin resistance in this mosquito species. Using reversetranscribed-polymerase chain reaction (RT-PCR) in combination with degenerate PCR primers whose sequences were based on CYP6 conserved amino acids, we have isolated CYP6AA3, CYP6P7, and CYP6P8 complete cDNAs from deltamethrin-resistant *An. minimus*  (Rongnoparut et al., 2003). The three genes showed elevated transcription level in deltamethrin resistant populations compared to the parent susceptible strain. We found that fold of mRNA increase of CYP6AA3 and CYP6P7 is correlated with increase of resistance during deltamethrin selection. However, this correlation was not observed for CYP6P8 (Rodpradit et al., 2005). The three mosquito P450s could thus be used as model enzymes for characterization of their metabolic activities toward insecticides and possibly for future development of tools for mosquito vector control. This can be accomplished by determining whether they possess catalytic activities toward pyrethroid insecticides, thus assuming a causal linkage of overexpression and increased pyrethroid detoxification leading to pyrethroid resistance. Equally important, elucidating properties of the *An. minimus* CYPOR and its influential role in P450 system is beneficial for understanding of P450 metabolisms of

We have heterologously expressed CYP6AA3, CYP6P7, and CYP6P8 in *Spodoptera frugiperda* (*Sf*9) insect cells via baculovirus-mediated expression system. The expression procedure employed full-length CYP6AA3, CYP6P7, and CYP6P8 cDNAs as templates to produce recombinant baculoviruses, and subsequently infected *Sf*9 cells for production of P450 proteins. RT-PCR amplification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were performed to verify expression of P450 mRNAs and proteins in the infected *Sf*9 cells. Expression of CYP6AA3, CYP6P7, and CYP6P8, each is predominantly detected in membrane fractions of infected cells after 72 hours of infection, with expected molecular size of approximately 59 kDa detected on SDS-PAGE (Kaewpa et al., 2007; Duangkaew et al., 2011b). The expressed proteins display CO-reduced difference spectrum of a characteristic peak at 450 nm (Omura & Sato, 1964). Total P450 content obtained from baculovirus-mediated expression of CYP6AA3, CYP6P7, and CYP6P8 ranges from 200 to 360 pmol/mg membrane protein. The expressed CYP6AA3, CYP6P7, and CYP6P8 proteins were used in enzymatic reaction assays testing against pyrethroids and other insecticide groups. Knowledge of the metabolic profile of these P450s could give us insight into functioning of these P450s within mosquitoes towards insecticide metabolisms, i.e. how mosquitoes detoxify against a spectrum of insecticide classes through P450-

In enzymatic assay, each P450 in the reaction was performed in the presence of NADPHregenerating system and was reconstituted with *An.minimus* CYPOR (Kaewpa et al., 2007), as CYPOR is required to supply electrons to P450 in the reaction cycle. Insecticide

contribute to strategies in control of mosquito vectors.

this mosquito species.

mediated metabolisms.

**2.1** *In vitro* **insecticide metabolisms** 

susceptible insect strains, and by identification of corresponding genes that display higher expression level in resistant insects (Bautista et al., 2007; Chareonviriyaphap et al., 2003; Tomita et al., 1995; Yaicharoen et al., 2005). Examinations in various insects such as house fly, cotton ballworm, and mosquito have implicated involvement of up-regulation of different P450 genes in pyrethroid resistance (Liu & Scott, 1998; Müller et al., 2007; Ranasinghe & Hobbs, 1998; Rodpradit et al., 2005; Tomita et al., 1995). Such P450 overexpression has been assumed constituting a defense mechanism against insecticides and responsible for insecticide resistance, presumably by virtue of enhanced insecticide detoxification.

Recent advanced methods employing microarray-based approach, when genomic sequence information for insects is available, have identified multiple genes involved in pyrethroid resistance in mosquitoes. Genes in CYP6 family, in particular, are reported to have an implication in insecticide resistance. In *Anopheles gambiae* malaria vector, microarray analyses reveal that several CYP6 P450 genes could contribute to pyrethroid resistance, these include CYP6M2, CYP6Z2 and CYP6P3 (Djouaka et al., 2008; Müller et al., 2007). These genes were observed up-regulated in pyrethroid resistant mosquitoes (Müller et al., 2008; Stevenson et al., 2011). CYP6M2 and CYP6P3 have shown ability to bind and metabolize pyrethroids, on the other hand CYP6Z2 is able to bind pyrethroids but does not degrade pyrethroids (Mclauglin et al., 2008). Genetic mapping of genes conferring pyrethroid resistance in *An. gambiae* also supports involvement of CYP6P3 in pyrethroid resistance (Wondji et al., 2007). Up-regulation of CYP6 genes has also been found in other resistant insects, for instance CYP6BQ9 in pyrethroid resistant *Tribolium castaneum* (Zhu et al., 2010), CYP6D1 in *Musca domestica* that is able to metabolize pyrethroids (Zhang & Scott, 1996), and CYP6BG1 in pyrethroid resistant *Plutella xylostella* (Bautista et al., 2007). In *T. castaneum* knockdown of CYP6BQ9 by dsRNA resulted in decreased resistance to deltamethrin (Zhu et al., 2010). Similar finding has been observed for CYP6BG1 in permethrin resistant *P. xylostella*, supporting the role of overexpression of these CYP6 genes in pyrethroid resistance (Bautista et al., 2009). In *An. minimus* mosquito, CYP6AA3 and CYP6P7 are upregulated and possess activities toward pyrethroid degradation (Duangkaew et al., 2011b; Rongnoparut et al., 2003).

## **2. Cytochrome P450 monooxygenase (P450) and NADPH-cytochrome P450 reductase (CYPOR) enzymes isolated from** *An. minimus*

In this chapter, we focus on investigation of the P450s that have been shown overexpressed in a laboratory-selected pyrethroid resistant *An. minimus* mosquito. We describe heterologous expression of the overexpressed P450s in baculovirus-mediated insect cell expression system and characterization of their catalytic roles toward pyrethroid insecticides. Tools utilized in functional investigation of *An. minimus* P450s have been developed and described. In parallel the *An. minimus* CYPOR has been cloned and protein expressed via bacterial expression system. Amino acid sequence of *An. minimus* CYPOR is intrigue in that several important residues that might play role in its functioning as P450 redox partner are different from those of previously reported enzymes from mammals and house fly. The *An. minimus* CYPOR is different in enzymatic properties and kinetic mechanisms from other CYPORs. In this context we speculate that *An. minimus* CYPOR could influence electron delivery to target mosquito P450 enzymes, and could act as a ratelimiting step in P450-mediated metabolisms. These results together could thus gain an insight into pyrethroid metabolisms in this mosquito species and knowledge obtained could contribute to strategies in control of mosquito vectors.

*An. minimus* is one of malaria vectors in Southeast Asia, including Thailand, Loas, Cambodia and Vietnam. We previously established a deltamethrin-selected mosquito strain of *An. minimus* species A, by exposure of subsequent mosquito generations to LD50 and LT50 values of deltamethrin (Chareonviriyaphap et al., 2002). Biochemical assays suggested that deltamethrin-resistant *An. minimus* predominantly employ P450s to detoxify pyrethroids (Chareonviriyaphap et al., 2003). We next set out on isolation of P450 genes that have a causal linkage in conferring deltamethrin resistance in this mosquito species. Using reversetranscribed-polymerase chain reaction (RT-PCR) in combination with degenerate PCR primers whose sequences were based on CYP6 conserved amino acids, we have isolated CYP6AA3, CYP6P7, and CYP6P8 complete cDNAs from deltamethrin-resistant *An. minimus*  (Rongnoparut et al., 2003). The three genes showed elevated transcription level in deltamethrin resistant populations compared to the parent susceptible strain. We found that fold of mRNA increase of CYP6AA3 and CYP6P7 is correlated with increase of resistance during deltamethrin selection. However, this correlation was not observed for CYP6P8 (Rodpradit et al., 2005). The three mosquito P450s could thus be used as model enzymes for characterization of their metabolic activities toward insecticides and possibly for future development of tools for mosquito vector control. This can be accomplished by determining whether they possess catalytic activities toward pyrethroid insecticides, thus assuming a causal linkage of overexpression and increased pyrethroid detoxification leading to pyrethroid resistance. Equally important, elucidating properties of the *An. minimus* CYPOR and its influential role in P450 system is beneficial for understanding of P450 metabolisms of this mosquito species.

## **2.1** *In vitro* **insecticide metabolisms**

266 Insecticides – Pest Engineering

susceptible insect strains, and by identification of corresponding genes that display higher expression level in resistant insects (Bautista et al., 2007; Chareonviriyaphap et al., 2003; Tomita et al., 1995; Yaicharoen et al., 2005). Examinations in various insects such as house fly, cotton ballworm, and mosquito have implicated involvement of up-regulation of different P450 genes in pyrethroid resistance (Liu & Scott, 1998; Müller et al., 2007; Ranasinghe & Hobbs, 1998; Rodpradit et al., 2005; Tomita et al., 1995). Such P450 overexpression has been assumed constituting a defense mechanism against insecticides and responsible for insecticide resistance, presumably by virtue of enhanced insecticide

Recent advanced methods employing microarray-based approach, when genomic sequence information for insects is available, have identified multiple genes involved in pyrethroid resistance in mosquitoes. Genes in CYP6 family, in particular, are reported to have an implication in insecticide resistance. In *Anopheles gambiae* malaria vector, microarray analyses reveal that several CYP6 P450 genes could contribute to pyrethroid resistance, these include CYP6M2, CYP6Z2 and CYP6P3 (Djouaka et al., 2008; Müller et al., 2007). These genes were observed up-regulated in pyrethroid resistant mosquitoes (Müller et al., 2008; Stevenson et al., 2011). CYP6M2 and CYP6P3 have shown ability to bind and metabolize pyrethroids, on the other hand CYP6Z2 is able to bind pyrethroids but does not degrade pyrethroids (Mclauglin et al., 2008). Genetic mapping of genes conferring pyrethroid resistance in *An. gambiae* also supports involvement of CYP6P3 in pyrethroid resistance (Wondji et al., 2007). Up-regulation of CYP6 genes has also been found in other resistant insects, for instance CYP6BQ9 in pyrethroid resistant *Tribolium castaneum* (Zhu et al., 2010), CYP6D1 in *Musca domestica* that is able to metabolize pyrethroids (Zhang & Scott, 1996), and CYP6BG1 in pyrethroid resistant *Plutella xylostella* (Bautista et al., 2007). In *T. castaneum* knockdown of CYP6BQ9 by dsRNA resulted in decreased resistance to deltamethrin (Zhu et al., 2010). Similar finding has been observed for CYP6BG1 in permethrin resistant *P. xylostella*, supporting the role of overexpression of these CYP6 genes in pyrethroid resistance (Bautista et al., 2009). In *An. minimus* mosquito, CYP6AA3 and CYP6P7 are upregulated and possess activities toward pyrethroid degradation (Duangkaew et al., 2011b; Rongnoparut et

**2. Cytochrome P450 monooxygenase (P450) and NADPH-cytochrome P450** 

In this chapter, we focus on investigation of the P450s that have been shown overexpressed in a laboratory-selected pyrethroid resistant *An. minimus* mosquito. We describe heterologous expression of the overexpressed P450s in baculovirus-mediated insect cell expression system and characterization of their catalytic roles toward pyrethroid insecticides. Tools utilized in functional investigation of *An. minimus* P450s have been developed and described. In parallel the *An. minimus* CYPOR has been cloned and protein expressed via bacterial expression system. Amino acid sequence of *An. minimus* CYPOR is intrigue in that several important residues that might play role in its functioning as P450 redox partner are different from those of previously reported enzymes from mammals and house fly. The *An. minimus* CYPOR is different in enzymatic properties and kinetic mechanisms from other CYPORs. In this context we speculate that *An. minimus* CYPOR could influence electron delivery to target mosquito P450 enzymes, and could act as a ratelimiting step in P450-mediated metabolisms. These results together could thus gain an

**reductase (CYPOR) enzymes isolated from** *An. minimus*

detoxification.

al., 2003).

We have heterologously expressed CYP6AA3, CYP6P7, and CYP6P8 in *Spodoptera frugiperda* (*Sf*9) insect cells via baculovirus-mediated expression system. The expression procedure employed full-length CYP6AA3, CYP6P7, and CYP6P8 cDNAs as templates to produce recombinant baculoviruses, and subsequently infected *Sf*9 cells for production of P450 proteins. RT-PCR amplification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were performed to verify expression of P450 mRNAs and proteins in the infected *Sf*9 cells. Expression of CYP6AA3, CYP6P7, and CYP6P8, each is predominantly detected in membrane fractions of infected cells after 72 hours of infection, with expected molecular size of approximately 59 kDa detected on SDS-PAGE (Kaewpa et al., 2007; Duangkaew et al., 2011b). The expressed proteins display CO-reduced difference spectrum of a characteristic peak at 450 nm (Omura & Sato, 1964). Total P450 content obtained from baculovirus-mediated expression of CYP6AA3, CYP6P7, and CYP6P8 ranges from 200 to 360 pmol/mg membrane protein. The expressed CYP6AA3, CYP6P7, and CYP6P8 proteins were used in enzymatic reaction assays testing against pyrethroids and other insecticide groups. Knowledge of the metabolic profile of these P450s could give us insight into functioning of these P450s within mosquitoes towards insecticide metabolisms, i.e. how mosquitoes detoxify against a spectrum of insecticide classes through P450 mediated metabolisms.

In enzymatic assay, each P450 in the reaction was performed in the presence of NADPHregenerating system and was reconstituted with *An.minimus* CYPOR (Kaewpa et al., 2007), as CYPOR is required to supply electrons to P450 in the reaction cycle. Insecticide

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 269

Fig. 1. Chemical structures of insecticides used in the study.

metabolism was determined by detection of disappearance of insecticide substrate at different times compared with that present at time zero as previously described (Booseupsakul et al., 2008). This time course degradation was detected through HPLC analysis. Table 1 summarizes enzyme activities of CYP6AA3 and CYP6P7 toward insecticides and metabolites detected. Insecticides that were tested by enzyme assays were type I pyrethroids (permethrin and bioallethrin), type II pyrethroids (deltamethrin, cypermethrin, and λ-cyhalothrin), organophosphate (chlorpyrifos), and carbamate (propoxur). Additional insecticides (bifenthrin, dichlorvos, fenitrothion, temephos, and thiodicarb) belonging to these four insecticide classes were tested by cytotoxicity assays (see Section 2.3). Chemical structures of these insecticides are shown in Fig. 1.


Table 1. Presence (+) and absence (-) of P450 activities in insecticide degradation and metabolites obtained. ND, products not determined

The results shown in Table 1 demonstrate that CYP6AA3 and CYP6P7 share overlapping metabolic profile against both type I and II pyrethroids, while no detectable activity was observed toward chlorpyrifos and propoxur (Duangkaew et al., 2011b), nor in the presence of piperonyl butoxide (a P450 inhibitor). Differences in activities of both enzymes could be noted, for CPY6AA3 could metabolize λ-cyhalothrin while CYP6P7 did not display activity against λ-cyhalothrin. For CYP6P8 we initially detected absence of pyrethroid degradation activity, further tests using cytotoxicity assays described in Section 2.3 suggest that CYP6P8 is not capable of degradation of pyrethroids, organophosphates and carbamates.

Determination of products obtained from CYP6AA3-mediated pyrethroid degradations using GC-MS analysis reveal multiple products for type II pyrethroid cypermethrin degradation and for earlier described deltamethrin metabolism (Boonseupsakul et al., 2008). These products were 3-phenoxybenzyaldehyde and two unknown products with chloride and bromide isotope distribution derived from cypermethrin and deltamethrin metabolisms, respectively. In contrast there was only one unknown product that was predominantly detected from CYP6AA3-mediated permethrin (type I pyrethroid) degradation, with mass spectrum profile showing characteristic chloride isotope distribution of permethrin-derived compound. Unlike cypermethrin and deltamethrin metabolisms, we did not obtain 3-phenoxybenzaldehyde from permethrin degradation (Boonseupsakul, 2008).

metabolism was determined by detection of disappearance of insecticide substrate at different times compared with that present at time zero as previously described (Booseupsakul et al., 2008). This time course degradation was detected through HPLC analysis. Table 1 summarizes enzyme activities of CYP6AA3 and CYP6P7 toward insecticides and metabolites detected. Insecticides that were tested by enzyme assays were type I pyrethroids (permethrin and bioallethrin), type II pyrethroids (deltamethrin, cypermethrin, and λ-cyhalothrin), organophosphate (chlorpyrifos), and carbamate (propoxur). Additional insecticides (bifenthrin, dichlorvos, fenitrothion, temephos, and thiodicarb) belonging to these four insecticide classes were tested by cytotoxicity assays (see

**Insecticides CYP6AA3 Activity (metabolites) CYP6P7 Activity** 

2 unknown products) +, ND

2 unknown products) +, ND

Bioallethrin - - Permethrin + (1 major unknown product) +, ND

λ - Cyhalothrin +, ND -

Chlorpyrifos - - **Carbamate** - - Propoxur - - Table 1. Presence (+) and absence (-) of P450 activities in insecticide degradation and

The results shown in Table 1 demonstrate that CYP6AA3 and CYP6P7 share overlapping metabolic profile against both type I and II pyrethroids, while no detectable activity was observed toward chlorpyrifos and propoxur (Duangkaew et al., 2011b), nor in the presence of piperonyl butoxide (a P450 inhibitor). Differences in activities of both enzymes could be noted, for CPY6AA3 could metabolize λ-cyhalothrin while CYP6P7 did not display activity against λ-cyhalothrin. For CYP6P8 we initially detected absence of pyrethroid degradation activity, further tests using cytotoxicity assays described in Section 2.3 suggest that CYP6P8

Determination of products obtained from CYP6AA3-mediated pyrethroid degradations using GC-MS analysis reveal multiple products for type II pyrethroid cypermethrin degradation and for earlier described deltamethrin metabolism (Boonseupsakul et al., 2008). These products were 3-phenoxybenzyaldehyde and two unknown products with chloride and bromide isotope distribution derived from cypermethrin and deltamethrin metabolisms, respectively. In contrast there was only one unknown product that was predominantly detected from CYP6AA3-mediated permethrin (type I pyrethroid) degradation, with mass spectrum profile showing characteristic chloride isotope distribution of permethrin-derived compound. Unlike cypermethrin and deltamethrin metabolisms, we did not obtain 3-phenoxybenzaldehyde from permethrin degradation

is not capable of degradation of pyrethroids, organophosphates and carbamates.

Section 2.3). Chemical structures of these insecticides are shown in Fig. 1.

Cypermethrin + (3-phenoxybenzaldehyde and

Deltamethrin + (3-phenoxybenzaldehyde and

**Type I pyrethroids**

**Organophosphate**

metabolites obtained. ND, products not determined

**Type II pyrethroids** 

(Boonseupsakul, 2008).

Fig. 1. Chemical structures of insecticides used in the study.

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 271

insects could be attributed to structural differences resulting in selectivity and different

In human, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 have been reported abilities to metabolize both type I and II pyrethroids (Godin et al., 2007; Scollon et al., 2009). The preference for type I pyrethroid in CYP6AA3 is similar to human CYP2C9 and CYP2C19, while similar metabolic activity toward both types of pyrethroids found for CYP6P7 is similar to that of human CYP2C8 enzyme (Scollon et al., 2009). Nevertheless efficiency of CYP6AA3 and CYP6P7 in deltamethrin degradation is 5- to 10-fold less effective than human CYP2C8 and CYP2C19. It is noteworthy that more than one P450s residing within an organism can metabolize pyrethroids as described for human and mosquito, multiple rat P450s are also found capable of pyrethroid metabolisms (Scollon et al., 2009). When comparing to *An. gambiae* CYP6P3, both CYP6AA3 and CYP6P7 possess at least 10 fold higher *K*m than CYP6P3, but *V*max values of both *An. minimus* CYP6AA3 and CYP6P7 are at least 20 fold higher (Müller et al., 2008). Higher values of *K*m and *V*max of CYP6AA3 and CYP6P7 than those values of *An. gambiae* CYP6M2 (Stevenson et al., 2011) are also observed.

**2.2.2 CYP6AA3 and CYP6P7 are inhibited differently by different compounds** 

further used for inhibition studies of both mosquito enzymes.

their chemical structures are shown in Fig. 2.

To obtain a potential fluorogenic substrate probe for fluorescent-based assays of CYP6AA3 and CYP6P7, we previously screened four resorufin fluorogenic substrates containing different alkyl groups (Duangkaew et al., 2011b) and results in Table 2 suggest that among test compounds, benzyloxyresorufin could be used as a fluorescent substrate probe since both CYP6P7 and CYP6AA3 could bind and metabolize benzyloxyresorufin with lowest *K*<sup>m</sup> (values of 1.92 for CYP6AA3 and 0.49 for CYP6P7) and with highest specific activities (Duangkaew et al., 2011b). The assays of benzyloxyresorufin-*O*-debenzylation activity were

Specific activity (pmole resorufin/min/pmole P450) Compounds CYP6AA3 CYP6P7 Benzyloxyresorufin 6.81 ± 0.65 4.99 ± 0.74 Ethyloxyresorufin 2.88 ± 0.21 3.61 ± 0.17 Methyloxyresorufin 0.02 ± 0.01 - Penthyloxyresorufin 0.01 ± 0.01 -

Using fluorescence-based assays, we could initially determine what compound types that give mechanism-based inhibition pattern by pre-incubation of enzyme with various concentrations of test inhibitors in the presence or absence of NADPH for 30 min before addition of substrates and IC50 values have been determined as described (Duangkaew et al., 2011b). As known, mechanism-based inactivation inhibits enzyme irreversibly, rendering this mechanism of inhibition more efficient than reversible inhibition. Nevertheless information on mode of inhibition for inhibitors is potential for understanding of catalytic nature of enzymes. We thus determined mode of inhibition for all compounds tested. As shown in Table 2, the compounds we have tested are phenolic compounds and

It is apparent that none of test flavonoids and furanocoumarins shows mechanism-based inhibition pattern, but piperonyl butoxide (PBO) and piperine that are methylenedioxyphenyl compounds show NADPH-dependent mechanism-based inhibition activities against both

Table 2. Specific activities of CYP6AA3 and CYP6P7 toward resorufin derivatives.

enzymatic activities against insecticides.

Type I and type II pyrethroids are different by the presence of cyano group (see Fig. 1). Thus our results implicate that presence of cyano group may play role in CYP6AA3-mediated pyrethroid degradations resulting in detection of 3-phenoxybenzaldehyde, possibly through oxidative cleavage reaction. In *An. gambiae* CYP6M2-mediated deltamethrin metabolism and house fly CYP6D1-mediated cypermethrin metabolism, 4'-hydroxylation of deltamethrin and cypermethrin is the major route of their metabolisms since 4'-hydroxylation products were predominantly detected (Stevenson et al., 2011; Zhang & Scott, 1996). The 4' hydroxylation and 3-phenoxybenzaldehyde products have been observed in *in vitro* pyrethroid metabolisms mediated by mammalian microsomal enzymes (Shono et al., 1979). The absence of detection of 3-phenoxybenzaldehyde in CYP6AA3-mediated permethrin degradation could be predicted that the reaction underwent monooxygenation of permethrin.

#### **2.2 Characterization of CYP6AA3 and CYP6P7 enzymes**

As described, both CYP6AA3 and CYP6P7 enzymes have enzymatic activities against pyrethroid insecticides and their metabolic profiles are different. Kinetics and inhibition studies further support their abilities to metabolize pyrethroids, however with different enzyme and kinetic properties that influence substrate and inhibitor selectivity. Such knowledge could have an implication in pyrethroid detoxification in *An. minimus* mosquito, for example how the two P450s redundantly metabolize overlapping sets of pyrethroids. Alongside investigation of pyrethroid metabolisms, we examined their activities toward fluorescent compounds for development of rapid enzymatic assays. Finally we performed cell-based MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assays for further determination of substrates and inhibitors of both P450 enzymes as reported herein.

#### **2.2.1 Determination of enzyme kinetics of CYP6AA3 and CYP6P7 enzymes**

We recently reported kenetic paremeters for CYP6AA3 and CYP6P7 enzymes (Duangkaew et al., 2011b). Kinetic results reveal that CYP6AA3 has preference in binding to and has higher rate in degradation of permethrin type I pyrethroid than type II pyrethroids (*K*<sup>m</sup> values toward permethrin, cypermethrin, deltamethrin, and λ-cyhalothrin of 41.0 ± 8.5, 70.0 ± 7.1, 80.2 ± 2.0, and 78.3 ± 7.0 µM, respectively and *V*max values of 124.2 ± 1.2, 40.0 ± 7.1, 60.2 ± 3.6, and 60.7 ± 1.1 pmol/min/pmol P450, respectively). In contradictory CYP6P7 does not have preference for type of pyrethroids (*K*m values toward permethrin, cypermethrin, and deltamethrin of 69.7 ± 10.5 , 97.3 ± 6.4, and 73.3 ± 2.9, respectively and *V*max values of 65.7 ± 1.6, 83.3 ± 7.6, and 55.3 ± 5.7 pmol/min/pmol P450, respectively) and does not metabolize λ-cyhalothrin. Thus although both enzymes are comparable in terms of capability to metabolize pyrethroids *in vitro*, their kinetic values are different. Enzyme structure could account for their differences in kinetic properties and substrate preference.

Since there has been no known crystal structure available for insect P450s, we initially constructed homology models for CYP6AA3, CYP6P7, and CYP6P8 in an attempt to increase our understanding of molecular mechanisms underlying their binding sites toward insecticide substrates and inhibitors. The three enzyme models are different in geometry of their active-site cavities and substrate access channels. Upon docking with various insecticide groups, results of its active site could predict and explain metabolic behavior toward pyrethroid, organophosphate, and carbamate insecticides (Lertkiatmongkol et al., 2011). These results suggest that differences in metabolic activities among P450 enzymes in

Type I and type II pyrethroids are different by the presence of cyano group (see Fig. 1). Thus our results implicate that presence of cyano group may play role in CYP6AA3-mediated pyrethroid degradations resulting in detection of 3-phenoxybenzaldehyde, possibly through oxidative cleavage reaction. In *An. gambiae* CYP6M2-mediated deltamethrin metabolism and house fly CYP6D1-mediated cypermethrin metabolism, 4'-hydroxylation of deltamethrin and cypermethrin is the major route of their metabolisms since 4'-hydroxylation products were predominantly detected (Stevenson et al., 2011; Zhang & Scott, 1996). The 4' hydroxylation and 3-phenoxybenzaldehyde products have been observed in *in vitro* pyrethroid metabolisms mediated by mammalian microsomal enzymes (Shono et al., 1979). The absence of detection of 3-phenoxybenzaldehyde in CYP6AA3-mediated permethrin degradation could be predicted that the reaction underwent monooxygenation of

As described, both CYP6AA3 and CYP6P7 enzymes have enzymatic activities against pyrethroid insecticides and their metabolic profiles are different. Kinetics and inhibition studies further support their abilities to metabolize pyrethroids, however with different enzyme and kinetic properties that influence substrate and inhibitor selectivity. Such knowledge could have an implication in pyrethroid detoxification in *An. minimus* mosquito, for example how the two P450s redundantly metabolize overlapping sets of pyrethroids. Alongside investigation of pyrethroid metabolisms, we examined their activities toward fluorescent compounds for development of rapid enzymatic assays. Finally we performed cell-based MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assays for further determination of substrates and inhibitors of both P450 enzymes as

**2.2.1 Determination of enzyme kinetics of CYP6AA3 and CYP6P7 enzymes** 

We recently reported kenetic paremeters for CYP6AA3 and CYP6P7 enzymes (Duangkaew et al., 2011b). Kinetic results reveal that CYP6AA3 has preference in binding to and has higher rate in degradation of permethrin type I pyrethroid than type II pyrethroids (*K*<sup>m</sup> values toward permethrin, cypermethrin, deltamethrin, and λ-cyhalothrin of 41.0 ± 8.5, 70.0 ± 7.1, 80.2 ± 2.0, and 78.3 ± 7.0 µM, respectively and *V*max values of 124.2 ± 1.2, 40.0 ± 7.1, 60.2 ± 3.6, and 60.7 ± 1.1 pmol/min/pmol P450, respectively). In contradictory CYP6P7 does not have preference for type of pyrethroids (*K*m values toward permethrin, cypermethrin, and deltamethrin of 69.7 ± 10.5 , 97.3 ± 6.4, and 73.3 ± 2.9, respectively and *V*max values of 65.7 ± 1.6, 83.3 ± 7.6, and 55.3 ± 5.7 pmol/min/pmol P450, respectively) and does not metabolize λ-cyhalothrin. Thus although both enzymes are comparable in terms of capability to metabolize pyrethroids *in vitro*, their kinetic values are different. Enzyme structure could account for their differences in kinetic properties and substrate preference. Since there has been no known crystal structure available for insect P450s, we initially constructed homology models for CYP6AA3, CYP6P7, and CYP6P8 in an attempt to increase our understanding of molecular mechanisms underlying their binding sites toward insecticide substrates and inhibitors. The three enzyme models are different in geometry of their active-site cavities and substrate access channels. Upon docking with various insecticide groups, results of its active site could predict and explain metabolic behavior toward pyrethroid, organophosphate, and carbamate insecticides (Lertkiatmongkol et al., 2011). These results suggest that differences in metabolic activities among P450 enzymes in

**2.2 Characterization of CYP6AA3 and CYP6P7 enzymes** 

permethrin.

reported herein.

insects could be attributed to structural differences resulting in selectivity and different enzymatic activities against insecticides.

In human, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 have been reported abilities to metabolize both type I and II pyrethroids (Godin et al., 2007; Scollon et al., 2009). The preference for type I pyrethroid in CYP6AA3 is similar to human CYP2C9 and CYP2C19, while similar metabolic activity toward both types of pyrethroids found for CYP6P7 is similar to that of human CYP2C8 enzyme (Scollon et al., 2009). Nevertheless efficiency of CYP6AA3 and CYP6P7 in deltamethrin degradation is 5- to 10-fold less effective than human CYP2C8 and CYP2C19. It is noteworthy that more than one P450s residing within an organism can metabolize pyrethroids as described for human and mosquito, multiple rat P450s are also found capable of pyrethroid metabolisms (Scollon et al., 2009). When comparing to *An. gambiae* CYP6P3, both CYP6AA3 and CYP6P7 possess at least 10 fold higher *K*m than CYP6P3, but *V*max values of both *An. minimus* CYP6AA3 and CYP6P7 are at least 20 fold higher (Müller et al., 2008). Higher values of *K*m and *V*max of CYP6AA3 and CYP6P7 than those values of *An. gambiae* CYP6M2 (Stevenson et al., 2011) are also observed.

## **2.2.2 CYP6AA3 and CYP6P7 are inhibited differently by different compounds**

To obtain a potential fluorogenic substrate probe for fluorescent-based assays of CYP6AA3 and CYP6P7, we previously screened four resorufin fluorogenic substrates containing different alkyl groups (Duangkaew et al., 2011b) and results in Table 2 suggest that among test compounds, benzyloxyresorufin could be used as a fluorescent substrate probe since both CYP6P7 and CYP6AA3 could bind and metabolize benzyloxyresorufin with lowest *K*<sup>m</sup> (values of 1.92 for CYP6AA3 and 0.49 for CYP6P7) and with highest specific activities (Duangkaew et al., 2011b). The assays of benzyloxyresorufin-*O*-debenzylation activity were further used for inhibition studies of both mosquito enzymes.


Table 2. Specific activities of CYP6AA3 and CYP6P7 toward resorufin derivatives.

Using fluorescence-based assays, we could initially determine what compound types that give mechanism-based inhibition pattern by pre-incubation of enzyme with various concentrations of test inhibitors in the presence or absence of NADPH for 30 min before addition of substrates and IC50 values have been determined as described (Duangkaew et al., 2011b). As known, mechanism-based inactivation inhibits enzyme irreversibly, rendering this mechanism of inhibition more efficient than reversible inhibition. Nevertheless information on mode of inhibition for inhibitors is potential for understanding of catalytic nature of enzymes. We thus determined mode of inhibition for all compounds tested. As shown in Table 2, the compounds we have tested are phenolic compounds and their chemical structures are shown in Fig. 2.

It is apparent that none of test flavonoids and furanocoumarins shows mechanism-based inhibition pattern, but piperonyl butoxide (PBO) and piperine that are methylenedioxyphenyl compounds show NADPH-dependent mechanism-based inhibition activities against both

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 273

Table 3. Mode of inhibition and inhibition constants of CYP6P7- or CYP6AA3-

without (w/o) NADPH. ND, not determined.

benzyloxyresorufin-*O*-debenzylation activities of flavonoids, furanocoumarins, and MDP compounds (Duangkaew et al., 2011b). Crude plant extracts reported herein are ethanolic extracts. Values marked with 'a' are significantly different between reactions with (w/) and

enzymes. Piperine has been commonly found in *Piper sp.* plant extracts, it possesses acute toxicity to mammals (Daware et al., 2000). Inhibition results shown in Table 3 also elucidate that -naphthoflavone displayed strongest inhibitory effect. Its inhibition pattern suggests that -naphthoflavone uncompetitively inhibit both enzymes by binding to CYP6AA3– and CYP6P7-benzyloxyresorufin complex. Moreover, a difference was noted for xanthotoxin as it uncompetitively inhibits CYP6AA3 but mixed-type inhibited CYP6P7. Thus inhibition results together with different metabolic profiles thus confirm that CYP6AA3 and CYP6P7 have different enzyme properties. We thus also tested crude extracts of two plants (*Citrus reticulate* and *Stemona spp.*) that were reported containing phenolic compounds (Kaltenegger et al., 2003; Jayaprakasha et al., 1997) and are found in Thailand. Initial results suggest that compounds within both plants may not possess mechanism-based activities against CYP6AA3 and CYP6P7, and both extracts did not inhibit both enzymes as efficient as flavonoids and methylenedioxyphenyl compounds.

Fig. 2. Chemical structures of different compound types used for inhibition assays of mosquito P450s.


Table 3. Mode of inhibition and inhibition constants of CYP6P7- or CYP6AA3 benzyloxyresorufin-*O*-debenzylation activities of flavonoids, furanocoumarins, and MDP compounds (Duangkaew et al., 2011b). Crude plant extracts reported herein are ethanolic extracts. Values marked with 'a' are significantly different between reactions with (w/) and without (w/o) NADPH. ND, not determined.

enzymes. Piperine has been commonly found in *Piper sp.* plant extracts, it possesses acute toxicity to mammals (Daware et al., 2000). Inhibition results shown in Table 3 also elucidate that -naphthoflavone displayed strongest inhibitory effect. Its inhibition pattern suggests that -naphthoflavone uncompetitively inhibit both enzymes by binding to CYP6AA3– and CYP6P7-benzyloxyresorufin complex. Moreover, a difference was noted for xanthotoxin as it uncompetitively inhibits CYP6AA3 but mixed-type inhibited CYP6P7. Thus inhibition results together with different metabolic profiles thus confirm that CYP6AA3 and CYP6P7 have different enzyme properties. We thus also tested crude extracts of two plants (*Citrus reticulate* and *Stemona spp.*) that were reported containing phenolic compounds (Kaltenegger et al., 2003; Jayaprakasha et al., 1997) and are found in Thailand. Initial results suggest that compounds within both plants may not possess mechanism-based activities against CYP6AA3 and CYP6P7, and both extracts did not inhibit both enzymes as efficient as

Fig. 2. Chemical structures of different compound types used for inhibition assays of

mosquito P450s.

flavonoids and methylenedioxyphenyl compounds.

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 275

degradation of insecticides tested in this report. Moreover, such cytotoxicity results implicate that the method could also be applied for primary screening of compounds that have an inhibitory effect towards CYP6AA3 and CYP6P7, as well as P450 enzymes that

Bioallethrinb 30.6 ± 2.1 32.7 ± 2.4 23.3 ± 3.9 29 Permethrinb 42.7 ± 1.8 406.7 ± 21.5a 214.7 ± 48.8a 78 Bifenthrin 45 ± 7.6 210 ± 12.4a 135 ± 51a 45 Cypermethrinb 21.8 ± 0.5 192.7 ± 30.4a 216.7 ± 21.4a 25 Deltamethrinb 27.5 ± 9.2 285.0 ± 27.8a 379.5 ± 21.9a 10 λ-Cyhalothrinb 38.4 ± 4.3 133.3 ± 37.5a 42.0 ± 1.8 ND

Chlorpyrifosb 40.3 ± 6.5 56.3 ± 8.5 41.7 ± 2.8 60 Fenitrothion 25.0 ± 5.3 30.0 ± 6.4 ND 25 Temephos 11.0 ± 3.9 19.0 ± 7.5 ND ND Dichlorvos 32.0 ± 8.9 39.0 ± 6.4 ND ND

Propoxurb 4.0 ± 6.6 4.7 ± 0.3 3.6 ± 0.2 ND Thiodicarb 28.6 ± 2.3 29.2 ± 4.7 ND ND Table 4. Cytotoxicity effects by insecticides on P450-infected cells and the parent SF9 cells using MTT assays. Values reported for CYP6P8 were average obtained from experiments performed in duplicate. Those marked with 'a' were significantly different from parent *Sf*9

To test whether inhibitors can be screened, MTT assays were performed with P450 expressing cells treated with 100 M deltamethin in the presence or absence of each test inhibitor. Concentrations of test inhibitory compounds were those of approximately LC20 values pre-determined by MTT assays on *Sf*9 cells. In cell-based inhibition assays, cell viability was determined upon co-incubation of test compound and deltamethrin, and normalized with viability of cells treated with test compound without deltamethrin. Inhibition experiments were performed with control *Sf*9 cells in the same manner as CYP6AA3-expressing cells and percent cell viability was plotted against test inhibitor concentrations, and results demonstrated that cell viability of parent *Sf*9 cells was not

The results shown in Fig. 3 indicate that cell viability of CYP6AA3-expressing cells was decreased upon increasing concentration of test inhibitors. Piperine, piperonyl butoxide, and -naphthoflavone could inhibit cytoprotective activity of CYP6AA3 more than xanthotoxin. This is thus in compliance with *in vitro* enzymatic inhibition assays, although piperonyl butoxide showed more potential than -naphthoflavone in inhibiting cytoprotective activity of CYP6AA3. Cell permeability of test compounds could be accounted for differences of cell-based MTT and *in vitro* enzymatic assays. The results however indicate usefulness of cells expressing P450 enzymes to primarily screen for P450 substrates and inhibitors. Our results indicated that PBO and piperine could inhibit P450s

cells and those marked with 'b' were reported in Duangkaew et al, 2011a. ND, not

*Sf*9 CYP6AA3 CYP6P7 CYP6P8

possess enzymatic activities against these insecticides.

**Pyrethroids** 

**Carbamates** 

determined.

affected by test compounds (data not shown).

Insecticides LC50 (M)

**Organophosphates**

#### **2.3 Use of cell-based MTT cytotoxicity assays to determine insecticide substrates and inhibitiors of** *An. minimus* **P450 enzymes**

Since *in vitro* reconstitution system demonstrated CYP6AA3 and CYP6P7 enzymatic activities against pyrethroids, further investigation of the ability of CYP6AA3 and CYP6P7 enzymes to eliminate pyrethroid toxicity from cells was assessed in P450-infected *Sf*9 cells. This can be accomplished because other than targeting on sodium channels of nervous system, pyrethroids possess toxic effects on cells such as inhibition of cell mitochondrial complex I or causing DNA damage and cell death (Gassner et al., 1997; Patel et al., 2007; Naravaneni & Jamil, 2005). Similar cell death and cytotoxic to cells caused by organophosphates and carbamate insecticides have also been reported (Maran et al., 2010; Schmuck & Mihail, 2004). This is supported by that we previously observed cytotoxic effects of deltamthrin on insect *Sf*9 cells. When using *Sf*9 cells that express CYP6AA3 in MTT assays, cell mortality was drastically decreased in the presence of insecticides due to degradation of deltamethrin by CYP6AA3 and thus posing cytoprotective role on *Sf*9 cells (Boonseupsakul et al., 2008). Use of insect cells to test for toxicity effects of compounds such as fungal metabolites (Fornelli et al., 2004) and pyridalyl insecticide (Saito et al., 2005) has been previously reported. Moreover, insect cells expressing P450 have also been successfully used to test detoxification capability of enzyme against cytotoxic xenochemicals (Grant et al., 1996; Greene et al., 2000). In this context, we used MTT assays to determine insecticide detoxification by P450 expressed in *Sf*9 cells. Insecticides tested were pyrethroids (deltamethrin, permethrin, cypermethrin, bifenthrin, bioallethrin and λ-cyhalothrin), organophosphates (chlorpyrifos, dichlorvos, fenitrothion and temephos), carbamates (thiodicarb and propoxur). Various concentrations (1-500 M) of insecticides were used for determination of cytotoxic effect of insecticides toward CYP6AA3-, CYP6P7-, and CYP6P8 expressing cells and compared to the control parent *Sf*9 cells. Cell viability of insecticide treated cells was measured by MTT assay as previously described (Boonseupsakul et al., 2008) and plotted against insecticide concentrations. The LC50 value of each insecticide was subsequently evaluated and obtained from each plot. Table 4 summarizes LC50 values of insecticides against *Sf*9 cells and cells with expression of P450s.

We observed that pyrethroids, organophosphates and carbamates are toxic to *Sf*9 parent cells. Since LC50 values of permethrin, bifenthrin, cypermethrin, and deltamethrin against CYP6AA3- and CYP6P7-expressing cells were approximately 4- to 19-folds significantly greater than those from parent *Sf*9 cells, these values imply that CYP6AA3 and CYP6P7 enzymes could cytoprotect *Sf*9 cells from pyrethroid toxicity. Conversely there was no significant difference of IC50 values between cells treated with organophosphate (chlorpyrifos, fenitrothion and temephos), carbamates (thiodicarb and propoxur) and bioallethrin pyrethroid insecticide, suggesting that expression of P450s did not cytoprotect cells from these insecticides. In addition CYP6P8 did not cytoprotect *Sf*9 cells against insecticides tested. It should be noted that LC50 value of λ-cyhalothrin in CYP6AA3 expressing cells was significantly greater than *Sf*9 parent cells, but not in CYP6P7-expressing cells. These results from MTT cytotoxicity assays are thus in agreement with *in vitro* enzymatic assays as described in Section 2.1. Thus abilities to cytoprotect against insecticide toxicity in infected *Sf*9 cells are due to P450-mediated enzymatic activity toward insecticides of CYP6AA3 and CYP6P7 (Duangkaew et al., 2011a). Together with *in vitro* enzymatic and cytotoxicity assays, we can conclude that both CYP6AA3 and CYP6P7 share metabolic activities against pyrethroids, but both enzymes play no role in degradations of organophosphates and carbamates. The results suggest that CYP6P8 plays no role in

**2.3 Use of cell-based MTT cytotoxicity assays to determine insecticide substrates and** 

Since *in vitro* reconstitution system demonstrated CYP6AA3 and CYP6P7 enzymatic activities against pyrethroids, further investigation of the ability of CYP6AA3 and CYP6P7 enzymes to eliminate pyrethroid toxicity from cells was assessed in P450-infected *Sf*9 cells. This can be accomplished because other than targeting on sodium channels of nervous system, pyrethroids possess toxic effects on cells such as inhibition of cell mitochondrial complex I or causing DNA damage and cell death (Gassner et al., 1997; Patel et al., 2007; Naravaneni & Jamil, 2005). Similar cell death and cytotoxic to cells caused by organophosphates and carbamate insecticides have also been reported (Maran et al., 2010; Schmuck & Mihail, 2004). This is supported by that we previously observed cytotoxic effects of deltamthrin on insect *Sf*9 cells. When using *Sf*9 cells that express CYP6AA3 in MTT assays, cell mortality was drastically decreased in the presence of insecticides due to degradation of deltamethrin by CYP6AA3 and thus posing cytoprotective role on *Sf*9 cells (Boonseupsakul et al., 2008). Use of insect cells to test for toxicity effects of compounds such as fungal metabolites (Fornelli et al., 2004) and pyridalyl insecticide (Saito et al., 2005) has been previously reported. Moreover, insect cells expressing P450 have also been successfully used to test detoxification capability of enzyme against cytotoxic xenochemicals (Grant et al., 1996; Greene et al., 2000). In this context, we used MTT assays to determine insecticide detoxification by P450 expressed in *Sf*9 cells. Insecticides tested were pyrethroids (deltamethrin, permethrin, cypermethrin, bifenthrin, bioallethrin and λ-cyhalothrin), organophosphates (chlorpyrifos, dichlorvos, fenitrothion and temephos), carbamates (thiodicarb and propoxur). Various concentrations (1-500 M) of insecticides were used for determination of cytotoxic effect of insecticides toward CYP6AA3-, CYP6P7-, and CYP6P8 expressing cells and compared to the control parent *Sf*9 cells. Cell viability of insecticide treated cells was measured by MTT assay as previously described (Boonseupsakul et al., 2008) and plotted against insecticide concentrations. The LC50 value of each insecticide was subsequently evaluated and obtained from each plot. Table 4 summarizes LC50 values of

**inhibitiors of** *An. minimus* **P450 enzymes** 

insecticides against *Sf*9 cells and cells with expression of P450s.

We observed that pyrethroids, organophosphates and carbamates are toxic to *Sf*9 parent cells. Since LC50 values of permethrin, bifenthrin, cypermethrin, and deltamethrin against CYP6AA3- and CYP6P7-expressing cells were approximately 4- to 19-folds significantly greater than those from parent *Sf*9 cells, these values imply that CYP6AA3 and CYP6P7 enzymes could cytoprotect *Sf*9 cells from pyrethroid toxicity. Conversely there was no significant difference of IC50 values between cells treated with organophosphate (chlorpyrifos, fenitrothion and temephos), carbamates (thiodicarb and propoxur) and bioallethrin pyrethroid insecticide, suggesting that expression of P450s did not cytoprotect cells from these insecticides. In addition CYP6P8 did not cytoprotect *Sf*9 cells against insecticides tested. It should be noted that LC50 value of λ-cyhalothrin in CYP6AA3 expressing cells was significantly greater than *Sf*9 parent cells, but not in CYP6P7-expressing cells. These results from MTT cytotoxicity assays are thus in agreement with *in vitro* enzymatic assays as described in Section 2.1. Thus abilities to cytoprotect against insecticide toxicity in infected *Sf*9 cells are due to P450-mediated enzymatic activity toward insecticides of CYP6AA3 and CYP6P7 (Duangkaew et al., 2011a). Together with *in vitro* enzymatic and cytotoxicity assays, we can conclude that both CYP6AA3 and CYP6P7 share metabolic activities against pyrethroids, but both enzymes play no role in degradations of organophosphates and carbamates. The results suggest that CYP6P8 plays no role in Insecticides LC50 (M) *Sf*9 CYP6AA3 CYP6P7 CYP6P8 **Pyrethroids**  Bioallethrinb 30.6 ± 2.1 32.7 ± 2.4 23.3 ± 3.9 29 Permethrinb 42.7 ± 1.8 406.7 ± 21.5a 214.7 ± 48.8a 78 Bifenthrin 45 ± 7.6 210 ± 12.4a 135 ± 51a 45 Cypermethrinb 21.8 ± 0.5 192.7 ± 30.4a 216.7 ± 21.4a 25 Deltamethrinb 27.5 ± 9.2 285.0 ± 27.8a 379.5 ± 21.9a 10 λ-Cyhalothrinb 38.4 ± 4.3 133.3 ± 37.5a 42.0 ± 1.8 ND **Organophosphates** Chlorpyrifosb 40.3 ± 6.5 56.3 ± 8.5 41.7 ± 2.8 60 Fenitrothion 25.0 ± 5.3 30.0 ± 6.4 ND 25 Temephos 11.0 ± 3.9 19.0 ± 7.5 ND ND Dichlorvos 32.0 ± 8.9 39.0 ± 6.4 ND ND **Carbamates**  Propoxurb 4.0 ± 6.6 4.7 ± 0.3 3.6 ± 0.2 ND Thiodicarb 28.6 ± 2.3 29.2 ± 4.7 ND ND

degradation of insecticides tested in this report. Moreover, such cytotoxicity results implicate that the method could also be applied for primary screening of compounds that have an inhibitory effect towards CYP6AA3 and CYP6P7, as well as P450 enzymes that possess enzymatic activities against these insecticides.

Table 4. Cytotoxicity effects by insecticides on P450-infected cells and the parent SF9 cells using MTT assays. Values reported for CYP6P8 were average obtained from experiments performed in duplicate. Those marked with 'a' were significantly different from parent *Sf*9 cells and those marked with 'b' were reported in Duangkaew et al, 2011a. ND, not determined.

To test whether inhibitors can be screened, MTT assays were performed with P450 expressing cells treated with 100 M deltamethin in the presence or absence of each test inhibitor. Concentrations of test inhibitory compounds were those of approximately LC20 values pre-determined by MTT assays on *Sf*9 cells. In cell-based inhibition assays, cell viability was determined upon co-incubation of test compound and deltamethrin, and normalized with viability of cells treated with test compound without deltamethrin. Inhibition experiments were performed with control *Sf*9 cells in the same manner as CYP6AA3-expressing cells and percent cell viability was plotted against test inhibitor concentrations, and results demonstrated that cell viability of parent *Sf*9 cells was not affected by test compounds (data not shown).

The results shown in Fig. 3 indicate that cell viability of CYP6AA3-expressing cells was decreased upon increasing concentration of test inhibitors. Piperine, piperonyl butoxide, and -naphthoflavone could inhibit cytoprotective activity of CYP6AA3 more than xanthotoxin. This is thus in compliance with *in vitro* enzymatic inhibition assays, although piperonyl butoxide showed more potential than -naphthoflavone in inhibiting cytoprotective activity of CYP6AA3. Cell permeability of test compounds could be accounted for differences of cell-based MTT and *in vitro* enzymatic assays. The results however indicate usefulness of cells expressing P450 enzymes to primarily screen for P450 substrates and inhibitors. Our results indicated that PBO and piperine could inhibit P450s

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control 277

aggregation and are unstable compared to rat and human CYPORs (Sarapusit et al., 2008, 2010)*.* While supplementation of FAD could increase activity of both full-length and membrane-deleted forms, FMN supplementation could increase activity of full-length form only (Sarapusit et al., 2008, 2010)*.* This behavior is different from membrane-deleted soluble CYPORs of rat and human in which exogenous FMN is readily incorporated into its FMNbinding site (Döhr et al., 2001; Shen et al., 1989). Due to loss of flavin cofactors and instability of *An. minimus* CYPOR, we have identified two key amino acids (Leu86 and Leu219 in FMN binding domain) by amino sequence alignment and shown that mutations of the two leucine residues into conserved phenylalanine residues that are found conserved among other CYPORs could rescue loss of FAD cofactor and increase protein stability of mosquito CYPOR (Sarapusit et al., 2008, 2010). These mutations do not affect kinetic mechanism and constants of enzyme. Double mutations of leucine to the conserved phenylalanine (L86F/L219F) in full-

CYP6AA3-mediated pyrethroid metabolism (Sarapusit et al., 2010), indicating that membranebound region of *An. minimus* CYPOR could influence both structural folding of FMN domain

The enzyme activity and kinetic mechanism of *flAnCYPOR* using cytochrome c as substrate are ionic strength dependent, with its mechanism following random Bi-Bi mechanism at low ionic strength and non-classical two-side Ping-Pong at high ionic strength. These mechanisms are different from rat, human, and house fly CYPORs (Murataliev et al., 2004; Sem & Kasper, 1994, 1995). In addition, *flAnCYPOR* could use extra flavins as additional substrates in which FAD binds at FAD/NADPH domain and FMN binds at FMN domain (as depicted in Fig. 4), resulting in an increase in its rate of electron transfer in CYP6AA3-

Fig. 4. Schematic representation of enzymatic reactions of CYPOR enzymes. CYPOR enzymes are represented in cartoon model of which FMN domain is in red color and FAD/NADPH domain is in green. Cofactors are represented in the stick mode; FMN is yellow colored, FAD is red, and NADP+ is cyan (rat CYOR: pdb code 1AMO). The cytochrome *c* substrate (cytychrome *c*: pdb code 1BBH) is in cyan cartoon model with an

In Figure 4, panel A illustrates common CYPOR (such as rat, human CYPORs) to which NADPH and cytochrome *c* substrate separately binds FAD/NADPH and FMN domains, while in panel B, *flAnCYPOR* could use extra flavins as additional substrates to which FAD

*55AnCYPOR*, could increase binding of FMN and increase

length *flAnCYPOR*, but not in

mediated pyrethroid degradation (Sarapusit et al., 2010).

orange heme group residing at the center.

and mediation of P450 catalysis (Murataliev et al., 2004; Wang et al., 1997).

and possess synergistic actions against deltamethrin cytotoxicity in Sf9 cells expressing P450. PBO has been used as pyrethroid synergist to enhance pyrethroid toxicity, as it can bind to P450s thereby inhibiting P450 activity (Fakoorziba et al., 2009; Kumar et al., 2002; Vijayan et al., 2007). Unfortunately PBO has been reported acutely toxic to mammals (Cox, 2002; Okamiya et al., 1998).

Fig. 3. Inhibition effect of test compounds against cell-based CYP6AA3-mediated deltamethrin detoxification measured by MTT assays.

#### **2.4** *An. minimus* **CYPOR and its possible role in regulation of P450-reaction cycle**

The NADPH-Cytochrome P450 oxidoreductase (CYPOR) enzyme is a member of di-flavin enzymes that transfers electrons, one by one, from NADPH through FAD and FMN to target enzymes to fulfill functioning of various cytochrome P450 enzymes as well as other enzymes (Murataliev et al., 2004). Other members of this class are those containing a flavoprotein subunit, such as nitric oxide synthase, sulfite reductase, methionine synthase reductase and protein NR 1. Detailed biochemical and structural studies of rat CYPOR reveal several conserved structural domains existed in this enzyme class, these are membrane-bound, FMN-binding, connecting, and FAD/NADPH binding domains (Wang et al., 1997).

The *An. minimus* CYPOR has been cloned and expressed in *E. coli*, and CYPOR could support CYP6AA3- and CYP6P7-mediated pyrethroid metabolisms *in vitro* (Duangkaew et al., 2011b; Kaewpa et al., 2007). However its expression has been of poor yield as a result of inclusion bodies formation. An attempt to obtain soluble protein by deletion of the first 55 amino acid residues comprising of membrane binding region (*55AnCYPOR*) has been successful (Sarapusit et al., 2008). However the protein could not be purified by 2'5'-ADP affinity column, indicating that NADPH binding capacity of mosquito CYPOR is low and this is different from CYPORs of other organisms such as rat and human (Sarapusit, 2009). Low binding affinity to 2'5'-ADP affinity column has also been recently reported in *An. gambiae* CYPOR (Lian et al., 2011). Only under specific condition was *55AnCYPOR* successfully expressed and purified to homogeneity by a combination of Ni2+NTA-affinity chromatography and G200-gel filtration chromatography (Sarapusit et al., 2008). Moreover both purified full-length (*flAnCYPOR*) and membrane-deleted *55AnCYPOR* proteins readily lose FAD and FMN cofactors, they undergo

and possess synergistic actions against deltamethrin cytotoxicity in Sf9 cells expressing P450. PBO has been used as pyrethroid synergist to enhance pyrethroid toxicity, as it can bind to P450s thereby inhibiting P450 activity (Fakoorziba et al., 2009; Kumar et al., 2002; Vijayan et al., 2007). Unfortunately PBO has been reported acutely toxic to mammals (Cox,

Fig. 3. Inhibition effect of test compounds against cell-based CYP6AA3-mediated

**2.4** *An. minimus* **CYPOR and its possible role in regulation of P450-reaction cycle**  The NADPH-Cytochrome P450 oxidoreductase (CYPOR) enzyme is a member of di-flavin enzymes that transfers electrons, one by one, from NADPH through FAD and FMN to target enzymes to fulfill functioning of various cytochrome P450 enzymes as well as other enzymes (Murataliev et al., 2004). Other members of this class are those containing a flavoprotein subunit, such as nitric oxide synthase, sulfite reductase, methionine synthase reductase and protein NR 1. Detailed biochemical and structural studies of rat CYPOR reveal several conserved structural domains existed in this enzyme class, these are membrane-bound, FMN-binding, connecting, and FAD/NADPH binding domains (Wang

The *An. minimus* CYPOR has been cloned and expressed in *E. coli*, and CYPOR could support CYP6AA3- and CYP6P7-mediated pyrethroid metabolisms *in vitro* (Duangkaew et al., 2011b; Kaewpa et al., 2007). However its expression has been of poor yield as a result of inclusion bodies formation. An attempt to obtain soluble protein by deletion of the first 55 amino acid

(Sarapusit et al., 2008). However the protein could not be purified by 2'5'-ADP affinity column, indicating that NADPH binding capacity of mosquito CYPOR is low and this is different from CYPORs of other organisms such as rat and human (Sarapusit, 2009). Low binding affinity to 2'5'-ADP affinity column has also been recently reported in *An. gambiae* CYPOR (Lian et al.,

homogeneity by a combination of Ni2+NTA-affinity chromatography and G200-gel filtration chromatography (Sarapusit et al., 2008). Moreover both purified full-length (*flAnCYPOR*) and

*55AnCYPOR* proteins readily lose FAD and FMN cofactors, they undergo

*55AnCYPOR*) has been successful

*55AnCYPOR* successfully expressed and purified to

deltamethrin detoxification measured by MTT assays.

residues comprising of membrane binding region (

2011). Only under specific condition was

2002; Okamiya et al., 1998).

et al., 1997).

membrane-deleted

aggregation and are unstable compared to rat and human CYPORs (Sarapusit et al., 2008, 2010)*.* While supplementation of FAD could increase activity of both full-length and membrane-deleted forms, FMN supplementation could increase activity of full-length form only (Sarapusit et al., 2008, 2010)*.* This behavior is different from membrane-deleted soluble CYPORs of rat and human in which exogenous FMN is readily incorporated into its FMNbinding site (Döhr et al., 2001; Shen et al., 1989). Due to loss of flavin cofactors and instability of *An. minimus* CYPOR, we have identified two key amino acids (Leu86 and Leu219 in FMN binding domain) by amino sequence alignment and shown that mutations of the two leucine residues into conserved phenylalanine residues that are found conserved among other CYPORs could rescue loss of FAD cofactor and increase protein stability of mosquito CYPOR (Sarapusit et al., 2008, 2010). These mutations do not affect kinetic mechanism and constants of enzyme. Double mutations of leucine to the conserved phenylalanine (L86F/L219F) in fulllength *flAnCYPOR*, but not in *55AnCYPOR*, could increase binding of FMN and increase CYP6AA3-mediated pyrethroid metabolism (Sarapusit et al., 2010), indicating that membranebound region of *An. minimus* CYPOR could influence both structural folding of FMN domain and mediation of P450 catalysis (Murataliev et al., 2004; Wang et al., 1997).

The enzyme activity and kinetic mechanism of *flAnCYPOR* using cytochrome c as substrate are ionic strength dependent, with its mechanism following random Bi-Bi mechanism at low ionic strength and non-classical two-side Ping-Pong at high ionic strength. These mechanisms are different from rat, human, and house fly CYPORs (Murataliev et al., 2004; Sem & Kasper, 1994, 1995). In addition, *flAnCYPOR* could use extra flavins as additional substrates in which FAD binds at FAD/NADPH domain and FMN binds at FMN domain (as depicted in Fig. 4), resulting in an increase in its rate of electron transfer in CYP6AA3 mediated pyrethroid degradation (Sarapusit et al., 2010).

Fig. 4. Schematic representation of enzymatic reactions of CYPOR enzymes. CYPOR enzymes are represented in cartoon model of which FMN domain is in red color and FAD/NADPH domain is in green. Cofactors are represented in the stick mode; FMN is yellow colored, FAD is red, and NADP+ is cyan (rat CYOR: pdb code 1AMO). The cytochrome *c* substrate (cytychrome *c*: pdb code 1BBH) is in cyan cartoon model with an orange heme group residing at the center.

In Figure 4, panel A illustrates common CYPOR (such as rat, human CYPORs) to which NADPH and cytochrome *c* substrate separately binds FAD/NADPH and FMN domains, while in panel B, *flAnCYPOR* could use extra flavins as additional substrates to which FAD

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## **3. Conclusion**

The results of this study on CYP6AA3 and CYP6P7 could lay groundwork into an understanding of the mechanisms that control substrates and reaction selectivity of both P450 enzymes, thereby increase an understanding of P450-mediated resistance mechanisms to various pesticides. The kinetic values, metabolic profile of pyrethroid insecticide metabolisms and inhibition patterns by different inhibitors of CYP6AA3 are different from CYP6P7. Future approach could aim at the strategy involving finding a collection of substrates together with structural models and mutation analyses of CYP6AA3 and CYP6P7 that affect specific P450 catalysis. Moreover, characterizing inhibitors and inhibition mechanisms of large collection of compounds with known chemical structures against CYP6AA3 and CYP6P7 enzymes could give insight into an understanding of mechanisms of cytochrome P450s that metabolize pyrethroids. It is conceivable that CYP6P8 does not play role in detoxification of pyrethroid, organophosphate, and carbamate insecticides. Further substrate search for CYP6P8 may help to learn about its overexpression in pyrethroidresistant mosquito. Together with knowledge obtained from enzymatic properties of *An. minimus* CYPOR, this could improve our understanding of P450-mediated detoxification of insecticides, as well as provide a foundation for rational design of P450 synergists specific for P450-mediated pesticide resistance and thus resistant management in mosquito vector control program.

## **4. Acknowledgment**

This work is supported by Grant BRG5380002 from Thailand Research Fund and Mahidol University; grant BT-B01-XG-14-4803 from BIOTEC, National Science and Technology Development Agency.

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The results of this study on CYP6AA3 and CYP6P7 could lay groundwork into an understanding of the mechanisms that control substrates and reaction selectivity of both P450 enzymes, thereby increase an understanding of P450-mediated resistance mechanisms to various pesticides. The kinetic values, metabolic profile of pyrethroid insecticide metabolisms and inhibition patterns by different inhibitors of CYP6AA3 are different from CYP6P7. Future approach could aim at the strategy involving finding a collection of substrates together with structural models and mutation analyses of CYP6AA3 and CYP6P7 that affect specific P450 catalysis. Moreover, characterizing inhibitors and inhibition mechanisms of large collection of compounds with known chemical structures against CYP6AA3 and CYP6P7 enzymes could give insight into an understanding of mechanisms of cytochrome P450s that metabolize pyrethroids. It is conceivable that CYP6P8 does not play role in detoxification of pyrethroid, organophosphate, and carbamate insecticides. Further substrate search for CYP6P8 may help to learn about its overexpression in pyrethroidresistant mosquito. Together with knowledge obtained from enzymatic properties of *An. minimus* CYPOR, this could improve our understanding of P450-mediated detoxification of insecticides, as well as provide a foundation for rational design of P450 synergists specific for P450-mediated pesticide resistance and thus resistant management in mosquito vector

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**Part 3** 

**Pest Management** 


## **Part 3**

**Pest Management** 

284 Insecticides – Pest Engineering

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**13** 

*Uruguay* 

Martina Díaz and Carmen Rossini

**Bioactive Natural Products from Sapindaceae** 

*Laboratorio de Ecología Química, Facultad de Química, Universidad de la República,* 

**Deterrent and Toxic Metabolites Against Insects** 

The Sapindaceae (soapberry family) is a family of flowering plants with about 2000 species occurring from temperate to tropical regions throughout the world. Members of this family have been widely studied for their pharmacological activities; being *Paullinia* and *Dodonaea* good examples of genera containing species with these properties. Besides, the family includes many species with economically valuable tropical fruits, and wood (Rodriguez

Antioxidant, anti-inflammatory and anti-diabetic properties are the pharmacological activities most commonly described for this family (Sofidiya et al. 2008; Simpson et al. 2010; Veeramani et al. 2010; Muthukumran et al. 2011). These activities are in some cases accounted for isolated phenolic compounds such as prenylated flavonoids (Niu et al. 2010), but in many cases, it is still unknown which are the active principles. Indeed, there are many studies of complex mixtures (crude aqueous or ethanolic extracts) from different species in which several other pharmacological activities have been described without characterization of the active compounds [*e.g.* antimigrane (Arulmozhi et al. 2005), anti-ulcerogenic (Dharmani et al. 2005), antimalarial (Waako et al. 2005), anti-microbial (Getie et al. 2003)]. Phytochemical studies on Sapindaceae species are abundant and various kinds of natural products have been isolated and elucidated. Examples of these are flavonoids from *Dodonaea* spp. (Getie et al. 2002; Wollenweber & Roitman 2007) and *Koelreuteria* spp. (Mahmoud et al. 2001), linear triterpenes from *Cupaniopsis* spp. (Bousserouel et al. 2005) and caffeine, xanthenes and cathequines from *Paullinia* spp. (Benlekehal et al. 2001; Sousa et al. 2009). All these compounds are naturally occurring in almost every plant family, however, the Sapindaceae do produce an unusual group of secondary metabolites: the cyanolipids (Avato et al. 2005). Eventhough these compounds exhibit a potential health hazard for humans and animals, for the plants, cyanolipids may have a protective physiological role. However, not many investigations have been developed involving the study of the ecological interactions among the plants producing them and other sympatric organisms. On the other hand, the toxicity of these cyanocompounds might be a potential source of pesticides. Indeed, not only cyanocompounds, but also a wide range of species of Sapindaceae have been tested on their anti-insect activity. Several extracts, fractions or pure compounds of different phenological stages, have been tested against diverse species of lepidopterans, dipterans and coleopterans of major importance in agriculture as well as in veterinary and medical applications. Examples of this include the larvicidal activity of *Magonia pubescens* against *Aedes aegypti*

1958), as well as many genera with reported anti-insect activity.

**1. Introduction** 

## **Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects**

Martina Díaz and Carmen Rossini

*Laboratorio de Ecología Química, Facultad de Química, Universidad de la República, Uruguay* 

## **1. Introduction**

The Sapindaceae (soapberry family) is a family of flowering plants with about 2000 species occurring from temperate to tropical regions throughout the world. Members of this family have been widely studied for their pharmacological activities; being *Paullinia* and *Dodonaea* good examples of genera containing species with these properties. Besides, the family includes many species with economically valuable tropical fruits, and wood (Rodriguez 1958), as well as many genera with reported anti-insect activity.

Antioxidant, anti-inflammatory and anti-diabetic properties are the pharmacological activities most commonly described for this family (Sofidiya et al. 2008; Simpson et al. 2010; Veeramani et al. 2010; Muthukumran et al. 2011). These activities are in some cases accounted for isolated phenolic compounds such as prenylated flavonoids (Niu et al. 2010), but in many cases, it is still unknown which are the active principles. Indeed, there are many studies of complex mixtures (crude aqueous or ethanolic extracts) from different species in which several other pharmacological activities have been described without characterization of the active compounds [*e.g.* antimigrane (Arulmozhi et al. 2005), anti-ulcerogenic (Dharmani et al. 2005), antimalarial (Waako et al. 2005), anti-microbial (Getie et al. 2003)].

Phytochemical studies on Sapindaceae species are abundant and various kinds of natural products have been isolated and elucidated. Examples of these are flavonoids from *Dodonaea* spp. (Getie et al. 2002; Wollenweber & Roitman 2007) and *Koelreuteria* spp. (Mahmoud et al. 2001), linear triterpenes from *Cupaniopsis* spp. (Bousserouel et al. 2005) and caffeine, xanthenes and cathequines from *Paullinia* spp. (Benlekehal et al. 2001; Sousa et al. 2009). All these compounds are naturally occurring in almost every plant family, however, the Sapindaceae do produce an unusual group of secondary metabolites: the cyanolipids (Avato et al. 2005). Eventhough these compounds exhibit a potential health hazard for humans and animals, for the plants, cyanolipids may have a protective physiological role. However, not many investigations have been developed involving the study of the ecological interactions among the plants producing them and other sympatric organisms. On the other hand, the toxicity of these cyanocompounds might be a potential source of pesticides. Indeed, not only cyanocompounds, but also a wide range of species of Sapindaceae have been tested on their anti-insect activity. Several extracts, fractions or pure compounds of different phenological stages, have been tested against diverse species of lepidopterans, dipterans and coleopterans of major importance in agriculture as well as in veterinary and medical applications. Examples of this include the larvicidal activity of *Magonia pubescens* against *Aedes aegypti*

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 289

For instance, a saponin extract from fruits from this species showed toxicity against adults of the greenhouse whitefly *Trialeurodes vaporariorum* (Hemiptera: Aleyrodidae) (Porras & Lopez-Avila 2009); and complete ethanolic extracts from fruits have shown larvicidal and morphological alterations effects on the mosquito *Aedes aegypti* (Diptera: Culicidae) (Ferreira Barreto et al. 2006). Some other saponins presenting other kinds of biological activity, isolated from the fruits of this species, are shown in Figure 1 (Lemos et al. 1992; Ribeiro et al.

*Sapindus emarginatus*, another tree from this genus, widely distributed in India, has also demonstrated larvicidal activity of its fruit extract against three important vector mosquitoes: *A. aegypti*, *Anopheles stephensi* and *Culex quimquefasciatus* (Diptera: Culicidae) (Koodalingam et al. 2009). Later, this group has also investigated the impact of the extracts on the activity of mosquito phosphatases and esterases to gain an insight into the extent of disturbance in metabolic homeostasis inflicted upon exposure to the extract (Koodalingam et al. 2011). Previous reports on this species have shown that the pericarps contain triterpene saponins (Figure 1), which are commonly used as antifertility, antipruritic and antiinflammatory agents in traditional Indian and Thai medicine (Jain 1976; Kanchanapoom et al. 2001). Perhaps, anti insect activity may be due to saponins in this plant similarly to the

Activity of members of this genus against other insect orders, further than dipterans and lepidopterans, has also been evaluated, including coleoptera and lice. That is the case of the extract from *Sapindus trifoliatus* fruit cortex which showed activity against the red flour beetle *Tribolium castaneum* (Herbst) (Coleoptera: Tenebrionidae) (Mukherjee & Joseph 2000). In this case, weight gain was significantly reduced when larvae were fed on diets including the extract at different doses; and females topically treated -upon emergence- with the extract laid fewer viable eggs. (Mukherjee & Joseph 2001). Another ethanolic fruit extracts, in this case from *Sapindus mukorossi*, also showed anti coleopteran activity against another pest of stored grains, *Sitophilus oryzae* (Coleoptera: Curculionidae) and also against *Pediculus humanus* (Phthiraptera: Pediculidae) (Rahman et al. 2007). Finally, from the methanolic extract of fruits of this species triterpenic saponins (Figure 1) have also been isolated and these natural products demonstrated their potential as growth regulators and antifeedants against *Spodoptera littura* (Lepidoptera: Noctuidae), both as glycosides and as free genines (Saha et al. 2009). In this particular study, it was verified that upon hydrolysis of the saponins, the growth regulatory activity was improved, whereas very little difference was

All in all, the genus *Sapindus* contains a variety of species which have been studied on their activity for some insects from different orders. In spite of the fact that not many reports do exist on the action of isolated compounds, the previous ethnobotanical uses of *Sapindus* spp. and the isolation of some active saponins from this genus, may suggest that this group of secondary metabolites might be related to the anti-insect activity. Saponins -glycosides of sapogenins containing a monosaccharide or a polysaccharide unit- reduce the surface tension becoming biological detergents. They are widely distributed secondary plant metabolites, found among almost 100 plant families (Bruneton 1995). Being effective defences for some insects (Plasman et al. 2001; Prieto et al. 2007), saponins have been implied in mechanisms of plant resistance against potential herbivores (Nielsen et al. 2010). The genus *Sapindus*, rich in this kind of compounds, may therefore be promissory raw material to develop plant pest control products. Further information can be found at recent works reviewing saponins from *Sapindus* spp. and their activity (Pelegrini et al. 2008;

1995).

case of *Sapindus saponaria* (Porras et al. 2009).

found in regard to the antifeedant activity.

Sharma et al. 2011).

(Diptera: Culicidae) (Arruda et al. 2003a,b); the toxicity of *Sapindus* spp. against *Sitophilus oryzae* (Coleoptera: Curculionidae) (Zidan et al. 1993; Rahman et al. 2007); and the toxicity of *Dodonaea* spp. against *Spodoptera* spp. (Lepidoptera: Noctuidae) (Abdel et al. 1995; El-Din & El-Gengaihi 2000; Deepa & Remadevi 2007; Malarvannan et al. 2008). The activity against different pest models of extracts from various South American species within the Sapindaceae was recently described. These species included *Allophylus edulis*, *Dodonaea viscosa* and *Serjania meridionalis*, from which isolated metabolites with anti-insect capacities have not yet been described (Castillo et al. 2009). This chapter will examine the available information on extracts and secondary metabolites from Sapindaceae focused on their defensive role for the plant against herbivory; and consequently this appraisal will also present a compilation of potential anti-insect agents from Sapindaceae.

## **2. Sapindaceae: its anti-insect potential**

Anti-insect activity has been described in at least 15 of the 202 genera (Anonymous 2011) belonging to this family. Among these findings, the cases in which the bioactive compounds were isolated represent the least. It has been in general tested the activity of aqueous or ethanolic extracts from different organs and from plants of different phenological stages against a variety of insect targets using different bioassay designs. As a consequence, different modes of action have been described. Extracts have revealed to be potentially deterrent agents, growth inhibitors and even toxic agents against different genera of insects. The following appraisal comprises the main Sapindaceae genera from which extracts or isolated compounds with anti-insect activity have been described.

#### **2.1** *Sapindus*

A vast number of species showing great potential as anti-insect agents belong to this genus. *Sapindus saponaria*, a tree widely distributed in Central and South America, is also frequently used as ornamental (Lorenzi 2004). Brazilian people commonly prepare homemade soap from this tree; and use its seeds to make handcrafts. Its wood is broadly used in construction. Its fruits and roots are popularly used as painkillers, astringents, expectorants and diuretics (Ferreira Barreto et al. 2006). Besides, its medicinal potential as healing and anti thrombotic agents has been studied. Research on that area has revealed that flavonoids in the leaf extracts are responsible for those activities (Meyer Albiero et al. 2002). On the other hand, much research has been devoted to the anti-insect capacity of extracts from different organs of this plant. Boiça Junior et al (2005), on their search for activity against larvae of the cabbage pest *Plutella xylostella* (Lepidoptera: Plutellidae) investigated eighteen plant species from a variety of families, finding that aqueous leaf extract of *S. saponaria* was one of the most active products. The extract produced 100 % of mortality in tests where the larvae were offered cabbage foliage disks coated with the extracts to be evaluated as a sole food (Boiça Junior et al. 2005). In another study, the aqueous fruit extract of this tree showed deterrent properties against another cabbage pest, *Ascia monuste orseis* (Lepidoptera: Pieridae*)*. In this case the activity was comparable to that showed by aqueous extracts of the neem tree, *Azadirachta indica*, the newest botanical pesticide in the market (Isman et al. 1996; Medeiros et al. 2007). The aqueous seeds´ extracts were evaluated against another lepidopteran, *Spodoptera frugiperda* (Lepidoptera: Noctuidae), showing strong effect on larvae development and midgut trypsin activity (dos Santos et al. 2008). Not only against lepidopterans has this tree revealed anti-insect potential, but also against other insect orders.

(Diptera: Culicidae) (Arruda et al. 2003a,b); the toxicity of *Sapindus* spp. against *Sitophilus oryzae* (Coleoptera: Curculionidae) (Zidan et al. 1993; Rahman et al. 2007); and the toxicity of *Dodonaea* spp. against *Spodoptera* spp. (Lepidoptera: Noctuidae) (Abdel et al. 1995; El-Din & El-Gengaihi 2000; Deepa & Remadevi 2007; Malarvannan et al. 2008). The activity against different pest models of extracts from various South American species within the Sapindaceae was recently described. These species included *Allophylus edulis*, *Dodonaea viscosa* and *Serjania meridionalis*, from which isolated metabolites with anti-insect capacities have not yet been described (Castillo et al. 2009). This chapter will examine the available information on extracts and secondary metabolites from Sapindaceae focused on their defensive role for the plant against herbivory; and consequently this appraisal will also

Anti-insect activity has been described in at least 15 of the 202 genera (Anonymous 2011) belonging to this family. Among these findings, the cases in which the bioactive compounds were isolated represent the least. It has been in general tested the activity of aqueous or ethanolic extracts from different organs and from plants of different phenological stages against a variety of insect targets using different bioassay designs. As a consequence, different modes of action have been described. Extracts have revealed to be potentially deterrent agents, growth inhibitors and even toxic agents against different genera of insects. The following appraisal comprises the main Sapindaceae genera from which extracts or

A vast number of species showing great potential as anti-insect agents belong to this genus. *Sapindus saponaria*, a tree widely distributed in Central and South America, is also frequently used as ornamental (Lorenzi 2004). Brazilian people commonly prepare homemade soap from this tree; and use its seeds to make handcrafts. Its wood is broadly used in construction. Its fruits and roots are popularly used as painkillers, astringents, expectorants and diuretics (Ferreira Barreto et al. 2006). Besides, its medicinal potential as healing and anti thrombotic agents has been studied. Research on that area has revealed that flavonoids in the leaf extracts are responsible for those activities (Meyer Albiero et al. 2002). On the other hand, much research has been devoted to the anti-insect capacity of extracts from different organs of this plant. Boiça Junior et al (2005), on their search for activity against larvae of the cabbage pest *Plutella xylostella* (Lepidoptera: Plutellidae) investigated eighteen plant species from a variety of families, finding that aqueous leaf extract of *S. saponaria* was one of the most active products. The extract produced 100 % of mortality in tests where the larvae were offered cabbage foliage disks coated with the extracts to be evaluated as a sole food (Boiça Junior et al. 2005). In another study, the aqueous fruit extract of this tree showed deterrent properties against another cabbage pest, *Ascia monuste orseis* (Lepidoptera: Pieridae*)*. In this case the activity was comparable to that showed by aqueous extracts of the neem tree, *Azadirachta indica*, the newest botanical pesticide in the market (Isman et al. 1996; Medeiros et al. 2007). The aqueous seeds´ extracts were evaluated against another lepidopteran, *Spodoptera frugiperda* (Lepidoptera: Noctuidae), showing strong effect on larvae development and midgut trypsin activity (dos Santos et al. 2008). Not only against lepidopterans has this tree revealed anti-insect potential, but also against other insect orders.

present a compilation of potential anti-insect agents from Sapindaceae.

isolated compounds with anti-insect activity have been described.

**2. Sapindaceae: its anti-insect potential** 

**2.1** *Sapindus* 

For instance, a saponin extract from fruits from this species showed toxicity against adults of the greenhouse whitefly *Trialeurodes vaporariorum* (Hemiptera: Aleyrodidae) (Porras & Lopez-Avila 2009); and complete ethanolic extracts from fruits have shown larvicidal and morphological alterations effects on the mosquito *Aedes aegypti* (Diptera: Culicidae) (Ferreira Barreto et al. 2006). Some other saponins presenting other kinds of biological activity, isolated from the fruits of this species, are shown in Figure 1 (Lemos et al. 1992; Ribeiro et al. 1995).

*Sapindus emarginatus*, another tree from this genus, widely distributed in India, has also demonstrated larvicidal activity of its fruit extract against three important vector mosquitoes: *A. aegypti*, *Anopheles stephensi* and *Culex quimquefasciatus* (Diptera: Culicidae) (Koodalingam et al. 2009). Later, this group has also investigated the impact of the extracts on the activity of mosquito phosphatases and esterases to gain an insight into the extent of disturbance in metabolic homeostasis inflicted upon exposure to the extract (Koodalingam et al. 2011). Previous reports on this species have shown that the pericarps contain triterpene saponins (Figure 1), which are commonly used as antifertility, antipruritic and antiinflammatory agents in traditional Indian and Thai medicine (Jain 1976; Kanchanapoom et al. 2001). Perhaps, anti insect activity may be due to saponins in this plant similarly to the case of *Sapindus saponaria* (Porras et al. 2009).

Activity of members of this genus against other insect orders, further than dipterans and lepidopterans, has also been evaluated, including coleoptera and lice. That is the case of the extract from *Sapindus trifoliatus* fruit cortex which showed activity against the red flour beetle *Tribolium castaneum* (Herbst) (Coleoptera: Tenebrionidae) (Mukherjee & Joseph 2000). In this case, weight gain was significantly reduced when larvae were fed on diets including the extract at different doses; and females topically treated -upon emergence- with the extract laid fewer viable eggs. (Mukherjee & Joseph 2001). Another ethanolic fruit extracts, in this case from *Sapindus mukorossi*, also showed anti coleopteran activity against another pest of stored grains, *Sitophilus oryzae* (Coleoptera: Curculionidae) and also against *Pediculus humanus* (Phthiraptera: Pediculidae) (Rahman et al. 2007). Finally, from the methanolic extract of fruits of this species triterpenic saponins (Figure 1) have also been isolated and these natural products demonstrated their potential as growth regulators and antifeedants against *Spodoptera littura* (Lepidoptera: Noctuidae), both as glycosides and as free genines (Saha et al. 2009). In this particular study, it was verified that upon hydrolysis of the saponins, the growth regulatory activity was improved, whereas very little difference was found in regard to the antifeedant activity.

All in all, the genus *Sapindus* contains a variety of species which have been studied on their activity for some insects from different orders. In spite of the fact that not many reports do exist on the action of isolated compounds, the previous ethnobotanical uses of *Sapindus* spp. and the isolation of some active saponins from this genus, may suggest that this group of secondary metabolites might be related to the anti-insect activity. Saponins -glycosides of sapogenins containing a monosaccharide or a polysaccharide unit- reduce the surface tension becoming biological detergents. They are widely distributed secondary plant metabolites, found among almost 100 plant families (Bruneton 1995). Being effective defences for some insects (Plasman et al. 2001; Prieto et al. 2007), saponins have been implied in mechanisms of plant resistance against potential herbivores (Nielsen et al. 2010). The genus *Sapindus*, rich in this kind of compounds, may therefore be promissory raw material to develop plant pest control products. Further information can be found at recent works reviewing saponins from *Sapindus* spp. and their activity (Pelegrini et al. 2008; Sharma et al. 2011).

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 291

From this genus, there are two species to which almost all research has been devoted: *Dodonaea angustifolia* and *Dodonaea viscosa*. These two species are considered by some taxonomists to be synonymous, while others recognize *D. angustifolia* as a sub-species of *D. viscosa* (cited in Omosa et al. 2010). *D. angustifolia* is widely distributed in Australia, Africa, Asia and South America; and it has been employed until present days in traditional medicine all over the world. It is traditionally used as analgesic, laxative, antipyretic, and to treat rheumatism, eczema, and skin ailments (Malarvannan et al. 2009; Omosa et al. 2010). *Dodonaea viscosa* is a shrub, rarely a small tree, widely distributed in tropical and subtropical areas of both hemispheres. It is used in folk medicine as a febrifuge, a diaphoretic drug, and also for the treatment of rheumatism, gout, inflammations, swelling and pain (Niu et al.

Anti-insect activity has been described for extracts from both plant species mostly against lepidopterans (Malarvannan & Subashini 2007; Malarvannan et al. 2008; 2009; Sharaby et al. 2009). For instance, extracts of leaves of *D. angustifolia* (obtained with hexane, petroleum ether, chloroform, acetone and water) were tested in field bioassays, showing to be effective biocontrol agents for the larvae of *Earias vitella* (Lepidoptera: Noctuidae) (Malarvannan et al. 2007). Besides, those extracts also showed ovicidal activity against *Helicoverpa armigera* (Lepidoptera: Noctuidae) (Malarvanan 2003). However, while in the case of extracts from different organs (fruits, leaves and twigs) of *D. viscosa* coming from Uruguay, none of these products proved to be active against the polyphagous *Spodoptera littoralis* (Lepidoptera: Noctuidae) (Castillo et al. 2009), insects from other orders were deterred by *D. viscosa* extracts. Interestingly, while extracts from leaves and twigs exhibited good activity against aphids (*Rhopalosiphum padi* and *Myzus persicae*) and a coleopteran (*Epilachna paenulata*), they were innocuous to beneficial insects (*Apis mellifera*) (Castillo et al. 2009). This selectivity makes *D. viscosa* a good candidate from which to develop botanical pesticides. Another independent study also showed a strong contact activity of the seed extracts against the

*D. angustifolia* is known to contain essential oils, flavonoids, terpenoids, phenols, coumarins, sterols and unidentified alcohols (Malarvannan et al. 2008). Meanwhile several flavonoids, diterpenoid acids and saponins have been isolated from *D. viscosa* (Niu et al. 2010). However, the chemical basis for the pesticide and antifeedant activities remains unclear as tests on individual compounds have not been performed. Nevertheless, a series of clerodane diterpenoids (Figure 2) and prenylated flavonoids (Figure 3) were isolated from the aerial parts of *D. viscosa* from China, having them not shown larvicidal activity against two mosquito species tested (Niu et al. 2010), however the authors stated that previous studies showed activity of these clerodanes against two lepidopterans (*Plutella xylostella* and *Pieris* 

At the same time, an investigation on this family of compounds from *D. angustifolia* from Kenya showed that the extracts from the leaf surface of this plant is composed mainly by clerodanes (Figure 2) and also by methylated flavones and flavonols (Figure 3) (Omosa et al.). Clerodanes isolated from *D. viscosa* and *D. angustifolia* belong to the *neo*-clerodane group. As it is well known, these secondary metabolites have a structure based on the carbon skeleton and absolute stereochemistry of clerodin (Klein Gebbinck et al. 2002) isolated first from *Clerodendron infortunatum* (Lamiaceae) (Banerjee 1936). This large group of plant secondary metabolites have been described mainly from Lamiaceae and Asteraceae, and they have exhibited a wide range of anti-insect properties as it has been reviewed

**2.2** *Dodonaea* 

2010).

coleopteran, *S. oryzae* (Zhao et al. 2006).

*rapae*) and against the coleopteran *Sitophilus oryzae*.

Fig. 1. Saponins isolated from *Sapindus* and *Nephelium* spp.

#### **2.2** *Dodonaea*

290 Insecticides – Pest Engineering

Fig. 1. Saponins isolated from *Sapindus* and *Nephelium* spp.

From this genus, there are two species to which almost all research has been devoted: *Dodonaea angustifolia* and *Dodonaea viscosa*. These two species are considered by some taxonomists to be synonymous, while others recognize *D. angustifolia* as a sub-species of *D. viscosa* (cited in Omosa et al. 2010). *D. angustifolia* is widely distributed in Australia, Africa, Asia and South America; and it has been employed until present days in traditional medicine all over the world. It is traditionally used as analgesic, laxative, antipyretic, and to treat rheumatism, eczema, and skin ailments (Malarvannan et al. 2009; Omosa et al. 2010). *Dodonaea viscosa* is a shrub, rarely a small tree, widely distributed in tropical and subtropical areas of both hemispheres. It is used in folk medicine as a febrifuge, a diaphoretic drug, and also for the treatment of rheumatism, gout, inflammations, swelling and pain (Niu et al. 2010).

Anti-insect activity has been described for extracts from both plant species mostly against lepidopterans (Malarvannan & Subashini 2007; Malarvannan et al. 2008; 2009; Sharaby et al. 2009). For instance, extracts of leaves of *D. angustifolia* (obtained with hexane, petroleum ether, chloroform, acetone and water) were tested in field bioassays, showing to be effective biocontrol agents for the larvae of *Earias vitella* (Lepidoptera: Noctuidae) (Malarvannan et al. 2007). Besides, those extracts also showed ovicidal activity against *Helicoverpa armigera* (Lepidoptera: Noctuidae) (Malarvanan 2003). However, while in the case of extracts from different organs (fruits, leaves and twigs) of *D. viscosa* coming from Uruguay, none of these products proved to be active against the polyphagous *Spodoptera littoralis* (Lepidoptera: Noctuidae) (Castillo et al. 2009), insects from other orders were deterred by *D. viscosa* extracts. Interestingly, while extracts from leaves and twigs exhibited good activity against aphids (*Rhopalosiphum padi* and *Myzus persicae*) and a coleopteran (*Epilachna paenulata*), they were innocuous to beneficial insects (*Apis mellifera*) (Castillo et al. 2009). This selectivity makes *D. viscosa* a good candidate from which to develop botanical pesticides. Another independent study also showed a strong contact activity of the seed extracts against the coleopteran, *S. oryzae* (Zhao et al. 2006).

*D. angustifolia* is known to contain essential oils, flavonoids, terpenoids, phenols, coumarins, sterols and unidentified alcohols (Malarvannan et al. 2008). Meanwhile several flavonoids, diterpenoid acids and saponins have been isolated from *D. viscosa* (Niu et al. 2010). However, the chemical basis for the pesticide and antifeedant activities remains unclear as tests on individual compounds have not been performed. Nevertheless, a series of clerodane diterpenoids (Figure 2) and prenylated flavonoids (Figure 3) were isolated from the aerial parts of *D. viscosa* from China, having them not shown larvicidal activity against two mosquito species tested (Niu et al. 2010), however the authors stated that previous studies showed activity of these clerodanes against two lepidopterans (*Plutella xylostella* and *Pieris rapae*) and against the coleopteran *Sitophilus oryzae*.

At the same time, an investigation on this family of compounds from *D. angustifolia* from Kenya showed that the extracts from the leaf surface of this plant is composed mainly by clerodanes (Figure 2) and also by methylated flavones and flavonols (Figure 3) (Omosa et al.). Clerodanes isolated from *D. viscosa* and *D. angustifolia* belong to the *neo*-clerodane group. As it is well known, these secondary metabolites have a structure based on the carbon skeleton and absolute stereochemistry of clerodin (Klein Gebbinck et al. 2002) isolated first from *Clerodendron infortunatum* (Lamiaceae) (Banerjee 1936). This large group of plant secondary metabolites have been described mainly from Lamiaceae and Asteraceae, and they have exhibited a wide range of anti-insect properties as it has been reviewed

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 293

O

OH O

OH

Fig. 3. Flavonoids from *Dodonaea* spp. (Niu et al. 2010; Omosa et al. 2010; Teffo et al. 2010).

*Paullinia* spp. is one of the few purine alkaloid-containing genera used to prepare stimulant drinks worldwide (Weckerle et al. 2003). *Paullinia clavigera* grows in primary forests and shores of the South American Amazonic aquatic ecosystems, and it has been studied mostly in Perú for the control of different insect pests (cited in Pérez et al. 2010). The toxicity of aqueous extracts of lianas from this species against larvae of *Anopheles benarrochi (*Diptera: Culicidae) (Pérez & Iannacone 2004) and afterwards the mortality and repellence of such extracts against larvae of *Rhynchophorus palmarum* (Coleoptera: Curculionidae) (Pérez & Iannacone 2006) were tested. Besides, the activity of aqueous extracts against *Eupalamides cyparissias* (Lepidoptera: Castniidae), and the activity of hydroalcoholic extracts against *Tuthillia cognata* (Hemiptera: Psyllidae) (Pérez et al. 2008) were also reported, showing the potential of this vegetal species as a biocontrol agent against different insect orders. However, it is still unclear which compounds are responsible for the anti-insect effects of *P. clavigera*. Flavonoids, phenols, triterpenes and saponins were detected in a phytochemical study in extracts from the stem cortex of *P. clavigera.* (Pérez et al. 2010); and other species from the same genus (*P. cururu*) contain saponins, tannins and polyphenols (Wilbert &

O

R4

OH

HO

O

O

O

 glucosyl H kaempferol 3-*O*--D-glucoside galactosyl H kaempferol 3-*O*--D-galactoside rhamnosyl H kaempferol 3-*O*--L-rhamnoside rhamnoglucosyl H kaempferol 3-*O*-rhamnoglucoside glucosyl OH quercetin 3-*O*--D-glucoside rhamnosyl OH quercetin 3-*O*--L-rhamnoside rhamnoglucosyl OH quercetin 3-*O*-rhamnoglucoside

OR1

R5

OH

**13** H H kaempferol

O

OH

OH O

O

OH

OH

OH

HO

R1 R5

O

OH O

O

OH O

 R1 R2 R3 R4 OH H OH OMe OMe OMe OH OH OMe OMe OH OMe OMe H OMe OMe OH H OH OH OMe H OMe OMe OH OMe OMe OMe OH H OMe OMe OMe H OMe OH OMe OMe OMe OH

Haiek 1991).

HO

O

R2

R3

O

R1

**2.3** *Magonia and Paullinia species* 

OH

R4

O

HO

O

O

O

R1

OH

 R1 R4 **11** OH OH **12** OMe OMe

Fig. 2. Clerodanes from *Dodonaea* spp. Structures shown were isolated from *D. viscosa* (Niu et al. 2010) with the exception of the indicated ones.

previously (Klein Gebbinck et al. 2002; Sosa & Tonn 2008). Worth to be noticed, an earlier work by Jefferies et al. (1973) reported the occurrence of various diterpenes in another species, *Dodonaea boroniaefolia*, of the opposite configuration in the main skeleton (Figure 3), that is *ent*-clerodanes.

Fig. 3. Flavonoids from *Dodonaea* spp. (Niu et al. 2010; Omosa et al. 2010; Teffo et al. 2010).

#### **2.3** *Magonia and Paullinia species*

292 Insecticides – Pest Engineering

O

O

O

O

O

O

OH

HO O

O

HO O

<sup>H</sup> HO

HO O

HO

<sup>H</sup> HO

H

O

O

O

H

OH

O

O

HO O

*D. viscosa* Niu et al., 2010

<sup>O</sup> <sup>O</sup> OCH3 <sup>O</sup>

H

*D. boroniaefolia* Jefferie et al., 1973

Fig. 2. Clerodanes from *Dodonaea* spp. Structures shown were isolated from *D. viscosa* (Niu

previously (Klein Gebbinck et al. 2002; Sosa & Tonn 2008). Worth to be noticed, an earlier work by Jefferies et al. (1973) reported the occurrence of various diterpenes in another species, *Dodonaea boroniaefolia*, of the opposite configuration in the main skeleton (Figure 3),

HO

H

H3CO

O O

H

O

*D. angustifolia* Omosa et al., 2010

that is *ent*-clerodanes.

H3CO

H

O

O

O

<sup>O</sup> <sup>O</sup> <sup>H</sup>

O O

O

H3CO

H

OH

HO O

H

et al. 2010) with the exception of the indicated ones.

O

O

*Paullinia* spp. is one of the few purine alkaloid-containing genera used to prepare stimulant drinks worldwide (Weckerle et al. 2003). *Paullinia clavigera* grows in primary forests and shores of the South American Amazonic aquatic ecosystems, and it has been studied mostly in Perú for the control of different insect pests (cited in Pérez et al. 2010). The toxicity of aqueous extracts of lianas from this species against larvae of *Anopheles benarrochi (*Diptera: Culicidae) (Pérez & Iannacone 2004) and afterwards the mortality and repellence of such extracts against larvae of *Rhynchophorus palmarum* (Coleoptera: Curculionidae) (Pérez & Iannacone 2006) were tested. Besides, the activity of aqueous extracts against *Eupalamides cyparissias* (Lepidoptera: Castniidae), and the activity of hydroalcoholic extracts against *Tuthillia cognata* (Hemiptera: Psyllidae) (Pérez et al. 2008) were also reported, showing the potential of this vegetal species as a biocontrol agent against different insect orders. However, it is still unclear which compounds are responsible for the anti-insect effects of *P. clavigera*. Flavonoids, phenols, triterpenes and saponins were detected in a phytochemical study in extracts from the stem cortex of *P. clavigera.* (Pérez et al. 2010); and other species from the same genus (*P. cururu*) contain saponins, tannins and polyphenols (Wilbert & Haiek 1991).

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 295

2002). *Koelreuteria paniculata* is popularly grown as an ornamental tree in temperate regions all across the world (Kamala-Kannan et al. 2009). From these two species of Sapindaceae, lectins have been isolated from their seeds (Macedo et al. 2002; Macedo et al. 2003). Lectins from *T. esculenta* inhibited larval growth of two bruchids *(Callobroschus maculatus* and *Zabrotes subfasciatus*) (Macedo et al. 2002). And in the case of *K. paniculata*, lectins not only showed insecticide activity against *C. maculatus* but also against *Anagasta kuehniella* (Lepidoptera: Pyralidae) (Macedo et al. 2003). Plant lectins are a large group of proteins dened as ''plant proteins that possess at least one non-catalytic domain that binds reversibly to a specic mono‐ or oligosaccharide'' (Peumans & Van Damme 1995). Plant lectines have been implied in many ecological roles, being their action as defences against insects one of the latest described (Murdock & Shade 2002; Van Damme et al. 2008). Their mechanisms of action as anti-insect agents are yet poorly understood, with many emerging hypotheses proposed (Van Damme et al. 2008). According to Macedo et al. (2003), the action of *K. paniculata* lectins on *C. maculatus* and *A. kuehniella* larvae may involve *(1) binding to glycoconjugates on the surface of epithelial cells along the digestive tract, (2) binding to glycosylated digestive enzymes, thereby inhibiting their activity, and (3) binding to the chitin component of the peritrophic membrane (or equivalent structures) in the insect midgut*. Finally, regarding *T. esculenta*, its aqueous seeds extracts were studied on its effect on *S. frugiperda* larvae which development was negatively affected, but the activity of the midgut trypsin was not

The red maple, *Acer rubrum*, is another Sapindaceae from which bioactive compounds have been isolated. This prominent maple occurs in hardwood forests, being avoided by several potential sympatric consumers [for instance, larvae of *Malacossoma disstria* (Lepidoptera: Lasiocampidae), and the North American beavers, *Castor canadensis*] (Abou-Zaid et al. 2001). The main constituents of an aqueous leaf extract have been phytochemically characterized as ellagic acid, gallate derivatives (structures **1-7** in Figure 5) and glycosides of flavonoids (quercetin and structures **13-20** in Figure 3). When these compounds were tested by themselves against *M. disstria* larvae, it was found that all gallate derivatives exhibited deterrent activity, but not the flavonoids. Among gallate derivatives, compounds **2** and **4-7** in Figure 5 were the five most active compounds. Perhaps in this case, the feeding deterrence effect of the extracts may be traced to the galloyl moiety in its secondary

*Blighia sapida*, commonly known as Ackee, is an evergreen tree, native from West African wild forests. In the late 18th century, the plant was introduced in Jamaica, where nowadays its fruit has been adopted as the national fruit (cited in Gaillard et al. 2011). Its bark is used as fish poison and also in folk medicine in the treatment of malaria, ulcers, back aches and headaches (Kayode 2006). The plant contains triterpenic and steroidal saponins, alkaloids, polyphenols and aminoacidic secondary metabolites (Mazzola et al. 2011). By ingestion, the unripe fruits can cause vomiting and circulatory collapse in humans due to the presence of hypoglycin-A (seeds and flesh) and hypoglycin-B (seeds) (Figure 6) (Hassall et al. 1954; Hassall & Reyle 1955; Gaillard et al. 2011). Acetone and ethanolic extracts of the fruits showed repellent properties against stored-product pests, namely, *C. maculatus*, *Cryptolestes ferrugineus* (Coleoptera: Cucujidae), and *Sitophilus zeamais* (Coleoptera: Curculionidae) (Khan & Gumbs 2003). Furthermore, ethanol, acetone, hexane, methanol, chloroform, and water extracts from the seeds were evaluated on their repellence against *T. castaneum,* demonstrating the aqueous extract to be the most active

inhibited (dos Santos et al. 2008)*.* 

metabolites.

(Khan et al. 2002).

*Magonia pubescens*, widely distributed in the Brazilian Cerrado, is commonly used in the construction industry. It has been mostly studied for its larvicidal activity against *A. aegypti* (Arruda et al. 2003; da Silva et al. 2003; Rodrigues et al. 2006). In this case, one of the most active fractions from the ethanolic extract of stem barks was shown to be rich in tannins, and specially in a proanthocyanidin (catequic tannin which structure is shown in Figure 4) (Silva et al. 2004). Tannins are largely distributed in nature, usually being the active principles of plants used in traditional medicine. Condensed tannins have a great ability to interact with metallic ions and macromolecules and to form soluble complexes with electron-donor groups such as the ones found in alkaloids and proteins. That may be one of the reasons explaining their toxicity against different organisms, including insects, fungi and bacteria. Morphological alterations caused by this active fraction on the epithelium of the midgut of larvae of *A. aegypti* resembled the ones recorded for tannic acid (Rey et al. 1999). In a side note, it is worth to notice that this vegetal species has also demonstrated potential on its ethanolic extract of stem barks, as acaricide against the larvae of the common cattle tick, *Rhipicephalus (Boophilus) sanguineus* (Acari: Ixodidae) (Fernandes et al. 2008).

Fig. 4. Catequic tannin with larvicidal activity against *A. aegypti* isolated from *Magonia pubescens* (Silva et al. 2004).

#### **2.4 Miscellaneous**

*Talisia esculenta,* locally known as pitomba occurs in northern and northeastern Brazil. Its fruits are edible to humans and birds which disperse the seeds. However, popular information also mentions that chickens die after ingesting the fruit (cited in Macedo et al.

*Magonia pubescens*, widely distributed in the Brazilian Cerrado, is commonly used in the construction industry. It has been mostly studied for its larvicidal activity against *A. aegypti* (Arruda et al. 2003; da Silva et al. 2003; Rodrigues et al. 2006). In this case, one of the most active fractions from the ethanolic extract of stem barks was shown to be rich in tannins, and specially in a proanthocyanidin (catequic tannin which structure is shown in Figure 4) (Silva et al. 2004). Tannins are largely distributed in nature, usually being the active principles of plants used in traditional medicine. Condensed tannins have a great ability to interact with metallic ions and macromolecules and to form soluble complexes with electron-donor groups such as the ones found in alkaloids and proteins. That may be one of the reasons explaining their toxicity against different organisms, including insects, fungi and bacteria. Morphological alterations caused by this active fraction on the epithelium of the midgut of larvae of *A. aegypti* resembled the ones recorded for tannic acid (Rey et al. 1999). In a side note, it is worth to notice that this vegetal species has also demonstrated potential on its ethanolic extract of stem barks, as acaricide against the larvae of the common cattle tick,

O

OH

O

OH

OH

OH

OH

OH

OH

OH

*Talisia esculenta,* locally known as pitomba occurs in northern and northeastern Brazil. Its fruits are edible to humans and birds which disperse the seeds. However, popular information also mentions that chickens die after ingesting the fruit (cited in Macedo et al.

Fig. 4. Catequic tannin with larvicidal activity against *A. aegypti* isolated from *Magonia* 

O

OH

HO

HO

HO

*pubescens* (Silva et al. 2004).

**2.4 Miscellaneous** 

OH

OH

*Rhipicephalus (Boophilus) sanguineus* (Acari: Ixodidae) (Fernandes et al. 2008).

O

2002). *Koelreuteria paniculata* is popularly grown as an ornamental tree in temperate regions all across the world (Kamala-Kannan et al. 2009). From these two species of Sapindaceae, lectins have been isolated from their seeds (Macedo et al. 2002; Macedo et al. 2003). Lectins from *T. esculenta* inhibited larval growth of two bruchids *(Callobroschus maculatus* and *Zabrotes subfasciatus*) (Macedo et al. 2002). And in the case of *K. paniculata*, lectins not only showed insecticide activity against *C. maculatus* but also against *Anagasta kuehniella* (Lepidoptera: Pyralidae) (Macedo et al. 2003). Plant lectins are a large group of proteins dened as ''plant proteins that possess at least one non-catalytic domain that binds reversibly to a specic mono‐ or oligosaccharide'' (Peumans & Van Damme 1995). Plant lectines have been implied in many ecological roles, being their action as defences against insects one of the latest described (Murdock & Shade 2002; Van Damme et al. 2008). Their mechanisms of action as anti-insect agents are yet poorly understood, with many emerging hypotheses proposed (Van Damme et al. 2008). According to Macedo et al. (2003), the action of *K. paniculata* lectins on *C. maculatus* and *A. kuehniella* larvae may involve *(1) binding to glycoconjugates on the surface of epithelial cells along the digestive tract, (2) binding to glycosylated digestive enzymes, thereby inhibiting their activity, and (3) binding to the chitin component of the peritrophic membrane (or equivalent structures) in the insect midgut*. Finally, regarding *T. esculenta*, its aqueous seeds extracts were studied on its effect on *S. frugiperda* larvae which development was negatively affected, but the activity of the midgut trypsin was not inhibited (dos Santos et al. 2008)*.* 

The red maple, *Acer rubrum*, is another Sapindaceae from which bioactive compounds have been isolated. This prominent maple occurs in hardwood forests, being avoided by several potential sympatric consumers [for instance, larvae of *Malacossoma disstria* (Lepidoptera: Lasiocampidae), and the North American beavers, *Castor canadensis*] (Abou-Zaid et al. 2001). The main constituents of an aqueous leaf extract have been phytochemically characterized as ellagic acid, gallate derivatives (structures **1-7** in Figure 5) and glycosides of flavonoids (quercetin and structures **13-20** in Figure 3). When these compounds were tested by themselves against *M. disstria* larvae, it was found that all gallate derivatives exhibited deterrent activity, but not the flavonoids. Among gallate derivatives, compounds **2** and **4-7** in Figure 5 were the five most active compounds. Perhaps in this case, the feeding deterrence effect of the extracts may be traced to the galloyl moiety in its secondary metabolites.

*Blighia sapida*, commonly known as Ackee, is an evergreen tree, native from West African wild forests. In the late 18th century, the plant was introduced in Jamaica, where nowadays its fruit has been adopted as the national fruit (cited in Gaillard et al. 2011). Its bark is used as fish poison and also in folk medicine in the treatment of malaria, ulcers, back aches and headaches (Kayode 2006). The plant contains triterpenic and steroidal saponins, alkaloids, polyphenols and aminoacidic secondary metabolites (Mazzola et al. 2011). By ingestion, the unripe fruits can cause vomiting and circulatory collapse in humans due to the presence of hypoglycin-A (seeds and flesh) and hypoglycin-B (seeds) (Figure 6) (Hassall et al. 1954; Hassall & Reyle 1955; Gaillard et al. 2011). Acetone and ethanolic extracts of the fruits showed repellent properties against stored-product pests, namely, *C. maculatus*, *Cryptolestes ferrugineus* (Coleoptera: Cucujidae), and *Sitophilus zeamais* (Coleoptera: Curculionidae) (Khan & Gumbs 2003). Furthermore, ethanol, acetone, hexane, methanol, chloroform, and water extracts from the seeds were evaluated on their repellence against *T. castaneum,* demonstrating the aqueous extract to be the most active (Khan et al. 2002).

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 297

also edible fruits and it has not been studied in its anti-insect activity. However, six saponins, namely, nepheliosides **1-6** (Figure 1) were isolated from a chloroform extract of its bark exhibiting cytotoxic activity when evaluated against a panel of human cancer cell lines

As stated, Sapindaceae are rich –in their seeds- in toxic cyanolipids (Figure 8), *e.g.* fatty acid esters of α- and - hydroxynitriles (Mikolajczak 1977; Seigler 1991). Although, some works report the occurrence of cyanolipids also in members of the Hippocastanaceae (Mikolajczak 1977; Bjarnholt & Møller 2008) and the Boraginaceae (Mikolajczak et al. 1969; Seigler et al. 1970), it appears that later investigations have confirmed that these metabolites are characteristic only of the Sapindaceae (Seigler 1976; Avato et al. 2005). The cyanolipids are usually extracted in the seed oils where the amounts vary broadly within the species (Dinesh & Hasan; Selmar et al. 1990; Hasan et al. 1994; Ucciani et al. 1994; Hasan & Roomi 1996; Sarita et al. 2002; Avato et al. 2005; Avato et al. 2006), ranging from only 3% in *Paullinia cupana var. sorbilis* (Guarana) (Avato et al. 2003), to 58% in *Schleichera trijuga* (Mikolajczak & Smith 1971).

O

NC R

NC

1-en-3-ol-ester

2-en-1-ol-ester

O

Type IV:1-cyano-2-methylprop-

Type II:1-cyano-2-methylprop-

NC O Glu

OH

O SO3 -

Cardiospermin

NC O Glu

Cardiospermin-5-sulfate

O

O

R

(Ito et al. 2004).

**3. Cyanocompounds** 

O

O

Type III:1-cyano-2-hydroxymethylprop-1-en-3-ol-diester

NC O Glu

Proacacipetalin

NC O Glu

Heterodendrin

Fig. 8. Cyanolipids and glucosinolates from Sapindaceae.

NC

O

O

O

R

NC O Glu

Glu O

NC

Sutherlandin

Epiproacacipetalin

OH

R

O

NC R

Type I:1-cyano-2-hydroxymethylprop-2-en-1-ol-diester

O

O

R

**8** ellagic acid

Fig. 5. Ellagic acid and gallate derivatives isolated from *A. rubrum.* (Abou-Zaid et al. 2001).

Hypoglycin-A Hypoglycin-B

Fig. 6. Hypoglycins isolated from the fruits of *B. sapida.*

*Serjania lethalis* is another species that has been studied in its anti-insect activity. The ethanolic stem bark extract showed larvicidal activity against *A. aegypti* (Rodrigues et al. 2006). In this study the active compounds were not identified. However, the presence in this species of tannins, flavonoids and of saponins (serjanosides) has been reported (Teixeira et al. 1984; de Sousa Araújo et al. 2008). *S. meridionalis,* a species phytochemically not described, exhibited deterrent activity against *E. paenulata* and *M. persicae* (Hemiptera: Aphididae) when its ethanolic leaf extracts were assayed. Disappointingly, this extract was also toxic against beneficial insects (honey bees) (Castillo et al. 2009).

From the genus *Nephelium*, the species *N. lappaceum* is commonly known for its edible fruit "rambutan". It is native from Southeast Asia where the fruits are an important commercial crop (Palanisamy et al. 2008). These fruits have shown potential on its ethanol seed extract, against *S. oryzae*, revealing to reduce esterase and glutathione-S-transferase activities from such insect (Bullangpoti et al. 2004). *N. maingayi,* native from Malasya and Indonesia, has also edible fruits and it has not been studied in its anti-insect activity. However, six saponins, namely, nepheliosides **1-6** (Figure 1) were isolated from a chloroform extract of its bark exhibiting cytotoxic activity when evaluated against a panel of human cancer cell lines (Ito et al. 2004).

## **3. Cyanocompounds**

296 Insecticides – Pest Engineering

O

HO

HO O

Fig. 6. Hypoglycins isolated from the fruits of *B. sapida.*

beneficial insects (honey bees) (Castillo et al. 2009).

HO

O

Fig. 5. Ellagic acid and gallate derivatives isolated from *A. rubrum.* (Abou-Zaid et al. 2001).

OH

*Serjania lethalis* is another species that has been studied in its anti-insect activity. The ethanolic stem bark extract showed larvicidal activity against *A. aegypti* (Rodrigues et al. 2006). In this study the active compounds were not identified. However, the presence in this species of tannins, flavonoids and of saponins (serjanosides) has been reported (Teixeira et al. 1984; de Sousa Araújo et al. 2008). *S. meridionalis,* a species phytochemically not described, exhibited deterrent activity against *E. paenulata* and *M. persicae* (Hemiptera: Aphididae) when its ethanolic leaf extracts were assayed. Disappointingly, this extract was also toxic against

From the genus *Nephelium*, the species *N. lappaceum* is commonly known for its edible fruit "rambutan". It is native from Southeast Asia where the fruits are an important commercial crop (Palanisamy et al. 2008). These fruits have shown potential on its ethanol seed extract, against *S. oryzae*, revealing to reduce esterase and glutathione-S-transferase activities from such insect (Bullangpoti et al. 2004). *N. maingayi,* native from Malasya and Indonesia, has

Hypoglycin-A Hypoglycin-B

**8** ellagic acid

NH2 <sup>O</sup> OH

O N

NH2

O

OH

O

RO

OH

OH

O

O

OH

**6** R = OH *m*-Digallate **7** R = OC2O5 Ethyl *m*-digallate

OH

OH

OH

H

O

O

HO

**1** R = H Gallic acid **2** R = Me Methyl gallate **3** R = Et Ethyl gallate

**4** R =-D-Glc 1-*O*-Galloyl-b-D-glucosa **5** R =-L-Rha 1-*O*-Galloyl-a-L-rhamnose

HO

O

OR

HO

As stated, Sapindaceae are rich –in their seeds- in toxic cyanolipids (Figure 8), *e.g.* fatty acid esters of α- and - hydroxynitriles (Mikolajczak 1977; Seigler 1991). Although, some works report the occurrence of cyanolipids also in members of the Hippocastanaceae (Mikolajczak 1977; Bjarnholt & Møller 2008) and the Boraginaceae (Mikolajczak et al. 1969; Seigler et al. 1970), it appears that later investigations have confirmed that these metabolites are characteristic only of the Sapindaceae (Seigler 1976; Avato et al. 2005). The cyanolipids are usually extracted in the seed oils where the amounts vary broadly within the species (Dinesh & Hasan; Selmar et al. 1990; Hasan et al. 1994; Ucciani et al. 1994; Hasan & Roomi 1996; Sarita et al. 2002; Avato et al. 2005; Avato et al. 2006), ranging from only 3% in *Paullinia cupana var. sorbilis* (Guarana) (Avato et al. 2003), to 58% in *Schleichera trijuga* (Mikolajczak & Smith 1971).

Fig. 8. Cyanolipids and glucosinolates from Sapindaceae.

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 299

cyanolipids stimulated aggregation in the cases of the saw toothed grain beetle, *Oryzaephilus surinamensis*, and of the rice weevil, *Sitophilus oryzae* (Mikolajczak et al. 1984). In this later work, when tested separately the four classes of cyanolipids (Figure 8) lost their repellent activity, suggesting synergism among them for this action. When the effect of cyanolipids was tested in a contact bioassay, only the ones belonging to classes I and IV showed a paralyzing effect against the saw toothed grain beetle, *Oryzaephilus surinamensis*. Eventhough one may be tempted to correlate this effect with the capacity of producing HCN by the pure compound tested, the conclusion is not again a general one since these cyanolipids did not have any effect on three other beetles (Mikolajczak et al. 1984). Finally, the European corn borer, *Ostrinia nubilalis*, was affected in its metamorphosis when cyanolipids of the classes II and IV were incorporated in its diet (Mikolajczak et al. 1984). On the whole, even though non- glycoside cyanogens were described in Sapindaceae as early as in the 1920` (cited by Mikolajczak et al. 1969), not many studies have been carried out in regard of either their ecological role as plant defences, or their potential as biopesticides. Although in the last case one can envisioned that cyanolipids of the classes I and IV will not be selective –because HCN is generated-, there is a chance that cyanolipids of the classes II

The family Sapindaceae includes many edible species, *e.g.* ackee, rambutan, longan and lychee (fruits from *Blighia sapida, Litchi chinensis*, *Nephelium lappaceum*, *Dimocarpus longan* respectively), which are widely consumed mainly in Asia and Australia (Diczbalis 2008; Vichitrananda & Somsri 2008; Diczbalis et al. 2010). Nevertheless, some species in this family produce in different phenological stages (including fruits in some cases) bioactive compounds with medicinal or toxicological properties. With reference to insect toxicity, up to now most of the studies carried out have found activity against species in the orders Lepidoptera and Diptera (mosquitoes). However, these results may be an artefact of the biodetection itself, as much of the research focuses on chewing armyworms and borers of economically significance, and on mosquitoes as important vectors of human diseases. In addition, products from Sapindaceae have revealed differential activity on insect targets from different orders, and even from the same order (Mikolajczak et al. 1984; Khan et al. 2002; Castillo et al. 2009); and have demonstrated selectivity when their activity was checked against beneficial insects while some other products have not (Castillo et al. 2009). These findings emphasise the need for widening

The Sapindaceae are well characterized for the presence in their seeds of toxic cyanolipids, and the occurrence of this group of secondary metabolites seems to be restricted to this family. However, it is difficult to foresee that the Sapindaceae will find their way into the development of botanicals based on their unique cyanogens due to the intrinsic general

This family takes its name from the soapberry tree *Sapindus saponaria* (Emanuel & Benkeblia 2011), mostly known for being rich in saponins. Those chemical constituents provide its extracts with tensoactive properties, having been widely used not only as a source of soap but also for the application of its biological effects in medicine as well as in pest control. Indeed, the tensoactivity has been a property that has found application in pest control as shown by the fact that soaps are probably among the oldest insecticides in use (Silva et al.

and III may have some interest in this regard.

the spectrum of biodetectors used in the tests.

toxicity of these compounds.

2007).

**4. Conclusion** 

Cyanolipids present in Sapindaceae belong to four types (Figure 8) which do not occur in all species. Although there is some controversy in the literature, it seems that each species has only one or two types of cyanolipids, but not all of them. The fatty acid moieties in cyanolipids vary within the species. In most of the studies, cyanolipids are esterified mostly by only one or two fatty acids. Eicosanoic acid (20:0) and eicosenoic acid (20:1 *n-9*) are present in high proportion in many species (Aichholz et al. 1997). In the genus *Paullinia*, the rare fatty acids paullinic acid (*Z*-13-eicosenoic acid) and cis-vaccenic acid (*Z*-11-octadecenoic acid) are ubiquitous constituents (Seigler 1974; Spitzer 1995; Spitzer 1996; Lago et al. 2000; Avato et al. 2003). Other fatty acids found go from dodecanoic (12:0) to docosanoic acids (22:0) (Mikolajczak et al. 1970a; 1970b; 1971; Aichholz et al. 1997; Avato et al. 2003).

Besides lipid derivatives, other cyanocompounds (the corresponding glycosides and salts) have also been isolated from aerial parts (Seigler et al. 1974; Bjarnholt et al. 2008) or roots of different species (Kumar et al. 2011). For example, cardiospermin (Figure 8) has been isolated from some species -and has been shown to be the responsible of the anxiolytic effects of ethanolic root extract from *Cardiospermum halicacabum* (Kumar et al. 2011); and in the particular case of *Cardiospermum grandiflorum* the corresponding sulphate-containing cyanogenic glucoside of cardiospermin was described (Hubel & Nahrstedt 1979).

Only cyanolipids of the type I and IV are cyanogenetic (Avato et al. 2005). Therefore cyanolipids do not work for all species as defensive compounds producing HCN. When HCN can be formed, its production works similarly to the one from glycosinolates, with a previous step of hydrolysis of the ester moieties catalyzed by estearases (Figure 9) (Wink et al. 1997). Cyanolipids can effectively work as plant defences, as it has been shown that their enzymatic breakdown produces the -hydroxynitriles, from which HCN is released in a similar way than the one from glycosinolates (Wink et al. 1997). Moreover, it has been shown that *in vivo* HCN is produced upon wounding by herbivores (Selmar et al. 1990). Besides, as any plant defence, these defensive metabolites have been overcome in their original function by herbivores. In that sense it is well known that some Heteroptera (*Leptocorus* and *Jadera* spp.) not only are specialists on Sapindaceae, but are also able to sequester cyanolipids as such from their food plants, and biotransform them to the glycosylated derivatives (Braekman et al. 1982; Aldrich et al. 1990). Moreover, such acquisition of cyanocompounds from their host renders these gregarious, aposematic insects unpalatable to a variety of predators (Aldrich et al. 1990).

Fig. 9. Hydrolysis of cyanolipids to produce HCN (Wink et al. 1997).

Cyanolipids have been investigated in their potential as control agents for insects. In that sense, insect repellent and insecticidal properties of some of these products have been described. For instance, when adults of the red flour beetles, *Tribolium castaneum,* were exposed to seed oils from two species of *Cardiospermum* (*C. canescens* and *C. belicacabum*), the beetles preferred the arena zones where no oil was applied (Khan et al. 1983). However this repellency effect seems not to be a general pattern as in a pitfall trap bioassay, oils reach in cyanolipids stimulated aggregation in the cases of the saw toothed grain beetle, *Oryzaephilus surinamensis*, and of the rice weevil, *Sitophilus oryzae* (Mikolajczak et al. 1984). In this later work, when tested separately the four classes of cyanolipids (Figure 8) lost their repellent activity, suggesting synergism among them for this action. When the effect of cyanolipids was tested in a contact bioassay, only the ones belonging to classes I and IV showed a paralyzing effect against the saw toothed grain beetle, *Oryzaephilus surinamensis*. Eventhough one may be tempted to correlate this effect with the capacity of producing HCN by the pure compound tested, the conclusion is not again a general one since these cyanolipids did not have any effect on three other beetles (Mikolajczak et al. 1984). Finally, the European corn borer, *Ostrinia nubilalis*, was affected in its metamorphosis when cyanolipids of the classes II and IV were incorporated in its diet (Mikolajczak et al. 1984). On the whole, even though non- glycoside cyanogens were described in Sapindaceae as early as in the 1920` (cited by Mikolajczak et al. 1969), not many studies have been carried out in regard of either their ecological role as plant defences, or their potential as biopesticides. Although in the last case one can envisioned that cyanolipids of the classes I and IV will not be selective –because HCN is generated-, there is a chance that cyanolipids of the classes II and III may have some interest in this regard.

## **4. Conclusion**

298 Insecticides – Pest Engineering

Cyanolipids present in Sapindaceae belong to four types (Figure 8) which do not occur in all species. Although there is some controversy in the literature, it seems that each species has only one or two types of cyanolipids, but not all of them. The fatty acid moieties in cyanolipids vary within the species. In most of the studies, cyanolipids are esterified mostly by only one or two fatty acids. Eicosanoic acid (20:0) and eicosenoic acid (20:1 *n-9*) are present in high proportion in many species (Aichholz et al. 1997). In the genus *Paullinia*, the rare fatty acids paullinic acid (*Z*-13-eicosenoic acid) and cis-vaccenic acid (*Z*-11-octadecenoic acid) are ubiquitous constituents (Seigler 1974; Spitzer 1995; Spitzer 1996; Lago et al. 2000; Avato et al. 2003). Other fatty acids found go from dodecanoic (12:0) to docosanoic acids

(22:0) (Mikolajczak et al. 1970a; 1970b; 1971; Aichholz et al. 1997; Avato et al. 2003).

cyanogenic glucoside of cardiospermin was described (Hubel & Nahrstedt 1979).

unpalatable to a variety of predators (Aldrich et al. 1990).

NC R NC OH

estearase

O

HO R

Fig. 9. Hydrolysis of cyanolipids to produce HCN (Wink et al. 1997).

Cyanolipids have been investigated in their potential as control agents for insects. In that sense, insect repellent and insecticidal properties of some of these products have been described. For instance, when adults of the red flour beetles, *Tribolium castaneum,* were exposed to seed oils from two species of *Cardiospermum* (*C. canescens* and *C. belicacabum*), the beetles preferred the arena zones where no oil was applied (Khan et al. 1983). However this repellency effect seems not to be a general pattern as in a pitfall trap bioassay, oils reach in

O

O

Besides lipid derivatives, other cyanocompounds (the corresponding glycosides and salts) have also been isolated from aerial parts (Seigler et al. 1974; Bjarnholt et al. 2008) or roots of different species (Kumar et al. 2011). For example, cardiospermin (Figure 8) has been isolated from some species -and has been shown to be the responsible of the anxiolytic effects of ethanolic root extract from *Cardiospermum halicacabum* (Kumar et al. 2011); and in the particular case of *Cardiospermum grandiflorum* the corresponding sulphate-containing

Only cyanolipids of the type I and IV are cyanogenetic (Avato et al. 2005). Therefore cyanolipids do not work for all species as defensive compounds producing HCN. When HCN can be formed, its production works similarly to the one from glycosinolates, with a previous step of hydrolysis of the ester moieties catalyzed by estearases (Figure 9) (Wink et al. 1997). Cyanolipids can effectively work as plant defences, as it has been shown that their enzymatic breakdown produces the -hydroxynitriles, from which HCN is released in a similar way than the one from glycosinolates (Wink et al. 1997). Moreover, it has been shown that *in vivo* HCN is produced upon wounding by herbivores (Selmar et al. 1990). Besides, as any plant defence, these defensive metabolites have been overcome in their original function by herbivores. In that sense it is well known that some Heteroptera (*Leptocorus* and *Jadera* spp.) not only are specialists on Sapindaceae, but are also able to sequester cyanolipids as such from their food plants, and biotransform them to the glycosylated derivatives (Braekman et al. 1982; Aldrich et al. 1990). Moreover, such acquisition of cyanocompounds from their host renders these gregarious, aposematic insects

2-hydroxy-3-methylbut-

+ HCN

O

3-enenitrile

The family Sapindaceae includes many edible species, *e.g.* ackee, rambutan, longan and lychee (fruits from *Blighia sapida, Litchi chinensis*, *Nephelium lappaceum*, *Dimocarpus longan* respectively), which are widely consumed mainly in Asia and Australia (Diczbalis 2008; Vichitrananda & Somsri 2008; Diczbalis et al. 2010). Nevertheless, some species in this family produce in different phenological stages (including fruits in some cases) bioactive compounds with medicinal or toxicological properties. With reference to insect toxicity, up to now most of the studies carried out have found activity against species in the orders Lepidoptera and Diptera (mosquitoes). However, these results may be an artefact of the biodetection itself, as much of the research focuses on chewing armyworms and borers of economically significance, and on mosquitoes as important vectors of human diseases. In addition, products from Sapindaceae have revealed differential activity on insect targets from different orders, and even from the same order (Mikolajczak et al. 1984; Khan et al. 2002; Castillo et al. 2009); and have demonstrated selectivity when their activity was checked against beneficial insects while some other products have not (Castillo et al. 2009). These findings emphasise the need for widening the spectrum of biodetectors used in the tests.

The Sapindaceae are well characterized for the presence in their seeds of toxic cyanolipids, and the occurrence of this group of secondary metabolites seems to be restricted to this family. However, it is difficult to foresee that the Sapindaceae will find their way into the development of botanicals based on their unique cyanogens due to the intrinsic general toxicity of these compounds.

This family takes its name from the soapberry tree *Sapindus saponaria* (Emanuel & Benkeblia 2011), mostly known for being rich in saponins. Those chemical constituents provide its extracts with tensoactive properties, having been widely used not only as a source of soap but also for the application of its biological effects in medicine as well as in pest control. Indeed, the tensoactivity has been a property that has found application in pest control as shown by the fact that soaps are probably among the oldest insecticides in use (Silva et al. 2007).

Bioactive Natural Products from Sapindaceae Deterrent and Toxic Metabolites Against Insects 301

Arruda, W., Cavasin Oliveira, G. M. & Garcia da Silva, I. (2003a). Morphological alterations

Arruda, W., Oliveira, G. M. C. & da Silva, I. G. (2003b). Toxicity of the ethanol extract of

Arulmozhi, D. K., Veeranjaneyulu, A., Bodhankar, S. L. & Arora, S. K. (2005).

Avato, P., Pesante, M. A., Fanizzi, F. P. & Santos, C. A. D. (2003). Seed oil composition of *Paullinia cupana* var. *sorbilis* (Mart.) Ducke. *Lipids* Vol. 38, 7, pp. 773-780 Avato, P., Rosito, I., Papadia, P. & Fanizzi, F. P. (2005). Cyanolipid-rich seed oils from *Allophylus natalensis* and *A. dregeanus*. *Lipids* Vol. 40, 10, pp. 1051-1056 Avato, P., Rosito, I., Papadia, P. & Fanizzi, F. P. (2006). Characterization of seed oil

Banerjee, H. N. (1936). Clerodin from *Clerodendron infortunatum*. *Science and Culture* Vol. 2,

Benlekehal, H., Clotteau, M., Dornier, M. & Reynes, M. (2001). An Amazonian product

Bjarnholt, N. & Møller, B. L. (2008). Hydroxynitrile glucosides. *Phytochemistry* Vol. 69, 10,

Boiça Junior, A. L., Medeiros, C. A. M., Torres, A. L. & Chagas Filho, N. R. (2005). Effect of

Bousserouel, H., Litaudon, M., Morleo, B., Martin, M. T., Thoison, O., Nosjean, O., Boutin, J.

Braekman, J. C., Daloze, D. & Pasteels, J. M. (1982). Cyanogenic and other glucosides in a

Bruneton, J. (1995). Pharmacognosy, phytochemistry, medicinal plants. Paris, Lavoisier

Bullangpoti, V., Visetson, S., Milne, J. & Pornbanlualap, S. (2004). Effects of Mangosteen's

Castillo, L., Gonzalez-Coloma, A., Gonzalez, A., Diaz, M., Alonso-Paz, E., Bassagoda, M. J. &

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(Mart.) Ducke]. *Fruits (Paris)* Vol. 56, 6, pp. 423-435

*Systematics and Ecology* Vol. 10, 4, pp. 355-364

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pp. 1947-1961

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Supplement, pp. 84-89

pp. 163

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of *Aedes aegypti* larvae exposed to *Magonia pubescens* extract. *Entomologia y Vectores*

*Magonia pubescens* on larvae *Aedes aegypti*. *Revista da Sociedade Brasileira de Medicina* 

Pharmacological studies of the aqueous extract of *Sapindus trifoliatus* on central nervous system: possible antimigraine mechanisms. *Journal of Ethnopharmacology*

components from *Nephelium lappaceum* L. *Natural Product Communications* Vol. 1, 9,

particularly rich in caffeine: the seed of guarana [*Paullinia cupana* H.B.K. var. *sorbilis*

plant aqueous extracts on the development of *Plutella xylostella* (L.) (Lepidoptera: Plutellidae), on collard greens. *Arquivos do Instituto Biologico Sao Paulo* Vol. 72, 1, pp.

A., Renard, P. & Sevenet, T. (2005). New biologically active linear triterpenes from the bark of three Newcaledonian *Cupaniopsis* species. *Tetrahedron* Vol. 61, 4, pp. 845-

neo-guinean bug *Leptocoris isolata*: Possible precursors in its host-plant. *Biochemical* 

peels and rambutan's seeds on toxicity, esterase and glutathione-S-transferase in rice weevil (*Sitophilus oryzae* L.). *Kasetsart Journal, Natural Sciences* Vol. 38, 5,

Rossini, C. (2009). Screening of Uruguayan plants for deterrent activity against

Many other bioactive compounds belonging to different chemical groups have also been isolated from members of this family. For instance, from species studied for their anti insect activity, it has been reported the occurrence of clerodane diterpenoids and prenylated flavonoids (*Dodonaea* spp.); flavonols, phenols and triterpenes (*Paullinia* spp.); tannins (*M. pubescens*); lectins (*Talisia esculenta)*; gallic acid, gallates and derivatives (*Acer rubrum*). Being confirmed in some cases that those compounds are the responsible for the activities found. Among these secondary metabolites, probably the ones with the strongest potential as antiinsect agents are the clerodanes. Eventhough the main sources of these diterpenes are species from Asteraceae and Lamiaceae, the Sapindaceae are showing to be also a good resource of them. So far, different studies have shown the antifeedant capability of clerodanes against many insects, including species from Lepidoptera, Coleoptera, and Orthoptera (Klein Gebbinck et al. 2002; Sosa et al. 2008).

All in all, the family Sapindaceae, which members are widely distributed in every continent and have been used since early days for different purposes –taking advantage of their medicinal and toxicological properties-, seems to be a promissory source of bioactive compounds to be used as biological control agents. However more extensive studies, not only on more species not yet prospected, but also concerning more diverse targets are still needed.

## **5. Acknowledgments**

We acknowledge financial support from the following agencies: OPCW (research grant L/ICA/ICB/111817/06); TWAS (research grant Nº 05-318); and LATU (Laboratorio Tecnológico de Uruguay), Facultad de Química (Universidad de la República) and Agencia Nacional de Investigación e Innovación (ANII, Uruguay) for doctoral fellowships to MD. Finally, we would like to thank the editors of this book for their kind invitation to participate in this endeavour.

#### **6. References**


Many other bioactive compounds belonging to different chemical groups have also been isolated from members of this family. For instance, from species studied for their anti insect activity, it has been reported the occurrence of clerodane diterpenoids and prenylated flavonoids (*Dodonaea* spp.); flavonols, phenols and triterpenes (*Paullinia* spp.); tannins (*M. pubescens*); lectins (*Talisia esculenta)*; gallic acid, gallates and derivatives (*Acer rubrum*). Being confirmed in some cases that those compounds are the responsible for the activities found. Among these secondary metabolites, probably the ones with the strongest potential as antiinsect agents are the clerodanes. Eventhough the main sources of these diterpenes are species from Asteraceae and Lamiaceae, the Sapindaceae are showing to be also a good resource of them. So far, different studies have shown the antifeedant capability of clerodanes against many insects, including species from Lepidoptera, Coleoptera, and

All in all, the family Sapindaceae, which members are widely distributed in every continent and have been used since early days for different purposes –taking advantage of their medicinal and toxicological properties-, seems to be a promissory source of bioactive compounds to be used as biological control agents. However more extensive studies, not only on more species not yet prospected, but also concerning more diverse targets are still

We acknowledge financial support from the following agencies: OPCW (research grant L/ICA/ICB/111817/06); TWAS (research grant Nº 05-318); and LATU (Laboratorio Tecnológico de Uruguay), Facultad de Química (Universidad de la República) and Agencia Nacional de Investigación e Innovación (ANII, Uruguay) for doctoral fellowships to MD. Finally, we would like to thank the editors of this book for their kind invitation to

Abdel, A., Shadia & Omer, E. A. (1995). Bio-evaluation of *Dodonaea viscosa* L. Jacq. extracts

Aichholz, R., Spitzer, V. & Lorbeer, E. (1997). Analysis of cyanolipids and triacylglycerols

Aldrich, J. R., Carroll, S. P., Lusby, W. R., Thompson, M. J., Kochansky, J. P. & Waters, R. M.

Anonymous (2011). Germplasm Resources Information Network - (GRIN) National

*Chromatography A* Vol. 787, 1-2, pp. 181-194

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**14** 

*USA* 

**Pest Management Strategies for Potato** 

This publication addresses pest management guidelines for insects that attack potatoes in the Pacific Northwest of the United States (Fig. 1). Non-chemical control options are strongly encouraged; however, proper use of insecticides has proven effective when used as an additional tool in integrated pest management programs. In general terms, Integrated Pest Management (IPM) is defined as a comprehensive approach of pest control (the term pest includes insects, weeds, diseases) that when combined, reduces the number of pest densities to a level tolerable by the crop. Traditional management practices include the use of host-plant resistance, cultural, mechanical, biological and chemical means of control. Overall, scientific research-based knowledge is required to implement a successful and

Fig. 1. The Pacific Northwest includes Washington and Oregon in the U.S. and the Canadian province of British Columbia. Geographical limits are indicated: California (south), the

**1. Introduction** 

functional management program.

Pacific Ocean (west), and Idaho (east).

**Insect Pests in the Pacific Northwest** 

*Oregon State University, Hermiston Agricultural Research and* 

**of the United States** 

*Extension Center, Hermiston, OR,* 

Silvia I. Rondon


## **Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States**

Silvia I. Rondon *Oregon State University, Hermiston Agricultural Research and Extension Center, Hermiston, OR, USA* 

## **1. Introduction**

308 Insecticides – Pest Engineering

Sousa, S. A., Alves, S. F., de Paula, J. A. M., Fiuza, T. S., Paula, J. R. & Bara, M. T. F. (2009).

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Teixeira, J. R. M., Lapa, A. J., Souccar, C. & Valle, J. R. (1984). Timbós: ichthyotoxic plants used by brazilian indians. *Journal of Ethnopharmacology* Vol. 10, 3, pp. 311-318 Ucciani, E., Mallet, J. F. & Zahra, J. P. (1994). Cyanolipids and fattyacids of *Sapindus trifoliatus* (Sapindaceae) seed oil. *Fat Science Technology* Vol. 96, 2, pp. 69-71 Van Damme, E. J. M., Lannoo, N., Peumans, W. J. & Jean-Claude Kader and Michel, D.

Veeramani, C., Pushpavalli, G. & Pugalendi, K. V. (2010). In vivo antioxidant and

Vichitrananda, S. & Somsri, S. (2008). Tropical fruit production in Thailand. *Acta* 

Waako, P. J., Gumede, B., Smith, P. & Folb, P. I. (2005). The in vitro and in vivo antimalarial

Weckerle, C. S., Stutz, M. A. & Baumann, T. W. (2003). Purine alkaloids in *Paullinia*.

Wilbert, W. & Haiek, G. (1991). Phytochemical screening of a Warao pharmacopoeia

Wink, M., Dey, P. M. & Harborne, J. B. (1997). Special nitrogen metabolism. In: *Plant* 

Wollenweber, E. & Roitman, J. N. (2007). New reports on surface flavonoids from

*Silphium* (Asteraceae). *Natural Product Communications* Vol. 2, 4, pp. 385-389 Zhao, H., Qin, X. & Yang, M. (2006). Preliminary study contact toxicity of extracts from

Zidan, Z. H., Gomaa, A. A., Afifi, F. A., Fam, E. Z. & Ahmed, S. M. S. (1993). Bioresidual

leaf extracts. *South African Journal of Botany* Vol. 76, 1, pp. 25-29

*Journal of Pharmacognosy* Vol. 20, 6, pp. 866-870

*Phytochemistry* Vol. 42, 5, pp. 1357-1360

21, 2, pp. 107-125

pp. 7-11

*Horticulturae* Vol. 787, pp. 33-46

*Phytochemistry* Vol. 64, 3, pp. 735-742

*University* Vol. 22, 6, pp. 942-944

*Journal of Ethnopharmacology* Vol. 99, 1, pp. 137-143

*Biochemistry*, pp. 439-486 Academic Press London

Determination of tannins and methylxanthines in powdered guarana (*Paullinia cupana* Kunth, Sapindaceae) by high performance liquid chromatography. *Brazilian* 

cyanolipid of *Paullinia elegans* (Sapindaceae): A rich source of cis-13-eicosenoic acid (paullinic acid). *Journal of High Resolution Chromatography* Vol. 18, 7, pp. 413-416 Spitzer, V. (1996). Fatty acid composition of some seed oils of the Sapindaceae.

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This publication addresses pest management guidelines for insects that attack potatoes in the Pacific Northwest of the United States (Fig. 1). Non-chemical control options are strongly encouraged; however, proper use of insecticides has proven effective when used as an additional tool in integrated pest management programs. In general terms, Integrated Pest Management (IPM) is defined as a comprehensive approach of pest control (the term pest includes insects, weeds, diseases) that when combined, reduces the number of pest densities to a level tolerable by the crop. Traditional management practices include the use of host-plant resistance, cultural, mechanical, biological and chemical means of control. Overall, scientific research-based knowledge is required to implement a successful and functional management program.

Fig. 1. The Pacific Northwest includes Washington and Oregon in the U.S. and the Canadian province of British Columbia. Geographical limits are indicated: California (south), the Pacific Ocean (west), and Idaho (east).

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 311

In the Pacific Northwest, potatoes are typically planted in the spring and harvested in late summer and fall. The growing season in most areas ranges from 120 to 140 days although high elevation areas have a 90 to 100 day season. In the Columbia Basin of Washington and Oregon, one of the most prolific regions in North America, potato production ranges between 160 to 180 days (University of California, 1986). Potatoes are produced as annual plants that grow from specialized underground stems called stolons later known as tubers (Gopal & Khurana, 2006). This vegetative propagation through the planting of tubers or part of tubers (seed pieces) is a common practice in the region (University of California, 1986; Wale et al., 2008). "Russet Burbank" is still a common variety in the region, followed by other Russet varieties such as Ranger Russet and Russet Norkotah (Table 1). Other varieties are available to producers in the region; many originated from the Tri-State potato breeding program (http://potatoes.wsu.edu/varieties/key.html). The Tri-State Program was created in the mid 1980's to intertwine researchers, scientists and stakeholders in the Pacific

Thirty-seven Tri State releases has been released since the mid 1980's including the popular "Premier Russet", Western Russet" (both dual purposes fry and fresh packs), "Blazer Russet", "Umatilla Russet", and "Ranger Russet" (processing for fries), and "Alturas" (dehydration/processing) (Potato Variety Management Institute http://www.pvmi.org/

Fig. 3. Top potato producing regions in the U.S.

Northwest to promote and develop new potato varieties.

varieties/varieties.htm).

Insects, both as pests and beneficials, have been studied by entomologists for a very long time. Of the millions of insects on the planet, only a fraction causes problems for crops and humans. Specific to this discussion, fewer than a dozen species cause most of the insect damage to potato production in the Pacific Northwest. Many of these pests infest at a specific stage of crop growth (e.g., seed corn maggot damage is more severe at planting), while others are permanent residents before planting, at planting and during crop development (e.g., wireworms) (Fig. 2). A number of pests are important to all western U.S. regions such as aphids, especially green peach aphid and potato aphid, both important vectors of leafroll virus, and more recently PVY. Other pests are important in specific regions such as beet leafhoppers in northeastern Oregon and southeastern Washington. This chapter will briefly provide a general profile of the potato crop in the region and will also discuss the most important insect pests affecting potato production. A general description of the pest, biology, ecology, monitoring and control will be provided. No specific recommendations are made regarding insecticide compounds. To obtain more information refer to the most current version of the Pacific Northwest Insect Control Handbook located at http://uspest.org/pnw/insects or consult your local extension office.

Fig. 2. Occurrence of potato insect pests in the Pacific Northwest of the United States.

## **2. Profile of the potato crop**

The potato is one of the world's most important food crops, domesticated approximately 8,000-10,000 years ago by native Peruvians in South America (Hawkes, 1990; Ministry of Agriculture, 2008). The potato arrived in Europe in the late 1500s (Salaman, 1985) and since then potatoes have been widely cultivated in Europe, Africa, Asia/Oceania and North America. The top ten potato producers worldwide are China, Russia, India, United States, Ukraine, Poland, Germany, Belarus, Netherlands, and France (http://www. potato2008.org/en/world/index.html; source http://faostat.fao.org/default .aspx).

In the U.S., all 50 states cultivate potatoes; however, including Idaho, the Pacific Northwest produces nearly two thirds of the potatoes grown in North America. Idaho is consistently ranked first in potato production and acreage (28% of total U.S. production), followed by Washington State (23% of total U.S. production). Other top U.S. potato producing areas are Oregon, California, Montana, Colorado, North Dakota, Michigan, Wisconsin, and Maine (Fig. 3) (http://www.agcensus.usda.gov/index.asp). Florida and Texas can be included as leading farm cash receipts (http://usda.mannlib.cornell.edu/MannUsda/view DocumentInfo.do?documentID=1235).

Fig. 3. Top potato producing regions in the U.S.

Insects, both as pests and beneficials, have been studied by entomologists for a very long time. Of the millions of insects on the planet, only a fraction causes problems for crops and humans. Specific to this discussion, fewer than a dozen species cause most of the insect damage to potato production in the Pacific Northwest. Many of these pests infest at a specific stage of crop growth (e.g., seed corn maggot damage is more severe at planting), while others are permanent residents before planting, at planting and during crop development (e.g., wireworms) (Fig. 2). A number of pests are important to all western U.S. regions such as aphids, especially green peach aphid and potato aphid, both important vectors of leafroll virus, and more recently PVY. Other pests are important in specific regions such as beet leafhoppers in northeastern Oregon and southeastern Washington. This chapter will briefly provide a general profile of the potato crop in the region and will also discuss the most important insect pests affecting potato production. A general description of the pest, biology, ecology, monitoring and control will be provided. No specific recommendations are made regarding insecticide compounds. To obtain more information refer to the most current version of the Pacific Northwest Insect Control Handbook located

at http://uspest.org/pnw/insects or consult your local extension office.

Fig. 2. Occurrence of potato insect pests in the Pacific Northwest of the United States.

potato2008.org/en/world/index.html; source http://faostat.fao.org/default .aspx).

The potato is one of the world's most important food crops, domesticated approximately 8,000-10,000 years ago by native Peruvians in South America (Hawkes, 1990; Ministry of Agriculture, 2008). The potato arrived in Europe in the late 1500s (Salaman, 1985) and since then potatoes have been widely cultivated in Europe, Africa, Asia/Oceania and North America. The top ten potato producers worldwide are China, Russia, India, United States, Ukraine, Poland, Germany, Belarus, Netherlands, and France (http://www.

In the U.S., all 50 states cultivate potatoes; however, including Idaho, the Pacific Northwest produces nearly two thirds of the potatoes grown in North America. Idaho is consistently ranked first in potato production and acreage (28% of total U.S. production), followed by Washington State (23% of total U.S. production). Other top U.S. potato producing areas are Oregon, California, Montana, Colorado, North Dakota, Michigan, Wisconsin, and Maine (Fig. 3) (http://www.agcensus.usda.gov/index.asp). Florida and Texas can be included as leading farm cash receipts (http://usda.mannlib.cornell.edu/MannUsda/view

**2. Profile of the potato crop** 

DocumentInfo.do?documentID=1235).

In the Pacific Northwest, potatoes are typically planted in the spring and harvested in late summer and fall. The growing season in most areas ranges from 120 to 140 days although high elevation areas have a 90 to 100 day season. In the Columbia Basin of Washington and Oregon, one of the most prolific regions in North America, potato production ranges between 160 to 180 days (University of California, 1986). Potatoes are produced as annual plants that grow from specialized underground stems called stolons later known as tubers (Gopal & Khurana, 2006). This vegetative propagation through the planting of tubers or part of tubers (seed pieces) is a common practice in the region (University of California, 1986; Wale et al., 2008). "Russet Burbank" is still a common variety in the region, followed by other Russet varieties such as Ranger Russet and Russet Norkotah (Table 1). Other varieties are available to producers in the region; many originated from the Tri-State potato breeding program (http://potatoes.wsu.edu/varieties/key.html). The Tri-State Program was created in the mid 1980's to intertwine researchers, scientists and stakeholders in the Pacific Northwest to promote and develop new potato varieties.

Thirty-seven Tri State releases has been released since the mid 1980's including the popular "Premier Russet", Western Russet" (both dual purposes fry and fresh packs), "Blazer Russet", "Umatilla Russet", and "Ranger Russet" (processing for fries), and "Alturas" (dehydration/processing) (Potato Variety Management Institute http://www.pvmi.org/ varieties/varieties.htm).

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 313

effective communication regarding the necessity of implementing a control tactic or not. In a modern definition, the goal is to prevent and suppress pests with minimum impact on human health, the environment and non-target organisms (Dreistadt, 2004). According to Pedigo (1986), in order to use an IPM approach, an understanding of the following factors is required: (1) the biology of the pest and its natural enemies, (2) the response of crops to management practices, (3) the effect of pesticide application on pests and non-target

Timing detection and monitoring is an essential long-term requirement against pest infestation in potatoes. Plants should be regularly checked for signs and symptoms of pest damage. During the height of the growing season, plants should be checked once or twice per week. The inspection of the undersides of leaves and the inner plant canopy is recommended since many pests prefer sheltered sites. A close look of any plant that has missing, absent or damaged leaves or flowers, or plants whose color, texture, or size looks

Some insect pests can be dislodged by laying a sheet, sturdy cardboard or paper below the infested plants and "beating" the canopy. Insects can be monitored with a hand lens or traps. Both techniques not only allow detection and monitoring of pest problems but also provide estimates of pest population density. The optimal timing of sampling depends upon the life history and behavior patterns of the pest and/or beneficial insects, as well as crop stage and state, and also environmental conditions. Some areas, such as field edges or fields

Different sampling procedures can be used depending on the crop, size of field, etc. In the Pacific Northwest, the majority of potatoes are planted in circles, under center-pivot irrigation, thus fields can be divided in quarters (e.g, north, east, west, south). Upon entering the field, quick visual examination of the field is recommended. Uncharacteristic areas with poor stands or patchy growth should be scouted thoroughly. In the Pacific North-western region traps are widely used. Some traps can be baited with a pheromone which is a chemical that usually attracts a single species (e.g., potato tuber moths). Traps can also be coated with adhesive material to "stick" the pest to the trap. Either natural (e.g., virgin females) or more often the synthetic pheromones are used to attract males (Roelofs et al., 1975; Persoons et al,. 1976; Voerman & Rothschild, 1978; Rothschild, 1986; Raman, 1988). Pheromones specifically disrupt the reproductive cycle of harmful insects. Pheromone traps are used extensively in commercial agriculture in the region helping farmers detect the presence of pest species (Merrill et al., 2011). Traps in general will work only for adult insects, as adults have developed wings and are more mobile. Yellow sticky traps attract fruit flies, winged aphids, thrips, psyllids, fungus gnats, wasps, numerous flies; many species collected in sticky cards belong to the same family or group (e.g., several moths in the Gelechiidae family can be found in traps meant to be for tuber moths, a member of the

Before planting, selecting field location can minimize pest damage and improve predictability of pest problems (Hoy et al., 2008). Also circumventing planting dates when insects will emerge and inflict most damage is recommended. Certified seed should be used at all times. If available, the selection of cultivars resistant or tolerant to important pests is

organisms, and (4) the action threshold or level of damage tolerable by the plant.

next to main roads are more prone to pest problems (personal observations).

Gelechiidae family), thus correct identification of target pest(s) is needed.

different than healthy ones, can be a signs of a problem.

**3.1 Monitoring** 

**3.2 Management** 

also desirable.


Table 1. Popular potato varieties (%) planted in Idaho, Washington and Oregon (Washington Potato Commission, 2007; National Agricultural Statistics Service, http://www.agcensus.usda.gov/index.asp).

After producers determine the nutritional needs of the crop (nitrogen, phosphorous, potassium), seed potatoes are cut uniformly, treated with fungicides and/or insecticides, and planted. Potatoes emerge 3-5 weeks after planting, depending on weather conditions, location and time of the year. After plant emergence, producers monitor crop needs regarding fertilization, pest control, and general management. Most potatoes in the area are grown under irrigation. Another common practice is desiccation or vine-killing (Hutchinson & Stall, 2007). Timely vine killing is essential for good tuber skin set, and efficient harvest. It is not clear if desiccation (naturally or artificially) of early season potatoes influences pest pressure in other nearby fields awaiting harvest. Johnson (2008) suggests a holistic plan for potato management combining management practices in the years prior to growing the potato crop, during the growing season, at harvest and during tuber storage. Miller and Hopkins (2008) specified that among many things, establishing appropriate long-term crop rotation, applying and alternating pesticides with different mode of action to avoid pesticide resistance, and improving soil microbial, physical and chemical health are necessary to achieve a healthy crop.

The potato plant develops through four clearly defined growth stages: (1) vegetative growth, (2) tuber initiation, (3) tuber growth and (4) plant maturation (Johnson, 2008). Each stage is affected by different groups of insect pests. The degree of the damage will depend on the timing of events, cultivar characteristics, and the intrinsic characteristics of each pest.

## **3. Managing insect pests in potatoes**

The first step for managing insect pests is the timely detection of pest infestation. The goal is to manage the crop as a whole system, keeping pests at acceptable levels by utilizing several harmonizing strategies (Johnson, 2008). Earlier in this chapter, IPM was defined as a comprehensive pest control. Heitefuss (1989) defines IPM as a system in which all economically, ecologically and toxicologically suitable procedures are utilized in maximum harmony to maintain pests below the economic threshold. In the late 1990's, Luckmann and Metcalf (1994) defined IPM as an intelligent selection and use of pest control actions (or tactics) that will ensure favorable economic, ecological, and sociological consequences. IPM techniques include monitoring of pest populations, the judicious use of pesticides, and the effective communication regarding the necessity of implementing a control tactic or not. In a modern definition, the goal is to prevent and suppress pests with minimum impact on human health, the environment and non-target organisms (Dreistadt, 2004). According to Pedigo (1986), in order to use an IPM approach, an understanding of the following factors is required: (1) the biology of the pest and its natural enemies, (2) the response of crops to management practices, (3) the effect of pesticide application on pests and non-target organisms, and (4) the action threshold or level of damage tolerable by the plant.

## **3.1 Monitoring**

312 Insecticides – Pest Engineering

Burbank 80 78 74 75 71 71 69 63 63 66 62

Norkotah 5 5 8 8 8 8 10 14 12 10 10

Russet 7 6 4 4 4 3 1 2 1 - 1 Shepody 4 7 9 8 11 12 13 13 15 13 14 Umatilla - - - 1 - - - - - - 2 Alturas - - - - - - 1 3 3 2 2 Others 4 5 4 4 6 6 5 5 6 9 9 Table 1. Popular potato varieties (%) planted in Idaho, Washington and Oregon (Washington

After producers determine the nutritional needs of the crop (nitrogen, phosphorous, potassium), seed potatoes are cut uniformly, treated with fungicides and/or insecticides, and planted. Potatoes emerge 3-5 weeks after planting, depending on weather conditions, location and time of the year. After plant emergence, producers monitor crop needs regarding fertilization, pest control, and general management. Most potatoes in the area are grown under irrigation. Another common practice is desiccation or vine-killing (Hutchinson & Stall, 2007). Timely vine killing is essential for good tuber skin set, and efficient harvest. It is not clear if desiccation (naturally or artificially) of early season potatoes influences pest pressure in other nearby fields awaiting harvest. Johnson (2008) suggests a holistic plan for potato management combining management practices in the years prior to growing the potato crop, during the growing season, at harvest and during tuber storage. Miller and Hopkins (2008) specified that among many things, establishing appropriate long-term crop rotation, applying and alternating pesticides with different mode of action to avoid pesticide resistance, and improving soil microbial, physical and chemical health are necessary to

The potato plant develops through four clearly defined growth stages: (1) vegetative growth, (2) tuber initiation, (3) tuber growth and (4) plant maturation (Johnson, 2008). Each stage is affected by different groups of insect pests. The degree of the damage will depend on the timing of events, cultivar characteristics, and the intrinsic characteristics of each pest.

The first step for managing insect pests is the timely detection of pest infestation. The goal is to manage the crop as a whole system, keeping pests at acceptable levels by utilizing several harmonizing strategies (Johnson, 2008). Earlier in this chapter, IPM was defined as a comprehensive pest control. Heitefuss (1989) defines IPM as a system in which all economically, ecologically and toxicologically suitable procedures are utilized in maximum harmony to maintain pests below the economic threshold. In the late 1990's, Luckmann and Metcalf (1994) defined IPM as an intelligent selection and use of pest control actions (or tactics) that will ensure favorable economic, ecological, and sociological consequences. IPM techniques include monitoring of pest populations, the judicious use of pesticides, and the

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Varieties Year

Potato Commission, 2007; National Agricultural Statistics Service,

http://www.agcensus.usda.gov/index.asp).

**3. Managing insect pests in potatoes** 

Russet

Russet

Ranger

achieve a healthy crop.

Timing detection and monitoring is an essential long-term requirement against pest infestation in potatoes. Plants should be regularly checked for signs and symptoms of pest damage. During the height of the growing season, plants should be checked once or twice per week. The inspection of the undersides of leaves and the inner plant canopy is recommended since many pests prefer sheltered sites. A close look of any plant that has missing, absent or damaged leaves or flowers, or plants whose color, texture, or size looks different than healthy ones, can be a signs of a problem.

Some insect pests can be dislodged by laying a sheet, sturdy cardboard or paper below the infested plants and "beating" the canopy. Insects can be monitored with a hand lens or traps. Both techniques not only allow detection and monitoring of pest problems but also provide estimates of pest population density. The optimal timing of sampling depends upon the life history and behavior patterns of the pest and/or beneficial insects, as well as crop stage and state, and also environmental conditions. Some areas, such as field edges or fields next to main roads are more prone to pest problems (personal observations).

Different sampling procedures can be used depending on the crop, size of field, etc. In the Pacific Northwest, the majority of potatoes are planted in circles, under center-pivot irrigation, thus fields can be divided in quarters (e.g, north, east, west, south). Upon entering the field, quick visual examination of the field is recommended. Uncharacteristic areas with poor stands or patchy growth should be scouted thoroughly. In the Pacific North-western region traps are widely used. Some traps can be baited with a pheromone which is a chemical that usually attracts a single species (e.g., potato tuber moths). Traps can also be coated with adhesive material to "stick" the pest to the trap. Either natural (e.g., virgin females) or more often the synthetic pheromones are used to attract males (Roelofs et al., 1975; Persoons et al,. 1976; Voerman & Rothschild, 1978; Rothschild, 1986; Raman, 1988). Pheromones specifically disrupt the reproductive cycle of harmful insects. Pheromone traps are used extensively in commercial agriculture in the region helping farmers detect the presence of pest species (Merrill et al., 2011). Traps in general will work only for adult insects, as adults have developed wings and are more mobile. Yellow sticky traps attract fruit flies, winged aphids, thrips, psyllids, fungus gnats, wasps, numerous flies; many species collected in sticky cards belong to the same family or group (e.g., several moths in the Gelechiidae family can be found in traps meant to be for tuber moths, a member of the Gelechiidae family), thus correct identification of target pest(s) is needed.

## **3.2 Management**

Before planting, selecting field location can minimize pest damage and improve predictability of pest problems (Hoy et al., 2008). Also circumventing planting dates when insects will emerge and inflict most damage is recommended. Certified seed should be used at all times. If available, the selection of cultivars resistant or tolerant to important pests is also desirable.

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 315

In the Pacific Northwest region, a number of arthropod species attack insect pests at the egg, immature, and/or adult stage. Species of Heteroptera (former Hemiptera, Family Pentatomidae) such as *Podisus maculiventris* (Say) (spined soldier bug) and the *Perillus bioculatus* (Fabricius) (two-spotted stink bug), *Opolomus dichrosus* L. (no common name) can be found in potato fields feeding on *Leptinotarsa decemlineata* (Say) (Colorado potato beetle) (Ferro, 1994; Lacey et al., 2001). Tamaki et al., (1983) and Lopez et al., (1995) indicated that *Myiopharus doryphorae* L. (Tachinidae fly) and *Edovum puttleri* L. (Eulophidae wasp) are moderately efficient parasitoids of Colorado potato beetles. Nabidae (damsel bugs), Neuroptera (lace wings), Coccinellidae (lady bugs), Carabidae (ground beetles) and spiders are also common inhabitants in potato fields behaving as generalists feeding on aphids,

Traditional IPM text books recommend considering chemical controls only if other techniques do not result in adequate pest control. However, chemical controls can be effectively used with other techniques (e.g., cultural, physical, biological, etc.). When choosing a chemical, be sure to read the label and choose the right product; chemical selection can be the breaking point for success or failure in controlling target pests. Worldwide, the potato requires more pesticides than any other major food crop (CIP, 1994). Intense pesticide dependency has led to the development of pesticide resistance in cases such as the Colorado potato beetles in almost all potato-growing areas in the U.S. (Weisz et al.,1995). To date, the only exception of documented Colorado potato resistance to chemicals is in the Pacific Northwest (Schreiber et al., 2010). Management tactics that preserve susceptible genotypes and allow them to interbreed with resistant individuals have the greatest potential for resistance management according to Tabashnik (1989) and Weisz et al.,

Wireworms are one of the most destructive insect pests in the Pacific Northwest. Nearly 40 species from 12 genera attack potato, but only a few are economically important (Hoy et al., 2008). In irrigated production, the most common wireworm species are the Pacific Coast wireworm *(Limonius canus* LeConte*),* the sugar beet wireworm *(L. californicus*  (Mannerheim)), the western field wireworm (*L. infuscatus* Motschulsky*)*, and the Columbia Basin wireworm (*L. subauratus* LeConte*)*. Areas with annual rainfall less than 15 inches may be infested with the Great Basin wireworm (*Ctenicera pruinina* (Horn)). West of the Cascade mountains *Agriotes* spp. is the most common pest. A complex of species may occur (Figure

Wireworms are the larval stage of click beetles. Adult click beetles are slender hard-shelled insects. They range in color from chocolate to dark brown and from about 0.8 – 1.9 cm long, depending on species. Click beetles get their name from their ability to snap a spine on their thorax that produces a "clicking" sound and allows them to jump in the air when distressed or disturbed. All beetles in the Elateridae family have this ability. This technique is used to avoid predation or to get back on their feet after falling on their backs. Depending on the

thrips, small larvae and potato beetles.

**4. Potato pests in the Pacific Northwest** 

**4.1 Wireworms (Order Coleoptera: Family Elateridae)** 

**3.2.5 Chemical methods** 

4) (Andrew et al., 2008).

**4.1.1 Pest description** 

(1996).

## **3.2.1 Seed quality and certification**

High quality seed is essential for the production of a profitable crop (Love et al., 2003). Several diseases, some transmitted by insects and especially aphids, can be transmitted to infected seed such as viruses, bacterial ring rot, blackleg, late blight, scab, and wilt diseases (University of California, 1986). Disease-free seed tubers are available from certified nurseries where seed potatoes are grown for several generations before being commercially offered to producers. This process is regulated by each state's seed certification programs. The use of appropriate production practices is only half of the process of growing high quality potatoes (Love t al. 2003). Samples from seed growing areas are grown in field trial to establish disease problems. There is a range of tolerance from zero to 6% for certain viruses (University of California, 1986). For instance, Idaho inspection tolerance for postharvest winter tests of seed destined for recertification range from 0.8% for leafroll, 2.0% mosaic, and 5% chemical injury (Love et al., 2003). Thus, (1) clean all equipment thoroughly before entering fields, (2) establish aphid control programs to prevent the development of aphid populations in potato seed fields, (3) control aphids in potato fields to prevent the movement of aphids to seed fields.

## **3.2.2 Resistant cultivars**

Cultivars tolerant or resistant to pests can provide long-term protection for the potato crop. For a long time it has been recognized that the evaluation of potato germplasm for resistance is a valuable tool to developing IPM programs (Horgan et al., 2007). Although no commercial potato cultivar is completely resistant to insect damage, there are variations in the susceptibility of cultivars to the pest (Hoy et al., 2008). For example, tubers of the transgenic clone Spunta G2 are resistant to the potato tuberworm (Douches et al., 2002). Rondon et al., (2009) tested several cultivars including Spunta G2 with excellent results. However the public perception regarding genetically-modified organisms is still "blocking" the widely use of this type of resources.

### **3.2.3 Cultural practices**

Proper management of field preparation, planting, harvesting and storage are essential for maximum yield and tuber quality (University of California, 1986). Pest infestation can be minimized by avoiding contaminated seed tubers, soil, and water, sanitation of machinery brought from infested areas, removing cull piles, and following rational fertilizer and irrigation programs. Crop rotation, which reduces certain pests by breaking their life cycle, should be an integral part of a holistic management program.

#### **3.2.4 Biological control**

In a sustainable ecosystem, insect pest populations may be kept in check by natural enemies such as other insects. Parasitoids, predators, pathogens, antagonists, or competitor populations that suppress pest populations are desirable in fields (Driesche & Bellows, 1996). Under current pest management programs in potatoes, especially with an intensive agricultural production system centered on frequent calendar sprays of broad-spectrum insecticides, the impact of natural enemies is relatively unknown (Koss, 2003; Rondon, 2010). In contrast, a lot of information regarding the biology and the potential of natural enemies (a.k.a., biological control agents) can be found in the literature (Rondon, 2010). The advantage of using biological control agents is that they have no pre-harvest intervals, and are safer for application personnel, consumers and non-target organisms.

In the Pacific Northwest region, a number of arthropod species attack insect pests at the egg, immature, and/or adult stage. Species of Heteroptera (former Hemiptera, Family Pentatomidae) such as *Podisus maculiventris* (Say) (spined soldier bug) and the *Perillus bioculatus* (Fabricius) (two-spotted stink bug), *Opolomus dichrosus* L. (no common name) can be found in potato fields feeding on *Leptinotarsa decemlineata* (Say) (Colorado potato beetle) (Ferro, 1994; Lacey et al., 2001). Tamaki et al., (1983) and Lopez et al., (1995) indicated that *Myiopharus doryphorae* L. (Tachinidae fly) and *Edovum puttleri* L. (Eulophidae wasp) are moderately efficient parasitoids of Colorado potato beetles. Nabidae (damsel bugs), Neuroptera (lace wings), Coccinellidae (lady bugs), Carabidae (ground beetles) and spiders are also common inhabitants in potato fields behaving as generalists feeding on aphids, thrips, small larvae and potato beetles.

## **3.2.5 Chemical methods**

314 Insecticides – Pest Engineering

High quality seed is essential for the production of a profitable crop (Love et al., 2003). Several diseases, some transmitted by insects and especially aphids, can be transmitted to infected seed such as viruses, bacterial ring rot, blackleg, late blight, scab, and wilt diseases (University of California, 1986). Disease-free seed tubers are available from certified nurseries where seed potatoes are grown for several generations before being commercially offered to producers. This process is regulated by each state's seed certification programs. The use of appropriate production practices is only half of the process of growing high quality potatoes (Love t al. 2003). Samples from seed growing areas are grown in field trial to establish disease problems. There is a range of tolerance from zero to 6% for certain viruses (University of California, 1986). For instance, Idaho inspection tolerance for postharvest winter tests of seed destined for recertification range from 0.8% for leafroll, 2.0% mosaic, and 5% chemical injury (Love et al., 2003). Thus, (1) clean all equipment thoroughly before entering fields, (2) establish aphid control programs to prevent the development of aphid populations in potato seed fields, (3) control aphids in potato fields to prevent the

Cultivars tolerant or resistant to pests can provide long-term protection for the potato crop. For a long time it has been recognized that the evaluation of potato germplasm for resistance is a valuable tool to developing IPM programs (Horgan et al., 2007). Although no commercial potato cultivar is completely resistant to insect damage, there are variations in the susceptibility of cultivars to the pest (Hoy et al., 2008). For example, tubers of the transgenic clone Spunta G2 are resistant to the potato tuberworm (Douches et al., 2002). Rondon et al., (2009) tested several cultivars including Spunta G2 with excellent results. However the public perception regarding genetically-modified organisms is still "blocking"

Proper management of field preparation, planting, harvesting and storage are essential for maximum yield and tuber quality (University of California, 1986). Pest infestation can be minimized by avoiding contaminated seed tubers, soil, and water, sanitation of machinery brought from infested areas, removing cull piles, and following rational fertilizer and irrigation programs. Crop rotation, which reduces certain pests by breaking their life cycle,

In a sustainable ecosystem, insect pest populations may be kept in check by natural enemies such as other insects. Parasitoids, predators, pathogens, antagonists, or competitor populations that suppress pest populations are desirable in fields (Driesche & Bellows, 1996). Under current pest management programs in potatoes, especially with an intensive agricultural production system centered on frequent calendar sprays of broad-spectrum insecticides, the impact of natural enemies is relatively unknown (Koss, 2003; Rondon, 2010). In contrast, a lot of information regarding the biology and the potential of natural enemies (a.k.a., biological control agents) can be found in the literature (Rondon, 2010). The advantage of using biological control agents is that they have no pre-harvest intervals, and

**3.2.1 Seed quality and certification** 

movement of aphids to seed fields.

the widely use of this type of resources.

should be an integral part of a holistic management program.

are safer for application personnel, consumers and non-target organisms.

**3.2.2 Resistant cultivars** 

**3.2.3 Cultural practices** 

**3.2.4 Biological control** 

Traditional IPM text books recommend considering chemical controls only if other techniques do not result in adequate pest control. However, chemical controls can be effectively used with other techniques (e.g., cultural, physical, biological, etc.). When choosing a chemical, be sure to read the label and choose the right product; chemical selection can be the breaking point for success or failure in controlling target pests. Worldwide, the potato requires more pesticides than any other major food crop (CIP, 1994). Intense pesticide dependency has led to the development of pesticide resistance in cases such as the Colorado potato beetles in almost all potato-growing areas in the U.S. (Weisz et al.,1995). To date, the only exception of documented Colorado potato resistance to chemicals is in the Pacific Northwest (Schreiber et al., 2010). Management tactics that preserve susceptible genotypes and allow them to interbreed with resistant individuals have the greatest potential for resistance management according to Tabashnik (1989) and Weisz et al., (1996).

## **4. Potato pests in the Pacific Northwest**

## **4.1 Wireworms (Order Coleoptera: Family Elateridae)**

Wireworms are one of the most destructive insect pests in the Pacific Northwest. Nearly 40 species from 12 genera attack potato, but only a few are economically important (Hoy et al., 2008). In irrigated production, the most common wireworm species are the Pacific Coast wireworm *(Limonius canus* LeConte*),* the sugar beet wireworm *(L. californicus*  (Mannerheim)), the western field wireworm (*L. infuscatus* Motschulsky*)*, and the Columbia Basin wireworm (*L. subauratus* LeConte*)*. Areas with annual rainfall less than 15 inches may be infested with the Great Basin wireworm (*Ctenicera pruinina* (Horn)). West of the Cascade mountains *Agriotes* spp. is the most common pest. A complex of species may occur (Figure 4) (Andrew et al., 2008).

## **4.1.1 Pest description**

Wireworms are the larval stage of click beetles. Adult click beetles are slender hard-shelled insects. They range in color from chocolate to dark brown and from about 0.8 – 1.9 cm long, depending on species. Click beetles get their name from their ability to snap a spine on their thorax that produces a "clicking" sound and allows them to jump in the air when distressed or disturbed. All beetles in the Elateridae family have this ability. This technique is used to avoid predation or to get back on their feet after falling on their backs. Depending on the

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 317

Wireworms can cause damage to potatoes by feeding upon potato seed pieces and sprouts in the spring, facilitating infection by pathogens or other insect pests. The latter damage can result in reduction in yield and/or rejection of the entire crop. In the U.S. there is zero tolerance for live larvae in tubers. Wireworms tend to be most damaging in potatoes that follow corn or small grains (wheat, barley) and on ground just entering cultivation. Wireworms damage potatoes both near planting time (damage to seed pieces) and during the growing season (damage to developing tubers). They can also be a problem at harvest

Potatoes, corn, wheat and grass are hosts for several species of wireworms in the Pacific Northwest. Also, beans, carrots, peas, and other annual crops may be infested; while

Adult click beetles emerge from pupae in the soil from late spring through late summer. In the Pacific Northwest, wireworms overwinter as larvae or adults. They can have up to a 7 year life cycle from egg to adult. Adults can fly, but usually remain in the areas where they developed as larvae. Eggs are laid in grassland or in cereal crops where larvae feed on grass roots and overwinter in the soil. Females tend to lay eggs in grassy areas. Larvae can live from 2-5 years in the soil, depending on the species. They require several years to mature and can overwinter at a depth of 30-100 cm or more in the soil, only to return near the surface in spring to resume feeding when soil temperatures exceed 50°F (10°C). Later in the season when temperatures reach 80°F (26.6°C) and above, the larvae tend to move deeper than 15 cm into the soil to escape the "heat" (Hoy et al., 2008; Schreiber et al., 2010). Wireworm larva can be confused with larvae of the family Tenebrionidae (false wireworms) or Tipulidae (crane fly). The Tenebrionidae group tends to be saprophagous

Trapping should start early, especially in areas with history of wireworm problems. In the Pacific Northwest trapping starts mid to late March until April to May. Horton (2006) modeled the relationship between bait trap counts and crop damage by *L. canus* in Wapato, WA. Horton's model predicts tuber damage based on number of wireworms collected. Wireworm presence or absence in a field should be determined before using control measures. Unfortunately, current monitoring methods are time consuming, laborious and often do not accurately reflect field populations of this pest. Historically, wireworms have been monitored by extracting and sifting through soil cores to locate larvae. Since the distribution of wireworms in a field tends to be patchy and unpredictable, large numbers of samples are required to accurately estimate population size. Baits have largely replaced random soil sampling, since they are less labor intensive and may detect low wireworm populations. Baited traps can be constructed by placing 3-4 tablespoons of a mixed of wheat and corn seeds or rolled oats inside a fine mesh bag or nylon. The seed mixture should be soaked in water 24 hours prior to placement in the hole to facilitate germination. Dig a hole about 20-25 cm deep and 3.5- 4 cm wide at the soil surface (Horton, 2006). Bury the mixture

melons, beet roots, and strawberry fruits are affected less frequently.

while Tipulidae are associated with grassy crops.

**4.1.2 Damage** 

**4.1.3 Hosts** 

**4.1.4 Biology** 

**4.1.5 Monitoring** 

and before entering storage.

Fig. 4. Wireworms in the Pacific Northwest, from left: *Limonius canus*, *L. californicus*, *L. infuscatus*, *L. subauratus*, *Ctenicera pruinina*, and *Agriotes lineatus* (Photo by C.J. Marshall, Oregon State Arthropod Collection http://osac.science.oregonstate.edu).

species, each female after mating lays an average of 80 eggs, singly or in small clusters in the soil. Immature stages have a hardened and shiny shell and very few hairs (Fig. 5). They have three body regions with a distinct head, a thorax with 3 pairs of legs and a segmented abdomen with a tail-end (Jensen et al., 2011). Characteristics of the tail-end serve for identification purposes. Depending on species and age, wireworm larvae range from about 2 mm after hatching to 4 cm long or more at maturity. Wireworm pupae are first white, but later change to reddish-brown; they pupae in the soil.

Fig. 5. Wireworm larva (Photo by A. Jensen, Washington Potato Commission).

## **4.1.2 Damage**

316 Insecticides – Pest Engineering

Fig. 4. Wireworms in the Pacific Northwest, from left: *Limonius canus*, *L. californicus*, *L. infuscatus*, *L. subauratus*, *Ctenicera pruinina*, and *Agriotes lineatus* (Photo by C.J. Marshall,

species, each female after mating lays an average of 80 eggs, singly or in small clusters in the soil. Immature stages have a hardened and shiny shell and very few hairs (Fig. 5). They have three body regions with a distinct head, a thorax with 3 pairs of legs and a segmented abdomen with a tail-end (Jensen et al., 2011). Characteristics of the tail-end serve for identification purposes. Depending on species and age, wireworm larvae range from about 2 mm after hatching to 4 cm long or more at maturity. Wireworm pupae are first white, but

Oregon State Arthropod Collection http://osac.science.oregonstate.edu).

Fig. 5. Wireworm larva (Photo by A. Jensen, Washington Potato Commission).

later change to reddish-brown; they pupae in the soil.

Wireworms can cause damage to potatoes by feeding upon potato seed pieces and sprouts in the spring, facilitating infection by pathogens or other insect pests. The latter damage can result in reduction in yield and/or rejection of the entire crop. In the U.S. there is zero tolerance for live larvae in tubers. Wireworms tend to be most damaging in potatoes that follow corn or small grains (wheat, barley) and on ground just entering cultivation. Wireworms damage potatoes both near planting time (damage to seed pieces) and during the growing season (damage to developing tubers). They can also be a problem at harvest and before entering storage.

## **4.1.3 Hosts**

Potatoes, corn, wheat and grass are hosts for several species of wireworms in the Pacific Northwest. Also, beans, carrots, peas, and other annual crops may be infested; while melons, beet roots, and strawberry fruits are affected less frequently.

## **4.1.4 Biology**

Adult click beetles emerge from pupae in the soil from late spring through late summer. In the Pacific Northwest, wireworms overwinter as larvae or adults. They can have up to a 7 year life cycle from egg to adult. Adults can fly, but usually remain in the areas where they developed as larvae. Eggs are laid in grassland or in cereal crops where larvae feed on grass roots and overwinter in the soil. Females tend to lay eggs in grassy areas. Larvae can live from 2-5 years in the soil, depending on the species. They require several years to mature and can overwinter at a depth of 30-100 cm or more in the soil, only to return near the surface in spring to resume feeding when soil temperatures exceed 50°F (10°C). Later in the season when temperatures reach 80°F (26.6°C) and above, the larvae tend to move deeper than 15 cm into the soil to escape the "heat" (Hoy et al., 2008; Schreiber et al., 2010). Wireworm larva can be confused with larvae of the family Tenebrionidae (false wireworms) or Tipulidae (crane fly). The Tenebrionidae group tends to be saprophagous while Tipulidae are associated with grassy crops.

## **4.1.5 Monitoring**

Trapping should start early, especially in areas with history of wireworm problems. In the Pacific Northwest trapping starts mid to late March until April to May. Horton (2006) modeled the relationship between bait trap counts and crop damage by *L. canus* in Wapato, WA. Horton's model predicts tuber damage based on number of wireworms collected. Wireworm presence or absence in a field should be determined before using control measures. Unfortunately, current monitoring methods are time consuming, laborious and often do not accurately reflect field populations of this pest. Historically, wireworms have been monitored by extracting and sifting through soil cores to locate larvae. Since the distribution of wireworms in a field tends to be patchy and unpredictable, large numbers of samples are required to accurately estimate population size. Baits have largely replaced random soil sampling, since they are less labor intensive and may detect low wireworm populations. Baited traps can be constructed by placing 3-4 tablespoons of a mixed of wheat and corn seeds or rolled oats inside a fine mesh bag or nylon. The seed mixture should be soaked in water 24 hours prior to placement in the hole to facilitate germination. Dig a hole about 20-25 cm deep and 3.5- 4 cm wide at the soil surface (Horton, 2006). Bury the mixture

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 319

This beetle can cause complete defoliation and nearly complete crop loss if allowed to reproduce unchecked. Both larvae and adults feed on potato foliage throughout the season.

Potatoes and other solanaceous plants such as eggplant, nightshade, horsenettle and

Pupation and overwintering occur in the soil. Adults emerge from the soil to lay eggs in the spring. Depending on the region, this insect may have three generations in a season. Adult beetles spend the winter buried 10-25 cm in the soil and emerge in the spring just as the first volunteer potatoes appear. Recently emerged beetles either mate close to the overwintering sites or fly to new potato fields to find a mate. Usually first infestations occur around field margins. Eggs are deposited on potato foliage in masses. CPB eggs resemble lady beetle eggs. Larvae pass through four life stages and then burrow into the

Start monitoring fields at crop emergence. There are no established treatment thresholds for CPB. Large CPB populations are harder to manage than small ones, thus the goal is to

Crop rotation may help in delaying or reducing CPB pressure. Colonizing beetles need to feed before laying eggs, so controlling volunteer potatoes and solanaceous weeds is important as are rotating crops and planting new potato fields far from the last year's potato fields (Schreiber et al., 2010). These practices will reduce the number of overwintering beetles migrating into the new field. This may not be a practical solution in the Pacific Northwest region since potatoes are use in rotation with other local crops such as wheat or corn. The use of "at planting" and systemic insecticides in early potatoes will contribute to the control of early-season CPB populations. The use of pyrethroid insecticides is not recommended since it has a direct effect on natural enemies. Targeting chemical

applications to control eggs and young larvae when possible is recommended.

**4.3 Green peach aphid and potato aphid (Order Heteroptera: Family Aphididae)**  The aphid population in western North America, north of Mexico, is comprised of 1,020 species in 178 genera in 15 subfamilies (Pike et al., 2003). Several aphid species are known to be pests of potatoes, but the green peach aphid, *Myzus persicae* (Sulzer), and potato aphid, *Macrosiphum euphorbiae* (Thomas), are two of the most important vectors of diseases in the Pacific Northwest. Aphids are important due to their ability to transmit viruses. According to Hoy et al., (2008) there are six commonly found potato viruses transmitted by aphids: *Potato leafroll virus* (PLRV), multiple strains of *Potato virus Y* (PVY), *Potato virus A* (PVA), *Potato virus S* (PVS), *Potato virus M* (PVM), and *alfalfa mosaic virus* (AMV). PLRV and PVY are transmitted by several species of aphids but primarily by green peach aphid. The potato

**4.2.2 Damage** 

**4.2.3 Hosts** 

**4.2.4 Biology** 

soil to pupate.

**4.2.6 Control** 

**4.2.5 Monitoring** 

control this pest early in the season.

aphid transmits PVY and PVA.

buffalobur are preferred hosts of this pest.

at the bottom of the hole. Fill the hole and mound a "soil dome" over the covered bait to serve as a solar collector and to prevent standing water. Cover each mound with a sheet of black plastic and cover the edges with soil to hold the plastic sheet down. The plastic collects solar radiation and speeds germination of the mixture. The germinating seeds attract wireworms. A few days later, remove the plastic and soil covering the bait and count the number of wireworm larvae found at each station. There are not specific recommendations as to how many traps per field should be placed, however, placement of the bait stations should represent different areas of a field (Campbell & Stone, 1939; Simmons et al., 1998)

## **4.1.6 Control**

There are no effective natural enemies for wireworm. If one suspects wireworms are present in a field based on trapping, chemical control is the best management option (http://potatoes.com/Research-IPM.cfm). Fumigants are effective on wireworms that are present at the time of fumigation and within the zone of fumigation (Schreiber et al., 2010). Fumigants are sensitive to soil temperatures. In furrow applications are also effective; however, some rotational restrictions may apply (Schreiber et al., 2010). Use of contemporary chemicals in other crops suggests that stand protection and wireworm reduction are not covered with current chemicals available (Vernon et al., 2009).

## **4.2 Colorado Potato Beetle (Order Coleoptera: Family Chrysomelidae)**

The Colorado potato beetle, *Leptinotarsa decemlineata* (Say), first described in 1824 by Thomas Say, is associated with potato plants and its solanaceous relatives such as nightshade. It is the most important defoliating insect pest of potato. Its remarkable ability to develop insecticide resistance, incredible reproductive potential and sustained feeding by larvae and adults, makes the management of this pest challenging (Hoy et al., 2008).

## **4.2.1 Pest description**

The Colorado Potato Beetle (CPB) is a yellow and black striped beetle, about 1.3 cm long and 0.6 cm wide. They can be found in almost all U.S. potato regions. Larvae are reddish orange, with two rows of black spots on each side. Orange egg clusters are found mainly on the undersides of leaves, mostly in the top third of the plant. Eggs resemble ladybug eggs (Fig. 6).

Fig. 6. Colorado potato beetle egg mass (left), larva (center), and adult (right). Photos by R. Marchosky (egg mass), L. Ketchum (larva), and S.I. Rondon (adult), OSU.

## **4.2.2 Damage**

318 Insecticides – Pest Engineering

at the bottom of the hole. Fill the hole and mound a "soil dome" over the covered bait to serve as a solar collector and to prevent standing water. Cover each mound with a sheet of black plastic and cover the edges with soil to hold the plastic sheet down. The plastic collects solar radiation and speeds germination of the mixture. The germinating seeds attract wireworms. A few days later, remove the plastic and soil covering the bait and count the number of wireworm larvae found at each station. There are not specific recommendations as to how many traps per field should be placed, however, placement of the bait stations should represent different areas of a field (Campbell & Stone, 1939; Simmons et al., 1998)

There are no effective natural enemies for wireworm. If one suspects wireworms are present in a field based on trapping, chemical control is the best management option (http://potatoes.com/Research-IPM.cfm). Fumigants are effective on wireworms that are present at the time of fumigation and within the zone of fumigation (Schreiber et al., 2010). Fumigants are sensitive to soil temperatures. In furrow applications are also effective; however, some rotational restrictions may apply (Schreiber et al., 2010). Use of contemporary chemicals in other crops suggests that stand protection and wireworm

The Colorado potato beetle, *Leptinotarsa decemlineata* (Say), first described in 1824 by Thomas Say, is associated with potato plants and its solanaceous relatives such as nightshade. It is the most important defoliating insect pest of potato. Its remarkable ability to develop insecticide resistance, incredible reproductive potential and sustained feeding by larvae and

The Colorado Potato Beetle (CPB) is a yellow and black striped beetle, about 1.3 cm long and 0.6 cm wide. They can be found in almost all U.S. potato regions. Larvae are reddish orange, with two rows of black spots on each side. Orange egg clusters are found mainly on the undersides of leaves, mostly in the top third of the plant. Eggs resemble ladybug eggs

Fig. 6. Colorado potato beetle egg mass (left), larva (center), and adult (right). Photos by R.

Marchosky (egg mass), L. Ketchum (larva), and S.I. Rondon (adult), OSU.

reduction are not covered with current chemicals available (Vernon et al., 2009).

**4.2 Colorado Potato Beetle (Order Coleoptera: Family Chrysomelidae)** 

adults, makes the management of this pest challenging (Hoy et al., 2008).

**4.1.6 Control** 

**4.2.1 Pest description** 

(Fig. 6).

This beetle can cause complete defoliation and nearly complete crop loss if allowed to reproduce unchecked. Both larvae and adults feed on potato foliage throughout the season.

## **4.2.3 Hosts**

Potatoes and other solanaceous plants such as eggplant, nightshade, horsenettle and buffalobur are preferred hosts of this pest.

## **4.2.4 Biology**

Pupation and overwintering occur in the soil. Adults emerge from the soil to lay eggs in the spring. Depending on the region, this insect may have three generations in a season. Adult beetles spend the winter buried 10-25 cm in the soil and emerge in the spring just as the first volunteer potatoes appear. Recently emerged beetles either mate close to the overwintering sites or fly to new potato fields to find a mate. Usually first infestations occur around field margins. Eggs are deposited on potato foliage in masses. CPB eggs resemble lady beetle eggs. Larvae pass through four life stages and then burrow into the soil to pupate.

## **4.2.5 Monitoring**

Start monitoring fields at crop emergence. There are no established treatment thresholds for CPB. Large CPB populations are harder to manage than small ones, thus the goal is to control this pest early in the season.

## **4.2.6 Control**

Crop rotation may help in delaying or reducing CPB pressure. Colonizing beetles need to feed before laying eggs, so controlling volunteer potatoes and solanaceous weeds is important as are rotating crops and planting new potato fields far from the last year's potato fields (Schreiber et al., 2010). These practices will reduce the number of overwintering beetles migrating into the new field. This may not be a practical solution in the Pacific Northwest region since potatoes are use in rotation with other local crops such as wheat or corn. The use of "at planting" and systemic insecticides in early potatoes will contribute to the control of early-season CPB populations. The use of pyrethroid insecticides is not recommended since it has a direct effect on natural enemies. Targeting chemical applications to control eggs and young larvae when possible is recommended.

## **4.3 Green peach aphid and potato aphid (Order Heteroptera: Family Aphididae)**

The aphid population in western North America, north of Mexico, is comprised of 1,020 species in 178 genera in 15 subfamilies (Pike et al., 2003). Several aphid species are known to be pests of potatoes, but the green peach aphid, *Myzus persicae* (Sulzer), and potato aphid, *Macrosiphum euphorbiae* (Thomas), are two of the most important vectors of diseases in the Pacific Northwest. Aphids are important due to their ability to transmit viruses. According to Hoy et al., (2008) there are six commonly found potato viruses transmitted by aphids: *Potato leafroll virus* (PLRV), multiple strains of *Potato virus Y* (PVY), *Potato virus A* (PVA), *Potato virus S* (PVS), *Potato virus M* (PVM), and *alfalfa mosaic virus* (AMV). PLRV and PVY are transmitted by several species of aphids but primarily by green peach aphid. The potato aphid transmits PVY and PVA.

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 321

In general, aphids injure plants directly by removing sap juices from phloem tissues. They also reduce the aesthetic quality of infested plants by secreting a sugary liquid called "honeydew" on which a black-colored fungus called "sooty mold" grows. The "sooty mold" reduces the photosynthetic potential of the plant. Most importantly, aphids transmit plant diseases, particularly viruses. Aphids on potato are serious pests because of their ability to transmit several plant diseases such as PLRV (transmitted mainly by green peach aphid) and PVY (transmitted by several species of aphids). PLRV causes necrosis while strains of PVY can cause internal brown lesions in the tubers. Srinivasan & Alvarez (2007) reported

The green peach aphid, also known as tobacco or spinach aphid, survives the winter in the egg stage on peach trees. They can also overwinter on various perennial, biennial, and winter annual weeds, such as tumble mustard, flixweed, shepherd's-purse, chickweed, mallow, horseweed, pennycress and redstem filaree. Besides potatoes and peaches, other hosts include lettuce, spinach, tomatoes, other vegetables and ornamentals (Dickson &

Green peach aphid migrates to potatoes in the spring from weeds and various crops where it has overwintered as nymphs and adults, or from peach and related trees where it overwinters as eggs. Most aphids reproduce sexually and develop through gradual metamorphosis (overwintering diapause egg, nymphs and winged or wingless adults) but also through a process called 'parthenogenesis' in which the production of offspring occurs without mating (Jensen et al., 2011). Potato aphids also overwinter as active nymphs, adults or eggs; eggs are laid on roses and sometimes other plants. Throughout the growing season aphids produce live young, all of which are female and can be either winged or wingless. In some instances, aphids undergo sexual, oviparous reproduction as a response of a change in photoperiod and temperature, or perhaps a lower food quantity or quality, where females produce sexual females and males. In the fall, winged males are produced which fly to overwintering hosts and mate with the egg-laying females produced on that host. Aphids found in the region undergo multiple overlapping generations per year (Jensen et al., 2011,

Fields should be checked for aphids at least once a week starting after emergence. The most effective scouting method is beating sheets, trays, buckets or white paper. There are no wellestablished treatment thresholds for aphids in potatoes in the Pacific Northwest but since aphids transmit viruses, producers are encouraged to control aphids early in the season, especially in seed potato producing areas. Schreiber et al., (2010) recommend a minimum sample size of ten locations per 100 acre field. For potatoes that are not to be stored, application of foliar aphidicide should begin when 5 aphids per 100 leaves or 5 aphids/plant are detected. Hoy et al., (2008) suggests some sampling methods and action thresholds for colonizing aphids on processing potatoes, table stock, and seed potato in

that mixed viral infections of heterologous viruses occur regularly in potatoes.

Laird, 1967; Wallis, 1967; Tamaki et al., 1980; Barry et al., 1982).

**4.3.2 Damage** 

**4.3.3 Hosts** 

**4.3.4 Biology** 

Schreiber et al., 2010).

different productions thresholds.

**4.3.5 Monitoring** 

## **4.3.1 Pest description**

Green peach aphids are small, usually less than 0.3 cm long. The body varies in color from pink to green with three darker stripes down the back. The head has long antennae which have an inward pointing projection or tubercle at its base (Fig. 7). Potato aphids are larger than green peach aphids with a body somewhat elongated and wedge-shaped (Fig. 8). The adults of both species may be winged (alatae) or wingless (apterous). Winged forms are usually triggered by environmental changes (e.g., decreasing photoperiod or temperature, deterioration of the host plant or overcrowding) (Branson et al., 1966). On the back of the fifth abdominal segment, a pair of tube-like structures called "siphunculi", "cornicles", or "pipes" are present on most aphid species. The green peach aphid present a "swollen" cornicles with a dark tip, while the cornicles on the potato aphid are 1/3 of the length of the body and are usually curved slightly outward (Alvarez et al., 2003).

Fig. 7. Green peach aphid wingless adult (left) and alatae (right). Photos by A. Jensen, Washington Potato Commission.

Fig. 8. Potato aphid wingless adult and nymphs (left) and alatae (right). Photos by A. Jensen, Washington Potato Commission.

## **4.3.2 Damage**

320 Insecticides – Pest Engineering

Green peach aphids are small, usually less than 0.3 cm long. The body varies in color from pink to green with three darker stripes down the back. The head has long antennae which have an inward pointing projection or tubercle at its base (Fig. 7). Potato aphids are larger than green peach aphids with a body somewhat elongated and wedge-shaped (Fig. 8). The adults of both species may be winged (alatae) or wingless (apterous). Winged forms are usually triggered by environmental changes (e.g., decreasing photoperiod or temperature, deterioration of the host plant or overcrowding) (Branson et al., 1966). On the back of the fifth abdominal segment, a pair of tube-like structures called "siphunculi", "cornicles", or "pipes" are present on most aphid species. The green peach aphid present a "swollen" cornicles with a dark tip, while the cornicles on the potato aphid are 1/3 of the length of the

Fig. 7. Green peach aphid wingless adult (left) and alatae (right). Photos by A. Jensen,

 Fig. 8. Potato aphid wingless adult and nymphs (left) and alatae (right). Photos by A. Jensen,

body and are usually curved slightly outward (Alvarez et al., 2003).

**4.3.1 Pest description** 

Washington Potato Commission.

Washington Potato Commission.

In general, aphids injure plants directly by removing sap juices from phloem tissues. They also reduce the aesthetic quality of infested plants by secreting a sugary liquid called "honeydew" on which a black-colored fungus called "sooty mold" grows. The "sooty mold" reduces the photosynthetic potential of the plant. Most importantly, aphids transmit plant diseases, particularly viruses. Aphids on potato are serious pests because of their ability to transmit several plant diseases such as PLRV (transmitted mainly by green peach aphid) and PVY (transmitted by several species of aphids). PLRV causes necrosis while strains of PVY can cause internal brown lesions in the tubers. Srinivasan & Alvarez (2007) reported that mixed viral infections of heterologous viruses occur regularly in potatoes.

## **4.3.3 Hosts**

The green peach aphid, also known as tobacco or spinach aphid, survives the winter in the egg stage on peach trees. They can also overwinter on various perennial, biennial, and winter annual weeds, such as tumble mustard, flixweed, shepherd's-purse, chickweed, mallow, horseweed, pennycress and redstem filaree. Besides potatoes and peaches, other hosts include lettuce, spinach, tomatoes, other vegetables and ornamentals (Dickson & Laird, 1967; Wallis, 1967; Tamaki et al., 1980; Barry et al., 1982).

## **4.3.4 Biology**

Green peach aphid migrates to potatoes in the spring from weeds and various crops where it has overwintered as nymphs and adults, or from peach and related trees where it overwinters as eggs. Most aphids reproduce sexually and develop through gradual metamorphosis (overwintering diapause egg, nymphs and winged or wingless adults) but also through a process called 'parthenogenesis' in which the production of offspring occurs without mating (Jensen et al., 2011). Potato aphids also overwinter as active nymphs, adults or eggs; eggs are laid on roses and sometimes other plants. Throughout the growing season aphids produce live young, all of which are female and can be either winged or wingless. In some instances, aphids undergo sexual, oviparous reproduction as a response of a change in photoperiod and temperature, or perhaps a lower food quantity or quality, where females produce sexual females and males. In the fall, winged males are produced which fly to overwintering hosts and mate with the egg-laying females produced on that host. Aphids found in the region undergo multiple overlapping generations per year (Jensen et al., 2011, Schreiber et al., 2010).

## **4.3.5 Monitoring**

Fields should be checked for aphids at least once a week starting after emergence. The most effective scouting method is beating sheets, trays, buckets or white paper. There are no wellestablished treatment thresholds for aphids in potatoes in the Pacific Northwest but since aphids transmit viruses, producers are encouraged to control aphids early in the season, especially in seed potato producing areas. Schreiber et al., (2010) recommend a minimum sample size of ten locations per 100 acre field. For potatoes that are not to be stored, application of foliar aphidicide should begin when 5 aphids per 100 leaves or 5 aphids/plant are detected. Hoy et al., (2008) suggests some sampling methods and action thresholds for colonizing aphids on processing potatoes, table stock, and seed potato in different productions thresholds.

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 323

2006). Potato is not a preferred host for BLH and will not spend much time on the crop

Among the favorite hosts are Kochia, Russian thistle, and various weedy mustard species such as tumble mustard. Beet leafhoppers are especially abundant on young, marginal, semi-dry and small weeds plants. They also thrive on radishes, sugar beet (Meyerdirk &

The beet leafhopper overwinters on rangeland weeds and migrates to potatoes as early as May. They overwinter as adult females in weedy and native vegetation throughout most of the dry production areas. The beet leafhopper has three life stages: egg, nymph and adult. The adult can have a "darker form", early or late in the season; and a "clear form (during the season) (Fig. 10). Beet leafhoppers can transmit BLTVA as adults and nymphs. Eggs are laid in stems of host plants, and a new spring generation begins developing in March and April. Beet leafhopper begins to move from weeds to potatoes and potentially affect potatoes during the first spring generation, which matures in late May to early June (Jensen et al., 2011). Potatoes are most seriously affected by BLTVA infections that occur early in the growing season (Rondon unpublished). Beet leafhopper remains common through the summer, during which it goes through 2 to 3 overlapping generations. The final generation for the year matures during late October-early November. Total number of beet leafhoppers

(however it does spend enough time to transmit BLTVA) (Schreiber et al., 2010).

Hessein, 1985), and carrots (Munyaneza, 2003).

varies from year to year (Crosslin et al., 2011).

mm. Photos by A. Jensen, Washington Potato Commission.

Fig. 10. "Clear form"(left) and "dark form" (right) of the beet leafhopper. Size of adults 2.5-3

Because potatoes are not a preferred host of the BLH, in-field sampling is problematic. Most recommendations suggest the use of yellow sticky cards around field margins. It is important to keep traps close to the ground where hoppers mostly move. Check and replace

**4.4.3 Hosts** 

**4.4.4 Biology** 

**4.4.5 Monitoring** 

## **4.3.6 Control**

Weed control and elimination of secondary hosts are critical. Early aphid infestations commonly occur on a number of weeds including species of mustards and nightshade; therefore, those weeds should be kept under control. Research in Idaho indicates that hairy nightshade is an excellent aphid and virus host (Srinivasan & Alvarez, 2007), thus, control of this weed is highly recommended. In some instances, the number of insects available to infest crops in the spring depends upon winter survival (DeBano et al., 2010). Thus, the elimination of overwintering sites is recommended if possible. Peach trees are the most common winter hosts, although apricots and several species of Prunus are sometimes infested (Schreiber et al., 2010). A large numbers of generalist predators feed on aphids including the minute pirate bugs, big-eyed bugs, damsel bugs, lady beetles and their larvae, lacewings, flower fly larvae, and aphid-specific parasitoid wasps. If aphids are present, use of insecticides in commercial fields should occur as soon as non-winged aphids are detected. In seed producing areas, preventive methods are recommended. Application of foliar aphidicide should begin just prior to the decline in performance of seed-treatment insecticides applied at planting (60 days after planting, Rondon unpublished). Schreiber et al., 2010 indicated that complete insect control from planting until aphid flights have ceased is the only means to manage diseases in full season potatoes.

## **4.4 Beet leafhopper (Order Heteroptera: Family Cicadellidae)**

The beet leafhopper, *Circulifer tenellus* Baker, is the carrier of the beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (a.k.a., Columbia Basin potato purple top phytoplasma) that causes significant yield losses and a reduction in potato tuber quality.

## **4.4.1 Pest description**

The beet leafhopper (BLH) is a wedge-shaped and pale green to gray or brown in color. It has several nymphal instars (Fig. 9). Adults may have dark markings on the upper surface of the body early and late in the season ("darker form") or clear during the season ("clear form") (Fig. 10).

Fig. 9. Beet leafhopper nymphs. Photos by A. Murphy, OSU.

## **4.4.2 Damage**

Beet leafhoppers must feed in the phloem of the plant. Direct feeding can cause relatively minor damage ("hopperburn"); however, BLTVA is a very destructive and detrimental disease affecting potatoes. BLTVA can cause a wide range of symptoms in potatoes, including leaf curling and purpling, aerial tubers, chlorosis, and early senescence. Most BLTVA infection occurs early in the season, during May and June (Munyaneza, 2003; Munyaneza & Crosslin, 2006). Potato is not a preferred host for BLH and will not spend much time on the crop (however it does spend enough time to transmit BLTVA) (Schreiber et al., 2010).

## **4.4.3 Hosts**

322 Insecticides – Pest Engineering

Weed control and elimination of secondary hosts are critical. Early aphid infestations commonly occur on a number of weeds including species of mustards and nightshade; therefore, those weeds should be kept under control. Research in Idaho indicates that hairy nightshade is an excellent aphid and virus host (Srinivasan & Alvarez, 2007), thus, control of this weed is highly recommended. In some instances, the number of insects available to infest crops in the spring depends upon winter survival (DeBano et al., 2010). Thus, the elimination of overwintering sites is recommended if possible. Peach trees are the most common winter hosts, although apricots and several species of Prunus are sometimes infested (Schreiber et al., 2010). A large numbers of generalist predators feed on aphids including the minute pirate bugs, big-eyed bugs, damsel bugs, lady beetles and their larvae, lacewings, flower fly larvae, and aphid-specific parasitoid wasps. If aphids are present, use of insecticides in commercial fields should occur as soon as non-winged aphids are detected. In seed producing areas, preventive methods are recommended. Application of foliar aphidicide should begin just prior to the decline in performance of seed-treatment insecticides applied at planting (60 days after planting, Rondon unpublished). Schreiber et al., 2010 indicated that complete insect control from planting until aphid flights have ceased

The beet leafhopper, *Circulifer tenellus* Baker, is the carrier of the beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (a.k.a., Columbia Basin potato purple top phytoplasma) that causes significant yield losses and a reduction in potato tuber quality.

The beet leafhopper (BLH) is a wedge-shaped and pale green to gray or brown in color. It has several nymphal instars (Fig. 9). Adults may have dark markings on the upper surface of the body early and late in the season ("darker form") or clear during the season ("clear

Beet leafhoppers must feed in the phloem of the plant. Direct feeding can cause relatively minor damage ("hopperburn"); however, BLTVA is a very destructive and detrimental disease affecting potatoes. BLTVA can cause a wide range of symptoms in potatoes, including leaf curling and purpling, aerial tubers, chlorosis, and early senescence. Most BLTVA infection occurs early in the season, during May and June (Munyaneza, 2003; Munyaneza & Crosslin,

is the only means to manage diseases in full season potatoes.

Fig. 9. Beet leafhopper nymphs. Photos by A. Murphy, OSU.

**4.4 Beet leafhopper (Order Heteroptera: Family Cicadellidae)** 

**4.3.6 Control** 

**4.4.1 Pest description** 

form") (Fig. 10).

**4.4.2 Damage** 

Among the favorite hosts are Kochia, Russian thistle, and various weedy mustard species such as tumble mustard. Beet leafhoppers are especially abundant on young, marginal, semi-dry and small weeds plants. They also thrive on radishes, sugar beet (Meyerdirk & Hessein, 1985), and carrots (Munyaneza, 2003).

## **4.4.4 Biology**

The beet leafhopper overwinters on rangeland weeds and migrates to potatoes as early as May. They overwinter as adult females in weedy and native vegetation throughout most of the dry production areas. The beet leafhopper has three life stages: egg, nymph and adult. The adult can have a "darker form", early or late in the season; and a "clear form (during the season) (Fig. 10). Beet leafhoppers can transmit BLTVA as adults and nymphs. Eggs are laid in stems of host plants, and a new spring generation begins developing in March and April. Beet leafhopper begins to move from weeds to potatoes and potentially affect potatoes during the first spring generation, which matures in late May to early June (Jensen et al., 2011). Potatoes are most seriously affected by BLTVA infections that occur early in the growing season (Rondon unpublished). Beet leafhopper remains common through the summer, during which it goes through 2 to 3 overlapping generations. The final generation for the year matures during late October-early November. Total number of beet leafhoppers varies from year to year (Crosslin et al., 2011).

Fig. 10. "Clear form"(left) and "dark form" (right) of the beet leafhopper. Size of adults 2.5-3 mm. Photos by A. Jensen, Washington Potato Commission.

#### **4.4.5 Monitoring**

Because potatoes are not a preferred host of the BLH, in-field sampling is problematic. Most recommendations suggest the use of yellow sticky cards around field margins. It is important to keep traps close to the ground where hoppers mostly move. Check and replace

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 325

Fig. 12. Potato tuberworm larva entering tuber. Photo by L. Ketchum, OSU.

excrement that can be a potential source for secondary infections.

several generations per year in the Pacific Northwest region.

found only on potatoes in the Pacific Northwest region (Rondon, 2010).

Tuberworm larvae behave as leaf miners. They can also live inside stems or within groups of leaves tied together with silk. The most important damage is to tubers, also a food source for the larvae, especially exposed tubers, or those within centimeters of the soil surface. Larvae can infest tubers when foliage is vine killed or desiccated right before harvest (Clough et al, 2010). Tunnels left by tuber worms in tubers can be full of droppings or

Although the potato tuberworm host range includes a wide array of Solanaceous crops such as tomatoes, peppers, eggplants, tobacco, and weeds such as nightshade, the pest has been

Potato tuberworm adults emerge as early as April in the Pacific Northwest, and continue to threaten the crop through November. Populations build sharply later in the growing season (September and October). The potato tuberworm has been detected in all potato growing regions of Oregon and throughout the Columbia Basin of Washington. A limited number of adults have been trapped in western Idaho. No tuber damage has been reported in Idaho (Rondon, 2010). A recent study suggests that locations with higher spring, summer, or fall temperatures are associated with increased trapping rates in most seasons (DeBano et al., 2010). Occasionally potato tuberworm pupae can be found on the surface of tubers, most commonly associated with indentations around the tuber eyes, but usually are not found inside tubers (Rondon et al., 2007). Considering the duration period of each instar and its relationship to abiotic factors such as temperature, the potato tuberworm can undergo

Pheromone-baited traps to catch adult male moths have been widely used in the region (Rondon et al., 2007). Unfortunately there are no established treatment thresholds. Another

**4.5.2 Damage** 

**4.5.3 Hosts** 

**4.5.4 Biology** 

**4.5.5 Monitoring** 

traps at least once a week. Rondon (unpublished data) suggests the use of DVAC (modified leaf blowers) to collect leafhoppers.

#### **4.4.6 Control**

Weed control in areas surrounding the potato field can help reduce initial sources of BLTVA inoculum. Due to the nature of the pest, few biological control efforts have been taking place in the Pacific Northwest. However, a species of *Anagrus* (Hymenoptera Mymaridae), has been reported as a common egg parasitoid in California (Meyerdirk & Moratorio, 1987). Foliar insecticides can reduce BLH populations and ergo, the incidence of the disease. Based on extensive research conducted in the Pacific Northwest, there are several foliar applied insecticides that are effective against BLH. Some evidence suggests that the use of some neonicotinoid insecticides at planting may provide control of BLTVA (Schreiber et al., 2010).

## **4.5 Potato Tuberworm (Order Lepidoptera: Family Gelechiidae)**

The potato tuberworm, *Phthorimaea operculella* Zeller, is one of the most economically significant insect pests of cultivated potatoes worldwide. The first significant economic damage to potato crops in the Columbia Basin region occurred in 2002, when a field in Oregon showed high levels of tuber damage associated with potato tuberworm. By 2003, the pest was a major concern to all producers in the region after potatoes from several fields were rejected by processors because of tuber damage. Since then, potato tuberworm has cost growers in the Columbia Basin millions of dollars through increased pesticide application and unmarketable potatoes (Rondon, 2010).

## **4.5.1 Pest description**

The potato tuberworm has four life stages: adult, egg, larva and pupa. Adults are small moths (approximately 0.94 cm long) with a wingspan of 1.27 cm. Forewings have dark spots (2-3 dots on males; "X" on females). Both pairs of wings have fringed edges (Rondon & Xue, 2010) (Fig. 11). Eggs are ≤ 0.1 cm spherical, translucent, and range in color from white or yellowish to light brown. Eggs are laid on foliage, soil and plant debris, or exposed tubers (Rondon et al., 2007); however, foliage is the preferred oviposition substrate (Varela, 1988). Adult female moths lays 150-200 eggs on the underside of leaves, on stems, and in tubers (Hoy et al., 2008). Larvae are usually light brown with a characteristic brown head. Mature larvae (approximately 0.94 cm long) may have a pink or greenish color (Fig. 12). Larvae close to pupation drop from infested foliage to the ground and may burrow into the tuber. Ultimately, larvae will spin silk cocoons and pupate on the soil surface or in debris under the plant.

Fig. 11. Forewings of potato tuberworm adult females present an "x" pattern (left); while male (right) present 2-3 dark spots. Photos by OSU (Rondon 2010).

Fig. 12. Potato tuberworm larva entering tuber. Photo by L. Ketchum, OSU.

## **4.5.2 Damage**

324 Insecticides – Pest Engineering

traps at least once a week. Rondon (unpublished data) suggests the use of DVAC (modified

Weed control in areas surrounding the potato field can help reduce initial sources of BLTVA inoculum. Due to the nature of the pest, few biological control efforts have been taking place in the Pacific Northwest. However, a species of *Anagrus* (Hymenoptera Mymaridae), has been reported as a common egg parasitoid in California (Meyerdirk & Moratorio, 1987). Foliar insecticides can reduce BLH populations and ergo, the incidence of the disease. Based on extensive research conducted in the Pacific Northwest, there are several foliar applied insecticides that are effective against BLH. Some evidence suggests that the use of some neonicotinoid insecticides at planting may provide control of BLTVA (Schreiber et al., 2010).

The potato tuberworm, *Phthorimaea operculella* Zeller, is one of the most economically significant insect pests of cultivated potatoes worldwide. The first significant economic damage to potato crops in the Columbia Basin region occurred in 2002, when a field in Oregon showed high levels of tuber damage associated with potato tuberworm. By 2003, the pest was a major concern to all producers in the region after potatoes from several fields were rejected by processors because of tuber damage. Since then, potato tuberworm has cost growers in the Columbia Basin millions of dollars through increased pesticide application

The potato tuberworm has four life stages: adult, egg, larva and pupa. Adults are small moths (approximately 0.94 cm long) with a wingspan of 1.27 cm. Forewings have dark spots (2-3 dots on males; "X" on females). Both pairs of wings have fringed edges (Rondon & Xue, 2010) (Fig. 11). Eggs are ≤ 0.1 cm spherical, translucent, and range in color from white or yellowish to light brown. Eggs are laid on foliage, soil and plant debris, or exposed tubers (Rondon et al., 2007); however, foliage is the preferred oviposition substrate (Varela, 1988). Adult female moths lays 150-200 eggs on the underside of leaves, on stems, and in tubers (Hoy et al., 2008). Larvae are usually light brown with a characteristic brown head. Mature larvae (approximately 0.94 cm long) may have a pink or greenish color (Fig. 12). Larvae close to pupation drop from infested foliage to the ground and may burrow into the tuber. Ultimately, larvae will spin silk cocoons and pupate on the soil surface or in debris under the plant.

Fig. 11. Forewings of potato tuberworm adult females present an "x" pattern (left); while

male (right) present 2-3 dark spots. Photos by OSU (Rondon 2010).

**4.5 Potato Tuberworm (Order Lepidoptera: Family Gelechiidae)** 

leaf blowers) to collect leafhoppers.

and unmarketable potatoes (Rondon, 2010).

**4.5.1 Pest description** 

**4.4.6 Control** 

Tuberworm larvae behave as leaf miners. They can also live inside stems or within groups of leaves tied together with silk. The most important damage is to tubers, also a food source for the larvae, especially exposed tubers, or those within centimeters of the soil surface. Larvae can infest tubers when foliage is vine killed or desiccated right before harvest (Clough et al, 2010). Tunnels left by tuber worms in tubers can be full of droppings or excrement that can be a potential source for secondary infections.

## **4.5.3 Hosts**

Although the potato tuberworm host range includes a wide array of Solanaceous crops such as tomatoes, peppers, eggplants, tobacco, and weeds such as nightshade, the pest has been found only on potatoes in the Pacific Northwest region (Rondon, 2010).

## **4.5.4 Biology**

Potato tuberworm adults emerge as early as April in the Pacific Northwest, and continue to threaten the crop through November. Populations build sharply later in the growing season (September and October). The potato tuberworm has been detected in all potato growing regions of Oregon and throughout the Columbia Basin of Washington. A limited number of adults have been trapped in western Idaho. No tuber damage has been reported in Idaho (Rondon, 2010). A recent study suggests that locations with higher spring, summer, or fall temperatures are associated with increased trapping rates in most seasons (DeBano et al., 2010). Occasionally potato tuberworm pupae can be found on the surface of tubers, most commonly associated with indentations around the tuber eyes, but usually are not found inside tubers (Rondon et al., 2007). Considering the duration period of each instar and its relationship to abiotic factors such as temperature, the potato tuberworm can undergo several generations per year in the Pacific Northwest region.

#### **4.5.5 Monitoring**

Pheromone-baited traps to catch adult male moths have been widely used in the region (Rondon et al., 2007). Unfortunately there are no established treatment thresholds. Another

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 327

Fig. 13 Two spotted spider mite adults range in size from 0.25 mm to 0.5 mm long; eggs are around 0.1 mm. Adults and nymphs are pale yellow or light green with two dark spots on

Northwest regions are listed below (Table 2). Cutworms feed on potato seeds, cut stems, and foliage; armyworms and loopers feed on foliage throughout the season. Cutworms and armyworms have three pairs of true legs and five pairs of prolegs behind; loopers have only three pair of true legs and three pair of prolegs behind. At planting insecticides protect potato seed from cutworms; however, after the residual effect is gone, the crop is unprotected; in some years, a foliar chemical application may be needed. Potatoes can tolerate some worm defoliation without loss in marketable yield. The period of full bloom is the most sensitive plant growth stage, but even then defoliation on the order of 10% appears to cause little if any yield loss. Applications should be targeted to control small larvae (1st

and 2nd instars), rather than larger larvae (Schreiber et al., 2010, Jensen et al., 2011).

**armyworm** Bertha armyworm *Mamestra configurata* Walker **Looper** Alfalfa looper *Autographa californica* (Speyer)

Table 2. Most common cutworm, armyworm, looper species in the Pacific Northwest (Zack.

Insecticides are the most powerful tool available for use in pest management (Metcalf, 1994). However the misuse, overuse and historically unnecessary use of insecticides have been some of the most important factors in the increasing interest in integrated pest management (Von Rumker & Horay, 1972; Metcalf, 1994). In the last decades, the Insecticide Resistance Action Committee (IRAC), a group of technical experts that coordinates responses to prevent or delay the development of resistance in insect and mite pests, defined resistance to insecticides as a "heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used

Cabbage looper *Trichoplusia ni* (Hübner)

**Group Common name Scientific name Cutworms** Spotted cutworms *Xestia c-nigrum* 

the abdomen (Photo by R.E. Berry, OSU).

**Western yellow striped** 

**4.7 Resistance to insecticides** 

et al., 2010).

way is to check leaf mining. Most mines are found in the upper third of the plant canopy, suggesting that efficient scouting for foliar damage should focus on the top third of the plant (DeBano et al., 2010). The number of mines gives a good indication of the history of potato tuberworm infestation in a plant, but it does not necessarily indicate the severity of larval infestation at a point in time. The study also found that reasonably precise estimates of foliar damage for areas of 23 ft x 30 ft can be made by sampling 9 plants (DeBano et al., 2010).

#### **4.5.6 Control**

Control efforts should be directed toward tuberworm populations right before or at harvest. Females prefer to lay eggs on potato foliage, but when potato foliage starts to degrade and change color, or when it is vine-killed, the risk of tuber infestation increases greatly. The greatest risk for tuber infestation occurs between desiccation and harvest (Clough et al., 2010; Rondon, 2010). If tuberworm populations appear to be building prior to late season, additional control measures may be necessary. Other means of control include the elimination of cull piles and the elimination of volunteer potatoes. Daily irrigation that keeps the soil surface moist can also aid in the control of tuberworm populations. Most chemical products aim to reduce larva population in foliage but that technique does not provide 100% protection for the tubers.

#### **4.6 Occasional pests**

#### **4.6.1 Mites**

The two-spotted spider mite, *Tetranichus urticae* Koch, is the most abundant mite species found in potatoes in the Pacific Northwest. They can occasionally be considered pests of potatoes when crops such as beans, corn, alfalfa or clover seed are planted nearby (Hoy et al., 2008). Mites in general prefer hot and dry conditions; they also prefer stressed plants where irrigation is poorly managed. They damage plants by puncturing the leaf tissue to extract plant juices. Plants respond by changing color from green to brown. Spider mites overwinter in the area as adults in debris around field edges (Jensen et al., 2011). Females are very prolific; after emerging from overwinter, they mate and lay eggs on the underside of leaves. If temperatures are warm (75-80oF or 23.8-26.6C), eggs can hatch in 3-5 days; nymphs to adults can take place in 7-9 days at those temperatures. When leaves get overcrowded, mites climb to the top of the plant and secrete silk that can be used as a "transport" device during light to moderate winds conditions (Fig. 13).

Sampling for mites requires a close visual inspection of leaves from different levels of the plants. Shaking potentially infested leaves above a piece of white paper helps to determine the presence of mites. Applications of miticides should be made upon early detection of mites. All potatoes should be surveyed for the presence of mites and mite eggs starting midseason (Schreiber et al., 2010). Thorough coverage is essential for good control and it is suggested that foliage should be dry at the time of application. While a single application of a miticide will suffice, if a second application of a miticide is required, the use of a miticide with different chemistry should be considered as a resistance prevention strategy (Jensen et al., 2011).

#### **4.6.2 Cutworm, armyworm and loopers**

These are several species of moth larvae that affect potato crops. Cutworms, armyworms and loopers are the immature stages of lepidopteran moths. Moths' typically have four defined life stages: egg, larva, pupa and adult. The most common species in the Pacific

Fig. 13 Two spotted spider mite adults range in size from 0.25 mm to 0.5 mm long; eggs are around 0.1 mm. Adults and nymphs are pale yellow or light green with two dark spots on the abdomen (Photo by R.E. Berry, OSU).

Northwest regions are listed below (Table 2). Cutworms feed on potato seeds, cut stems, and foliage; armyworms and loopers feed on foliage throughout the season. Cutworms and armyworms have three pairs of true legs and five pairs of prolegs behind; loopers have only three pair of true legs and three pair of prolegs behind. At planting insecticides protect potato seed from cutworms; however, after the residual effect is gone, the crop is unprotected; in some years, a foliar chemical application may be needed. Potatoes can tolerate some worm defoliation without loss in marketable yield. The period of full bloom is the most sensitive plant growth stage, but even then defoliation on the order of 10% appears to cause little if any yield loss. Applications should be targeted to control small larvae (1st and 2nd instars), rather than larger larvae (Schreiber et al., 2010, Jensen et al., 2011).


Table 2. Most common cutworm, armyworm, looper species in the Pacific Northwest (Zack. et al., 2010).

## **4.7 Resistance to insecticides**

326 Insecticides – Pest Engineering

way is to check leaf mining. Most mines are found in the upper third of the plant canopy, suggesting that efficient scouting for foliar damage should focus on the top third of the plant (DeBano et al., 2010). The number of mines gives a good indication of the history of potato tuberworm infestation in a plant, but it does not necessarily indicate the severity of larval infestation at a point in time. The study also found that reasonably precise estimates of foliar damage for areas of 23 ft x 30 ft can be made by sampling 9 plants (DeBano et al., 2010).

Control efforts should be directed toward tuberworm populations right before or at harvest. Females prefer to lay eggs on potato foliage, but when potato foliage starts to degrade and change color, or when it is vine-killed, the risk of tuber infestation increases greatly. The greatest risk for tuber infestation occurs between desiccation and harvest (Clough et al., 2010; Rondon, 2010). If tuberworm populations appear to be building prior to late season, additional control measures may be necessary. Other means of control include the elimination of cull piles and the elimination of volunteer potatoes. Daily irrigation that keeps the soil surface moist can also aid in the control of tuberworm populations. Most chemical products aim to reduce larva population in foliage but that technique does not

The two-spotted spider mite, *Tetranichus urticae* Koch, is the most abundant mite species found in potatoes in the Pacific Northwest. They can occasionally be considered pests of potatoes when crops such as beans, corn, alfalfa or clover seed are planted nearby (Hoy et al., 2008). Mites in general prefer hot and dry conditions; they also prefer stressed plants where irrigation is poorly managed. They damage plants by puncturing the leaf tissue to extract plant juices. Plants respond by changing color from green to brown. Spider mites overwinter in the area as adults in debris around field edges (Jensen et al., 2011). Females are very prolific; after emerging from overwinter, they mate and lay eggs on the underside of leaves. If temperatures are warm (75-80oF or 23.8-26.6C), eggs can hatch in 3-5 days; nymphs to adults can take place in 7-9 days at those temperatures. When leaves get overcrowded, mites climb to the top of the plant and secrete silk that can be used as a

Sampling for mites requires a close visual inspection of leaves from different levels of the plants. Shaking potentially infested leaves above a piece of white paper helps to determine the presence of mites. Applications of miticides should be made upon early detection of mites. All potatoes should be surveyed for the presence of mites and mite eggs starting midseason (Schreiber et al., 2010). Thorough coverage is essential for good control and it is suggested that foliage should be dry at the time of application. While a single application of a miticide will suffice, if a second application of a miticide is required, the use of a miticide with different chemistry should be considered as a resistance prevention strategy (Jensen et

These are several species of moth larvae that affect potato crops. Cutworms, armyworms and loopers are the immature stages of lepidopteran moths. Moths' typically have four defined life stages: egg, larva, pupa and adult. The most common species in the Pacific

"transport" device during light to moderate winds conditions (Fig. 13).

**4.5.6 Control** 

provide 100% protection for the tubers.

**4.6.2 Cutworm, armyworm and loopers** 

**4.6 Occasional pests** 

**4.6.1 Mites** 

al., 2011).

Insecticides are the most powerful tool available for use in pest management (Metcalf, 1994). However the misuse, overuse and historically unnecessary use of insecticides have been some of the most important factors in the increasing interest in integrated pest management (Von Rumker & Horay, 1972; Metcalf, 1994). In the last decades, the Insecticide Resistance Action Committee (IRAC), a group of technical experts that coordinates responses to prevent or delay the development of resistance in insect and mite pests, defined resistance to insecticides as a "heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used

Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 329

Andrews N., M. Ambrosino, G. Fisher, and S.I. Rondon. (2008). Wireworm biology and non-

Barry, A., R.E. Berry, and G. Tamaki. (1982). Host preference of the green peach aphid, *Myzus persicae* (Hemiptera: Aphididae). *Environmental Entomology*. 11(4): 824-827. Branson, T., F. Terry, and G. Robert. (1966). Effects of a nitrogen deficient host and crowding on the corn leaf aphid. *Journal of Economic Entomology*. 59(2): 290-294. Cambell, R.E. & M.W Stone. (1939). Trapping elaterid beetles as a control measure against

CIP (Centro Internacional de la Papa or International Potato Center). (1994). *International* 

Clough, G.H., S.I. Rondon, S.J. DeBano, N. David, and P.B. Hamm. (2010). Reducing tuber

practices and insecticides. *Journal of Economic Entomology* 103(4): 1306-1311. Crosslin, J.M., S.I. Rondon and P.B. Hamm. (2011). Population dynamics of the beet

DeBano, S.J., P.B. Hamm, A. Jensen, S.I. Rondon, and P.J. Landolt. (2010). Spatial and

Dickson, R., & E.F. Laird. (1967). Fall dispersal of green peach aphids to desert valleys.

Douches, D.S, W. Li, K. Zarka, J. Coombs, W. Pett, E. Grafius, T. El-Nasr. (2002).

Dreistadt, S.H., J.K. Clark and M.L. Flint. (2004)*. Pests of Landscape Trees and Shrubs: An* 

California Division of Agriculture and Natural Resources, Davis. Pp 501. Driesche, R.G., & T.S. Bellows. (1996). *Biological control*. Chapman and Hall. International

Ferro, D. N. (1994). Biological control of the Colorado potato beetle. *In*: G. W. Zehnder, M. L.

Gopal, J. & S.M.P. Khurana. (2006). Potato production, improvement and postharvest

Hamm, P.B.., C,W. Hy, P.J. Hutchinson, W.R. Stevenson, R.A. Boydston, J.M. Alvarez, A.

Hawkes, J.G. (1990). *The potato, evolution, biodiversity, and genetic resources*. Belhaven Press,

Heitefuss, R. (1989). *Crop and plant protection: the practical foundations*. Ellis Horwood Ltd,

Hoy, C.W., G. Boiteau, A. Alyokhin, G. Dively, and J.M. Alvarez. (2008). Managing insect

and mite pests. In (Ed D. Johnson) *Potato Health Management. Plant Health* 

top phytoplasma. American Journal of Potato Research (In press).

*Annals of the Entomological Society of America*. 60(5): 1088-1091.

Thomson Publishing Company. New York. Pp 539.

*Management*. APS Press, St. Paul. Pp. 357-375.

management. The Haworth press. Pp 587.

damage by the potato tuberworm (Lepidoptera: Gelechiidae) with cultural

leafhopper in northern Oregon and incidence of the Columbia Basin potato purple

temporal dynamics of potato tuberworm (Lepidoptera: Gelechiidae) in the Columbia Basin of the Pacific Northwest. *Journal of Economic Entomology* 39(1): 1-14.

Development of Bt-cry5 insect-resistant potato lines 'Spunta-G2' and 'Spunta-G3'.

*Integrated Pest Management Guide*. 2nd edition, Publication 3359. University of

Powelson, R. K. Jansson and K. V. Raman (eds), *Advances in Potato Pest Biology and* 

Alyokin, G. Dively, N.C. Gudmestad, and W.W. Kirk. (2008). Managing pesticide resistance. *In Potato Health Management. Plant Health Management Series* (Ed. D.A. Johnson). American Phytopathological Society, St Paul, Minnesota, USA. Second

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wireowmrs. *Journal of Economic Entomology*. 32: 47-53.

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chemical management in potatoes in the Pacific Northwest. *Oregon State University* 

according to the label recommendations for that pest species" (http://www.iraconline.org/). In other words, it is the inherited ability of a pest population to survive a pesticide which is a result of a process of selection (Hamm et al., 2008). Some potato pests have developed resistance to certain groups of pesticides; however, significant insecticide resistance is not yet known to occur in the Pacific Northwest (University of California, 1986). For instance, while spider mite infesting potatoes has demonstrated the ability to readily develop resistance to miticides there appears to be no evidence of this problem developing in the U.S. Pacific region (Schreiber et al., 2010).

Pesticides such as pyrethroids that disturb natural enemies can cause a resurgence of primary or secondary pests, especially when applied mid to late season. In the past few years, package mixes of insecticide, some including pyrethroids have been available for use on potatoes. More research is needed to evaluate the real impact of this pesticide in the Pacific Northwest potato region. Seed and soil treatments with systemic insecticides have become a standard approach to control early "invaders" (Hoy et al., 2008). This approach may be less disruptive to predator and non-target insects than traditional foliar or ground chemical applications.

There are several key components to developing a resistance management program for insect pests: first, producers must employ non-chemical control tactics for control of pest problems, including irrigation, cultivation and proper fertilization management; second, producers must rotate insecticidal modes of action. This integrated pest management approach will lead producers to a sustainable production system with long term economic benefits. Alvarez et al., (2003) suggest keeping good records of chemical applications, rotating insecticide use changing not only the product but also the class of compound, applying insecticides at labeled rates, using newer insecticides with chemistries that are safer for applicators and non-target organisms, and reducing insecticide applications by scouting and making applications only as needed.

## **5. Conclusions**

Potato is one of the most important food crops widely grown over many latitudes and elevations over the world. Increasing potato production in a sustainable manner requires an integrated approach covering a range of strategies. Combating pests is a continuous challenge that producers have to face as they intensify their production techniques to satisfy the increasing demands of the global market.

## **6. Acknowledgements**

The author would like to thank A. Smith, A. Murphy, and R. Marchosky, the author's staff at Oregon State University, for their help providing tables, figures and pictures. Special thanks to A. Goyer, A. Murphy, M. Corp, and G. Clough also from Oregon State University, for peer proofing the manuscript.

## **7. References**

Alvarez, J.M., R.L. Stotlz, C.R. Baird, and L.E. Sandoval. (2003). Insect pest and their management. In *Potato Production System*s (ed) J.C. Stark and S.L. Love. University of Idaho Extension. Pp 205-239.

Andrews N., M. Ambrosino, G. Fisher, and S.I. Rondon. (2008). Wireworm biology and nonchemical management in potatoes in the Pacific Northwest. *Oregon State University Extension Service Publication*. Dec. PNW 607. Available at

http://extension.oregonstate.edu/catalog/pdf/pnw/pnw607.pdf.

328 Insecticides – Pest Engineering

according to the label recommendations for that pest species" (http://www.iraconline.org/). In other words, it is the inherited ability of a pest population to survive a pesticide which is a result of a process of selection (Hamm et al., 2008). Some potato pests have developed resistance to certain groups of pesticides; however, significant insecticide resistance is not yet known to occur in the Pacific Northwest (University of California, 1986). For instance, while spider mite infesting potatoes has demonstrated the ability to readily develop resistance to miticides there appears to be no evidence of this problem developing

Pesticides such as pyrethroids that disturb natural enemies can cause a resurgence of primary or secondary pests, especially when applied mid to late season. In the past few years, package mixes of insecticide, some including pyrethroids have been available for use on potatoes. More research is needed to evaluate the real impact of this pesticide in the Pacific Northwest potato region. Seed and soil treatments with systemic insecticides have become a standard approach to control early "invaders" (Hoy et al., 2008). This approach may be less disruptive to predator and non-target insects than traditional foliar or ground

There are several key components to developing a resistance management program for insect pests: first, producers must employ non-chemical control tactics for control of pest problems, including irrigation, cultivation and proper fertilization management; second, producers must rotate insecticidal modes of action. This integrated pest management approach will lead producers to a sustainable production system with long term economic benefits. Alvarez et al., (2003) suggest keeping good records of chemical applications, rotating insecticide use changing not only the product but also the class of compound, applying insecticides at labeled rates, using newer insecticides with chemistries that are safer for applicators and non-target organisms, and reducing insecticide applications by

Potato is one of the most important food crops widely grown over many latitudes and elevations over the world. Increasing potato production in a sustainable manner requires an integrated approach covering a range of strategies. Combating pests is a continuous challenge that producers have to face as they intensify their production techniques to satisfy

The author would like to thank A. Smith, A. Murphy, and R. Marchosky, the author's staff at Oregon State University, for their help providing tables, figures and pictures. Special thanks to A. Goyer, A. Murphy, M. Corp, and G. Clough also from Oregon State University,

Alvarez, J.M., R.L. Stotlz, C.R. Baird, and L.E. Sandoval. (2003). Insect pest and their

management. In *Potato Production System*s (ed) J.C. Stark and S.L. Love. University

in the U.S. Pacific region (Schreiber et al., 2010).

scouting and making applications only as needed.

the increasing demands of the global market.

of Idaho Extension. Pp 205-239.

chemical applications.

**5. Conclusions** 

**6. Acknowledgements** 

**7. References** 

for peer proofing the manuscript.


Pest Management Strategies for Potato Insect Pests in the Pacific Northwest of the United States 331

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**15** 

*Tunisia* 

**Management of** *Tuta absoluta* **(Lepidoptera, Gelechiidae) with Insecticides on Tomatoes** 

Tomato, *Lycopersicon esculentum* Mill is a vegetable crop of large importance throughout the world. Its annual production accounts for 107 million metric tons with fresh market tomato representing 72 % of the total (FAO, 2002). It is the first horticultural crop in Tunisia with a production area of 25,000 hectares and a total harvest of 1.1 million metric tons (DGPA, 2009) of which nearly 70 % are processed (Tomatonews, 2011). Tomatoes are grown both

The tomato leafminer, *Tuta absoluta* Meyrick, (Lepidoptera : Gelechiidae) is a serious pest of both outdoor and greenhouse tomatoes. The insect deposits eggs usually on the underside of leaves, stems and to a lesser extent on fruits (photo 1). After hatching, young larvae penetrate into tomato fruits (photo 2), leaves (photo 3) on which they feed and develop creating mines and galleries. On leaves, larvae feed only on mesophyll leaving the epidermis intact (OEPP, 2005). Tomato plants can be attacked at any developmental stage,

Photo 1. *T. absoluta* eggPhoto 2. Larvae on fruit Photo 3. Larva of *T. absoluta*

Originated from South America, *T. absoluta* was reported since the early 1980s from Argentina, Brazil and Bolivia (Estay, 2000); the insect rapidly invaded many European and

**1. Introduction** 

from seedlings to mature stage.

under plastic covered greenhouses and in open field.

*Centre régional de recherche en Horticulture et Agriculture Biologique;* 

Mohamed Braham and Lobna Hajji

*Laboratoire d'Entomologie – Ecologie; Chott-Mariem,* 


## **Management of** *Tuta absoluta* **(Lepidoptera, Gelechiidae) with Insecticides on Tomatoes**

Mohamed Braham and Lobna Hajji

*Centre régional de recherche en Horticulture et Agriculture Biologique; Laboratoire d'Entomologie – Ecologie; Chott-Mariem, Tunisia* 

## **1. Introduction**

332 Insecticides – Pest Engineering

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leafroll virus) on biology and preference of vectors *Myzus persicae* and *Macrosiphum euphorbiae* (Hemiptera: Aphididae). *Journal of Economic Entomology*. 100(3): 646-655. Tabashnick, B.E. (1989). Managing resistance with multiple pesticides tactics: theory, evidence, and recommendations. *Journal of Economic Entomology.* 92: 1263-1269. Tamaki, G., L. Fox, and R.L. Chauvin. (1980). Green peach aphid: orchard weeds are host to

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Pp 685.

Tomato, *Lycopersicon esculentum* Mill is a vegetable crop of large importance throughout the world. Its annual production accounts for 107 million metric tons with fresh market tomato representing 72 % of the total (FAO, 2002). It is the first horticultural crop in Tunisia with a production area of 25,000 hectares and a total harvest of 1.1 million metric tons (DGPA, 2009) of which nearly 70 % are processed (Tomatonews, 2011). Tomatoes are grown both under plastic covered greenhouses and in open field.

The tomato leafminer, *Tuta absoluta* Meyrick, (Lepidoptera : Gelechiidae) is a serious pest of both outdoor and greenhouse tomatoes. The insect deposits eggs usually on the underside of leaves, stems and to a lesser extent on fruits (photo 1). After hatching, young larvae penetrate into tomato fruits (photo 2), leaves (photo 3) on which they feed and develop creating mines and galleries. On leaves, larvae feed only on mesophyll leaving the epidermis intact (OEPP, 2005). Tomato plants can be attacked at any developmental stage, from seedlings to mature stage.

Photo 1. *T. absoluta* eggPhoto 2. Larvae on fruit Photo 3. Larva of *T. absoluta*

Originated from South America, *T. absoluta* was reported since the early 1980s from Argentina, Brazil and Bolivia (Estay, 2000); the insect rapidly invaded many European and

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 335

water

300 cc/hl

Science 100 cc/hl

Corporation 100 cc/hl

Nemours 150 cc/hl

Science 150 cc/hl

Agroscience 60 cc/hl

Agricola 100 cc/hl

SL Basf 50 cc/hl

ingredients Trade name Companies Dose cc/ hl

abamectin Vertimec Syngenta 30 cc/hl

Table 1. Insecticides and natural plant extracts used in the laboratory trial in 2009.

weeks later). The Insecticides and natural plant extracts used are given in table 2.

returned to the laboratory and put in wooden cages for insecticide trials. Leaves were examined under binocular microscope and *T. absoluta* larvae were counted just before insecticide spray (April 3, 2010) and regularly after 2 to 3 days post-treatment. Dead larvae following trial were recorded. The second insecticide spray was done on April 19, 2010 (two

Active ingredients Trade name Companies Dose cc/ hl water diafenthiuron Pegasus Syngenta 125 cc/hl triflumuron Alystin SC 480 Bayer Crop science 50cc/hl

benzoate Proclaim® Syngenta 2500 grams/hl Plant extracts Tutafort AltincoAgro (Spain) 125 cc/hl

Field experiments using botanical extracts, Spinosad and Kaolin Clay were conducted from March 2010 to May 2010 in a half commercial tomato greenhouse (34 meters long x 8 meters width) in Saheline region, Tunisia (35°42' North, 10°40'East). Tomato seeds (cv Sahel) were sown on 27 October 2009 in an expanded polyester tray under plastic protected nursery bed. Four double rows of tomato were transplanted on 23 November 2009. The plot (greenhouse) was prepared according to usual cropping practices in the region. Ploughing, tillage and second tillage to incorporate manure, bed formation, irrigation device establishment and

Table 2. Insecticides and natural plant extracts used in the laboratory in 2010.

Atlantica Agricola (Spain)

deltamethrin Decis EC25 Bayer Crop

bifenthrin Talstar FMC

methomyl Lannate 25 Dupont de

metamidophos Tamaron 40 Bayer Crop

Spinosad Tracer Dow-

Neem extract Oleargan Atlantica

acetamiprid Mospilan 200

Rotenone Rotargan

emamectin

**2.2 Field trials** 

drip irrigation.

**2.2.1 Trials using natural products** 

Active

Mediterranean countries. It was first recorded from eastern Spain in late 2006 (Urbaneja, 2007), then Morocco, Algeria, France, Greece, Malta, Egypt and other countries (for a complete list see www.tutaabsoluta.com; Roditakis *et al*., 2010, Mohammed, 2010).

Chemical control using synthetic insecticides is the primary method to manage the pest, but it has serious drawbacks, including reduced profits from high insecticide costs, destruction of natural enemy populations (Campbell *et al*., 1991), build-up of insecticide residues on tomato fruits (Walgenbach *et al.,* 1991) and in the environment and fundamentally the rapid development of insecticide resistance. For example, resistance development has been reported against abamectin, cartap, methamidophos and permethrin in Brazil (Siqueira *et al*., 2000a, Siqueira *et al*., 2000b) and against deltamethrin and abamectin in Argentina (Lietti *et al*., 2005). Thus, in order to avoid selection of resistant biotypes, a careful management with frequent changes of active ingredients is desirable. Furthermore, modern integrated pest management recommends effective pesticides that have low mammalian toxicity, low persistence in the environment and high degree of selectivity. Since insecticide control currently remains an indispensable tool, the goal is to minimize the amount and impact of pesticides through the diversification of active ingredients used.

In this paper, we present the data from insecticides trials conducted in 2009 and 2010 under laboratory and field conditions, in which the efficacy of several hitherto untested insecticides and natural products was compared with that the widely used insecticides to manage *T. absoluta* in Tunisia such as spinosad, indoxacarb and pyrethroids compounds.

## **2. Material and methods**

#### **2.1 Laboratory trials**

#### **2.1.1 Laboratory assays in 2009**

Tomato seeds (cv Topsun) were sown on 30 January 2009. Seeds were deposited in 110 cm3 cells in a rectangular polyester tray of 60 cm x 40 cm x 5 cm filled with peat (Potgrond H, Germany). On March 3, 2009, seedlings were transplanted into 1 liter plastic flowerpot (bottom diameter =8 cm, top diameter = 12 cm and height = 12 cm) filled with peat without fertilization and watered as required. The tomato plants were maintained in the laboratory until use. Three days before the assay, plants (having four to six true leaves) were deposited in a tomato crop situated in the vicinity of the laboratory to permit *T. absoluta* egg-laying then transferred to the laboratory. Leaves were examined under binocular microscope and *T. absoluta* larvae were counted. Insecticides were sprayed using a hand sprayer (1 liter of capacity). After drying, the treated plants were kept in an unsealed empty greenhouse bordering the laboratory. There were four replications (plants) for each product and an untreated plant was used as a check. The efficacies of the products were tested twice: 48 hours following sprays and 12 days later. The Insecticides and natural plant extracts used are given in table 1.

#### **2.1.2 Laboratory assays in 2010**

A colony of *T. absoluta* was established from larvae and pupae collected from tomato infested field in the Chott-Mariem region. The insect was reared and maintained in a small greenhouse (10\*6 m). From time to time, tomato leaves harboring *T. absoluta* pre-imaginal stages collected in the field were introduced in the rearing greenhouse.

Tomato seeds (cultivar Riogrande) were sown on February 13, 2010 in a rectangular polyester tray as mentioned before. Plants having four to six true leaves were transferred to the rearing greenhouse and remained there for 2 to 3 days to allow egg-laying. Thereafter


Table 1. Insecticides and natural plant extracts used in the laboratory trial in 2009.

returned to the laboratory and put in wooden cages for insecticide trials. Leaves were examined under binocular microscope and *T. absoluta* larvae were counted just before insecticide spray (April 3, 2010) and regularly after 2 to 3 days post-treatment. Dead larvae following trial were recorded. The second insecticide spray was done on April 19, 2010 (two weeks later). The Insecticides and natural plant extracts used are given in table 2.


Table 2. Insecticides and natural plant extracts used in the laboratory in 2010.

## **2.2 Field trials**

334 Insecticides – Pest Engineering

Mediterranean countries. It was first recorded from eastern Spain in late 2006 (Urbaneja, 2007), then Morocco, Algeria, France, Greece, Malta, Egypt and other countries (for a

Chemical control using synthetic insecticides is the primary method to manage the pest, but it has serious drawbacks, including reduced profits from high insecticide costs, destruction of natural enemy populations (Campbell *et al*., 1991), build-up of insecticide residues on tomato fruits (Walgenbach *et al.,* 1991) and in the environment and fundamentally the rapid development of insecticide resistance. For example, resistance development has been reported against abamectin, cartap, methamidophos and permethrin in Brazil (Siqueira *et al*., 2000a, Siqueira *et al*., 2000b) and against deltamethrin and abamectin in Argentina (Lietti *et al*., 2005). Thus, in order to avoid selection of resistant biotypes, a careful management with frequent changes of active ingredients is desirable. Furthermore, modern integrated pest management recommends effective pesticides that have low mammalian toxicity, low persistence in the environment and high degree of selectivity. Since insecticide control currently remains an indispensable tool, the goal is to minimize the amount and impact of

In this paper, we present the data from insecticides trials conducted in 2009 and 2010 under laboratory and field conditions, in which the efficacy of several hitherto untested insecticides and natural products was compared with that the widely used insecticides to manage *T. absoluta* in Tunisia such as spinosad, indoxacarb and pyrethroids compounds.

Tomato seeds (cv Topsun) were sown on 30 January 2009. Seeds were deposited in 110 cm3 cells in a rectangular polyester tray of 60 cm x 40 cm x 5 cm filled with peat (Potgrond H, Germany). On March 3, 2009, seedlings were transplanted into 1 liter plastic flowerpot (bottom diameter =8 cm, top diameter = 12 cm and height = 12 cm) filled with peat without fertilization and watered as required. The tomato plants were maintained in the laboratory until use. Three days before the assay, plants (having four to six true leaves) were deposited in a tomato crop situated in the vicinity of the laboratory to permit *T. absoluta* egg-laying then transferred to the laboratory. Leaves were examined under binocular microscope and *T. absoluta* larvae were counted. Insecticides were sprayed using a hand sprayer (1 liter of capacity). After drying, the treated plants were kept in an unsealed empty greenhouse bordering the laboratory. There were four replications (plants) for each product and an untreated plant was used as a check. The efficacies of the products were tested twice: 48 hours following sprays and 12 days later. The Insecticides and natural plant extracts used

A colony of *T. absoluta* was established from larvae and pupae collected from tomato infested field in the Chott-Mariem region. The insect was reared and maintained in a small greenhouse (10\*6 m). From time to time, tomato leaves harboring *T. absoluta* pre-imaginal

Tomato seeds (cultivar Riogrande) were sown on February 13, 2010 in a rectangular polyester tray as mentioned before. Plants having four to six true leaves were transferred to the rearing greenhouse and remained there for 2 to 3 days to allow egg-laying. Thereafter

stages collected in the field were introduced in the rearing greenhouse.

complete list see www.tutaabsoluta.com; Roditakis *et al*., 2010, Mohammed, 2010).

pesticides through the diversification of active ingredients used.

**2. Material and methods** 

**2.1.1 Laboratory assays in 2009** 

**2.1 Laboratory trials** 

are given in table 1.

**2.1.2 Laboratory assays in 2010** 

## **2.2.1 Trials using natural products**

Field experiments using botanical extracts, Spinosad and Kaolin Clay were conducted from March 2010 to May 2010 in a half commercial tomato greenhouse (34 meters long x 8 meters width) in Saheline region, Tunisia (35°42' North, 10°40'East). Tomato seeds (cv Sahel) were sown on 27 October 2009 in an expanded polyester tray under plastic protected nursery bed. Four double rows of tomato were transplanted on 23 November 2009. The plot (greenhouse) was prepared according to usual cropping practices in the region. Ploughing, tillage and second tillage to incorporate manure, bed formation, irrigation device establishment and drip irrigation.

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 337

variance. Wherever significant difference occurred, Tukey's multiple comparison test was

In the laboratory trial of 2010, due to the low number of live larvae in the control, a one way-

i. Abbott formula : the percentage of efficacy = (Ca-Ta)/Ca\*100 where Ca is the average

ii. The percentage of larval mortality = mean number of dead larvae/( mean number of

One day before the assay, the mean number of total live larvae (L1 to L4 instars) per plant varied from 0.75 to 3. There is no significant difference between treatments (GLM-ANOVA. F= 0.99, df= 9,30; P = 0.47, table 5). Three days after the first application, the mean number of live larvae per plant decreases in all treatments except in the control (Table 5). All insecticides significantly reduced *T. absoluta* larvae when compared with non treated control (F= 4.24, df = 9,30; P= 0.001, Table 5). However, the level of suppression by acetamiprid and

Mean number of larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT) Insecticides ! 1DBT1!! 3DAT1 5DAT1 8DAT1 12DAT1 spinosad(1) 1.75a 0.5a(86.66)\* 0.50a(85.71)\* 0.5a (87.5)\* 0.25a(93.75)\* neem extract(2) 1.5a 0.75a(80) 0.75a(78.50) 0.5a(87.5) 0.5a(87.5) rotenone(3) 0.75a 0.25a(93.33) 0.25a(92.90) 0.5a(87.5) 0.75a(81.25) deltamethrin(4) 0.5a 0a(100) 1a(71.42) 0.75a(81.25) 1.5ab(62.5) acetamiprid(5) 2a 1.25ab(66.66) 1.25ab(64.28) 1.25ab(68.75) 0.50a(87.5) methomyl(6) 3a 0.5a(87) 0.5a(86) 0.50a(88) 0.75a(81) metamidophos(7) 2a 0.75a(80) 0.75a(79) 0.75a(81) 1.00a(75) abamectin(8) 2.25a 0.75a(80) 0.75a(79) 0.5a(88) 0.25a(94) bifenthrin(9) 2a 1.25ab(67) 2ab(43) 1.25ab(69) 1.00a(75) Control 2.5a 3.75b 3.5b 4b 4b Statistical analysis F= 0.99 F= 4.24 F= 3.69 F= 4.20 F= 4.66 ANOVA- df = 9,30 df = 9,30 df = 9,30 df = 9,30 df = 9,30 GLM P = 0.47 P= 0.001 P= 0.003 P= 0.001 P=0.003 ! denote commercial compounds: (1): Tracer, (2): Oleargan, (3): Rotargan, (4): Decis, (5): Mospilan, (6):

!! Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-

Table 5. Mean number of *T. absoluta* total live larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT) (the first treatment was done on April 1,

live larvae in the control and Ta is the mean survival score in the treatment.

ANOVA percentage of mortality was used instead of corrected mortality. The percentages of efficacies of insecticides were evaluated either:

dead larvae + mean number of live larvae)\*100.

bifenthrin did not differ significantly from the control (Table 5).

Lannate (7): Tamaran, (8): Vertimec, (9): Talstar

2009).

GLM procedure) followed by Tukey multiple comparison \* Data in brackets denote percent Abbott mortality (Abbott, 1925)

applied for mean separation.

**3. Results** 

**3.1 Laboratory trials 3.1.1 Assays in 2009** 


Table 3. Natural products experimented in 2010.

Plots measured 4 m2 each (10 plants) arranged in a randomized block design with four replications. The active ingredients, the trade name and doses of the natural products are given in table 3. The products were diluted with tap water and applied at field rates based on the recommended label dilutions without surfactants.

## **2.2.2 Trials using insecticides**

Trials using insecticides were undertaken during the same period in the second half greenhouse. Plot measured 8 square meters each (20 plants) arranged in a randomized block design with four replications. Three chemical compounds were used (table 4).


Table 4. Insecticides compounds experimented under tomato greenhouse in 2010.

#### **Insect monitoring**

To assess the *T. absoluta* infestation prior to the trial, thirty leaf samples, taken from about 30 different plants were weekly collected (from January to March 2010) at random from the entire greenhouse. The sample was placed in a plastic bag and taken to the laboratory. Leaves were examined under binocular microscope (Leica MZ12.5); eggs, larvae pupae, of *T. absoluta* live or dead as well as mines were recorded. However, only larvae (live or dead) were presented in this study.

### **2.3 Statistical analysis**

Data on the effectiveness of various insecticides were analyzed using the Minitab Software for Windows (Minitab 13.0). The mean number of live larvae per plant or per leaf was tested for Normality assumption by Kolmogorov-Smirnov test then the data were square root transformed. General linear model procedures were used to perform the analysis of variance. Wherever significant difference occurred, Tukey's multiple comparison test was applied for mean separation.

In the laboratory trial of 2010, due to the low number of live larvae in the control, a one way-ANOVA percentage of mortality was used instead of corrected mortality.

The percentages of efficacies of insecticides were evaluated either:


## **3. Results**

336 Insecticides – Pest Engineering

Spinosad Tracer 240 Dow- Agroscience 60 cc/hl Neem extract Oleargan Atlantica Agricola (Spain) 100 cc/hl

Botanical extracts Deffort AltincoAgro (Spain) 350 cc/hl

*amara* and Neem) Conflic Atlantica Agricola (Spain) 250 cc/hl

Plots measured 4 m2 each (10 plants) arranged in a randomized block design with four replications. The active ingredients, the trade name and doses of the natural products are given in table 3. The products were diluted with tap water and applied at field rates based

Trials using insecticides were undertaken during the same period in the second half greenhouse. Plot measured 8 square meters each (20 plants) arranged in a randomized block

ingredients Trade name Companies Dose cc/ hl water

science 50 cc/hl

design with four replications. Three chemical compounds were used (table 4).

indoxacarb Avaunt 150EC Dupont 50 cc/hl

diafenthiuron Pegasus 500SC Syngenta 125 cc/hl

Table 4. Insecticides compounds experimented under tomato greenhouse in 2010.

To assess the *T. absoluta* infestation prior to the trial, thirty leaf samples, taken from about 30 different plants were weekly collected (from January to March 2010) at random from the entire greenhouse. The sample was placed in a plastic bag and taken to the laboratory. Leaves were examined under binocular microscope (Leica MZ12.5); eggs, larvae pupae, of *T. absoluta* live or dead as well as mines were recorded. However, only larvae (live or dead)

Data on the effectiveness of various insecticides were analyzed using the Minitab Software for Windows (Minitab 13.0). The mean number of live larvae per plant or per leaf was tested for Normality assumption by Kolmogorov-Smirnov test then the data were square root transformed. General linear model procedures were used to perform the analysis of

Kaolin Clay Surround WPTM Engelhard Corporation

Orange extract Prev-amTM ORO Agri International

Botanical extracts Armorex Soil Technologies Corp

Botanical extracts (*Quassia* 

**2.2.2 Trials using insecticides** 

Active

**Insect monitoring** 

were presented in this study.

**2.3 Statistical analysis** 

Table 3. Natural products experimented in 2010.

on the recommended label dilutions without surfactants.

triflumuron Alsystin SC 480 Bayer Crop

Active ingredients Trade name Companies Dose cc/ hl water

(NJ.U.S.A) 5 kg/hl

Ltd 300 cc/hl

(U.S.A) 60 cc/hl

## **3.1 Laboratory trials**

#### **3.1.1 Assays in 2009**

One day before the assay, the mean number of total live larvae (L1 to L4 instars) per plant varied from 0.75 to 3. There is no significant difference between treatments (GLM-ANOVA. F= 0.99, df= 9,30; P = 0.47, table 5). Three days after the first application, the mean number of live larvae per plant decreases in all treatments except in the control (Table 5). All insecticides significantly reduced *T. absoluta* larvae when compared with non treated control (F= 4.24, df = 9,30; P= 0.001, Table 5). However, the level of suppression by acetamiprid and bifenthrin did not differ significantly from the control (Table 5).


! denote commercial compounds: (1): Tracer, (2): Oleargan, (3): Rotargan, (4): Decis, (5): Mospilan, (6): Lannate (7): Tamaran, (8): Vertimec, (9): Talstar

!! Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

\* Data in brackets denote percent Abbott mortality (Abbott, 1925)

Table 5. Mean number of *T. absoluta* total live larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT) (the first treatment was done on April 1, 2009).

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 339

However, the percentage of larval mortality (number of dead larvae/sum of dead and live larvae) following the first and second insecticide application shows the best performance of

Fig. 1. Overall percentage of efficacy according to Abbott formula (1925). DAT1 = days after

the first treatment, DAT2 = days after the second treatment (laboratory trial, 2009).

% larval mortality

neem extract

Rotenone

deltamethrin (Decis)

acetamiprid(Mospilan)

Fig. 2. Percentage of larval morality following the first (T1) and the second treatment (T2) (mean number of four dates after the fist treatment and 3 dates after the second treatment).

methomyl (Lannate)

metamidophos(Tamaran)

% mortalityT1 % mortalityT2 Average

abamectin (Vertimec)

bifenthrin(Talstar)

Control

spinosad (Tracer)

spinosad (91 %), neem extract (71 %) and abamectin (71%).

Five days following the first application, all the products performed well except acetamiprid and bifenthrin which show no significant difference compared with the control (Table 5). Eight days after the first application, the mean number of total live larvae per plant varied from 0.5 to 4. All the tested products reduced significantly the density of live larvae per plant compared with the control (F= 4.20; df = 9,30; P= 0.001). Still, acetamiprid and bifenthrin showed mild efficacy (table 5). At 12 days following treatments, all the products performed well (F= 4.66, df = 9,30 ; P= 0.003), yet the plants treated with deltamethrin show increasing mean live larvae per plant (table 5).

Regarding the corrected mortality according to Abbott formula, Spinosad and rotenone gave satisfactory results post-treatment (88.4 % and 88.7% respectively) followed by Lannate (85%), Vertimec (85%), neem extract (83. 22), and Tamaran (79%). However, Decis (78.8%), Mospilan (71.8) and Talstar (63%) showed mild efficacy. Though, Decis performed well till 8 days following the first application (84.2%).


\* Data in brackets denote percent Abbott mortality (Abbott, 1925)

!! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 6. Mean number of total *T. absoluta* live larvae/plant the day of the second treatment and thereafter (DAT2) (the treatment was undertaken on April 21)

Just before the second application, the mean number of live larvae in treated plants remained low compared with the control. It varied between zero (Tracer) and 3.5 (control) (table 6). Two days following the second insecticide application, all tested compounds show good efficacy compared with control (F=4.24; df = 9,30; P<0.001). Spinosad (Tracer) performed well (100 % efficacy according to Abbott corrected mortality formula). However, bifenthrin (Talstar) shows mild efficacy (table 6). The same conclusion can be formulated four days following treatments (table 6). At eight days after trial, the insecticide spinosad remains active and performed well (83.33 % efficacy) (table 6).

The overall efficacy according to Abbott formula (1925) shows the good performance of spinosad (Tracer), rotenone (Rotargan), methomyl (Lannate), abamectin (Vertimec) (Fig. 1.).

Five days following the first application, all the products performed well except acetamiprid and bifenthrin which show no significant difference compared with the control (Table 5). Eight days after the first application, the mean number of total live larvae per plant varied from 0.5 to 4. All the tested products reduced significantly the density of live larvae per plant compared with the control (F= 4.20; df = 9,30; P= 0.001). Still, acetamiprid and bifenthrin showed mild efficacy (table 5). At 12 days following treatments, all the products performed well (F= 4.66, df = 9,30 ; P= 0.003), yet the plants treated with deltamethrin show

Regarding the corrected mortality according to Abbott formula, Spinosad and rotenone gave satisfactory results post-treatment (88.4 % and 88.7% respectively) followed by Lannate (85%), Vertimec (85%), neem extract (83. 22), and Tamaran (79%). However, Decis (78.8%), Mospilan (71.8) and Talstar (63%) showed mild efficacy. Though, Decis performed well till 8

Mean number of total live larvae/plant on indicated days after the second treatment (DAT)

> F= 6.07 F= 7.24 F=5.84 F= 4.39 df = 9,30 df = 9,30 df = 9,30 df = 9,30 P = 0.00 P= 0.00 P= 0.00 P= 0.001

Insecticides 0DBT2!! 2DAT2 4DAT2 8DAT2 spinosad 0a 0a(100)\* 0a(100)\* 0.75a(83.33)\* neem extract 0.5a 0.5a(92.85) 0.75a(78.60) 1.75a(61.11) rotenone 0.25a 0.5a(85.71) 0.5a(85.71) 1.25a(72.22) deltamethrin 0.75a 0.75a(78.60) 1a(71.42) 1.25a(72.22) acetamiprid 0.5a 0.5a(85.71) 0.75a(78.60) 1.5a(66.66) methomyl 1.25a 0.75a(78.60) 0.75a(78.60) 2a(83.33) metamidophos 0.75a 0.75a(78.60) 0.5a(85.71) 1a(77.71) abamectin 0.5a 0.5a(85.71) 0.5a(85.71) 1.5a(66.66) bifenthrin 1a 1a(64.28) 1.5ab(57.14) 2a(55.55) Control 3.5b 3.5b 3.5b 4.5b

!! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 6. Mean number of total *T. absoluta* live larvae/plant the day of the second treatment

Just before the second application, the mean number of live larvae in treated plants remained low compared with the control. It varied between zero (Tracer) and 3.5 (control) (table 6). Two days following the second insecticide application, all tested compounds show good efficacy compared with control (F=4.24; df = 9,30; P<0.001). Spinosad (Tracer) performed well (100 % efficacy according to Abbott corrected mortality formula). However, bifenthrin (Talstar) shows mild efficacy (table 6). The same conclusion can be formulated four days following treatments (table 6). At eight days after trial, the insecticide spinosad

The overall efficacy according to Abbott formula (1925) shows the good performance of spinosad (Tracer), rotenone (Rotargan), methomyl (Lannate), abamectin (Vertimec) (Fig. 1.).

increasing mean live larvae per plant (table 5).

days following the first application (84.2%).

\* Data in brackets denote percent Abbott mortality (Abbott, 1925)

(ANOVA-GLM procedure) followed by Tukey multiple comparison

and thereafter (DAT2) (the treatment was undertaken on April 21)

remains active and performed well (83.33 % efficacy) (table 6).

Statistical analysis

However, the percentage of larval mortality (number of dead larvae/sum of dead and live larvae) following the first and second insecticide application shows the best performance of spinosad (91 %), neem extract (71 %) and abamectin (71%).

Fig. 1. Overall percentage of efficacy according to Abbott formula (1925). DAT1 = days after the first treatment, DAT2 = days after the second treatment (laboratory trial, 2009).

Fig. 2. Percentage of larval morality following the first (T1) and the second treatment (T2) (mean number of four dates after the fist treatment and 3 dates after the second treatment).

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 341

ANOVA F= 4.95 df = 4, 120; P= 0.001). The compound Proclaim® shows the highest

At three days after the second insecticide application, there is no significant difference regarding the mean number of live larvae per leaf (GLM-ANOVA). Nevertheless, plants treated with the product Proclaim® harbour zero live larvae per leaf suggesting the good efficacy of this insecticide. This is confirmed by the high percentage of mortality (100 %) as well as the significant difference between treated and control plants (One way ANOVA, F=

Mean number of live larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT)(µ) Insecticides 16DAT1! 3DAT2 5DAT2 8DAT2 10DAT2 (1) 0.33(12.55)a 0.19(51.71)a 0.16(59.91)ac 0.15(59.4)ac 0.15(59.14)ac (2) 0.31(21.47)a 0.06(80.15)a 0.06(83.64)ac 0.06(83.35)ac 0.06(83.35)ac (3) 0.20(54.83)a 0(100)a 0(100)bc 0(100)bc 0(100)bc (4) 0.20(19.60)a 0.16(0)a 0.1(59.55)ac 0.09(59)ac 0.09(59)ac (5) 0.20(0)a 0.16(0)a 0.16(0)a 0.16(0)a 0.06(0)a

GLM- P= 0.89 P= 0.096 P= 0.123 P= 0.096 P=0.189

! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 7. (continued). Mean number of live *T. absoluta* larvae on indicated days before

Five days after the second spray, the mean number of live larvae did not vary among treated and untreated plants (table 7. Cont.). But the percentage of mortality significantly varies (ANOVA one factor F= 3.98 df = 4, 120; P= 0.03) showing again the good performance of

At eight days after the second spray, there is no significant difference between treated plants and control regarding the mean number of live larvae (table 7.Cont.). However, the percentage of mortality varies (ANOVA, one factor, F= 3.88 df = 4, 120; P= 0.005). The compounds Proclaim® and Tutafort are the best (100 % and 83.35 respectively, table

At 10 days after the second insecticide application, there is no significant difference between treated plants and control (GLM-ANOVA, Table 7.Cont.). Concerning the percentage of mortality, there is a significant difference between treated and control plants (ANOVA, one factor, F= 3.99 df = 4, 120; P= 0.006). Proclaim® followed by Tutafort performed well (100 %

The first spray was undertaken on March 26, 2010, then on April 8 and on April 19, 2010. At three days following the first application, the mean live larvae (small and old larvae) per leaf did not significantly vary between treated and control plots (GLM-ANOVA Procedure,

F= 0.27 F= 2.02 F= 1.85 F= 1.85 F= 1.56 df =4, 116 df =4, 116 df =4, 116 df =4, 116 df =4, 116

mortality percentage (54.83 % table 7.Cont.).

µ : Data under brackets denote percentage of mortality

(ANOVA-GLM procedure) followed by Tukey multiple comparison

treatments and days after treatments (laboratory trial, 2010)

**3.2.1 Natural products experimented in 2010 under greenhouse** 

4.51 df = 4, 120; P= 0.002).

Statistical analysis

ANOVA

7.Cont.).

**3.2 Field trials** 

Proclaim® (table 7.Cont.).

and 83.35 respectively, table 7.Cont.).

## **3.1.2 Assays in 2010**

Just before the first spray (April 3, 2010), the mean number of live larvae (first to fourth instars) per leaf varied from 0.12 (Control) to 0.52 (Proclaim®). Although there is no significant difference between treatments (ANOVA-GLM F= 1.37, df = 4, 116; P=0.24), the control plants harboured less live larvae (table 7). There is no larval mortality.

Two days following the first spray (April 5), there is no significant difference between treatments regarding live larvae (GLM; F= 0.93, df = 4, 116; P= 0.46. Table 7). However, the percentage of larval mortality did vary (ANOVA, 1 factor, F = 4.17; df = 4, 120; P= 0.003) showing the best performance of Proclaim® (57.14 %; Table 7).

Nine days after the first insecticide application (April 12), the mean number of live larvae per leaf did not significantly vary between treatments (ANOVA-GLM procedure Table 7). However, the percentage of mortality significantly varies between treated and untreated plants (ANOVA 1 factor, F= 3.07; df = 4, 120; P= 0.021). The maximum percentage of mortality is given by Proclaim® (45.70%, table 7).

At 11 days after the first insecticide application (on April 14), the mean number of live larvae did not significantly vary among treated and untreated plants (ANOVA - GLM procedures Table 7). However, the percentage of mortality did vary according to treatments (F = 3.16, df = 4, 120; P= 0.017) showing the good efficacy of Proclaim® (52.93 % Table 7).


! :(1):triflumuron(Alystin), (2) plant extract (Tutafort), (3) emamectin benzoate (Proclaim®) (4) diafenthiuron (Pegasus ) and (5) Control.

µ: Data under brackets denote percentage of mortality

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 7. Mean number of live *T. absoluta* larvae on indicated days before treatments and days after treatments (laboratory trial, 2010)

At 13 days after the first application, the mean number of live larvae did not significantly vary between treatments and control (Table 7). However, the percentage of mortality significantly varies between treated and control plants (F = 3.53 df = 4, 120; P= 0.009) showing the good efficacy of the compound Proclaim® (51.51 %, table 7).

At 16DAT1 and just before the second spray, the mean number of live larvae shows no significant difference between treated and control plants (table 7. continued). However, the percentage of mortality did significantly vary between treated and control plants (One way ANOVA F= 4.95 df = 4, 120; P= 0.001). The compound Proclaim® shows the highest mortality percentage (54.83 % table 7.Cont.).

At three days after the second insecticide application, there is no significant difference regarding the mean number of live larvae per leaf (GLM-ANOVA). Nevertheless, plants treated with the product Proclaim® harbour zero live larvae per leaf suggesting the good efficacy of this insecticide. This is confirmed by the high percentage of mortality (100 %) as well as the significant difference between treated and control plants (One way ANOVA, F= 4.51 df = 4, 120; P= 0.002).


µ : Data under brackets denote percentage of mortality

! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 7. (continued). Mean number of live *T. absoluta* larvae on indicated days before treatments and days after treatments (laboratory trial, 2010)

Five days after the second spray, the mean number of live larvae did not vary among treated and untreated plants (table 7. Cont.). But the percentage of mortality significantly varies (ANOVA one factor F= 3.98 df = 4, 120; P= 0.03) showing again the good performance of Proclaim® (table 7.Cont.).

At eight days after the second spray, there is no significant difference between treated plants and control regarding the mean number of live larvae (table 7.Cont.). However, the percentage of mortality varies (ANOVA, one factor, F= 3.88 df = 4, 120; P= 0.005). The compounds Proclaim® and Tutafort are the best (100 % and 83.35 respectively, table 7.Cont.).

At 10 days after the second insecticide application, there is no significant difference between treated plants and control (GLM-ANOVA, Table 7.Cont.). Concerning the percentage of mortality, there is a significant difference between treated and control plants (ANOVA, one factor, F= 3.99 df = 4, 120; P= 0.006). Proclaim® followed by Tutafort performed well (100 % and 83.35 respectively, table 7.Cont.).

## **3.2 Field trials**

340 Insecticides – Pest Engineering

Just before the first spray (April 3, 2010), the mean number of live larvae (first to fourth instars) per leaf varied from 0.12 (Control) to 0.52 (Proclaim®). Although there is no significant difference between treatments (ANOVA-GLM F= 1.37, df = 4, 116; P=0.24), the

Two days following the first spray (April 5), there is no significant difference between treatments regarding live larvae (GLM; F= 0.93, df = 4, 116; P= 0.46. Table 7). However, the percentage of larval mortality did vary (ANOVA, 1 factor, F = 4.17; df = 4, 120; P= 0.003)

Nine days after the first insecticide application (April 12), the mean number of live larvae per leaf did not significantly vary between treatments (ANOVA-GLM procedure Table 7). However, the percentage of mortality significantly varies between treated and untreated plants (ANOVA 1 factor, F= 3.07; df = 4, 120; P= 0.021). The maximum percentage of

At 11 days after the first insecticide application (on April 14), the mean number of live larvae did not significantly vary among treated and untreated plants (ANOVA - GLM procedures Table 7). However, the percentage of mortality did vary according to treatments (F = 3.16, df = 4, 120; P= 0.017) showing the good efficacy of Proclaim® (52.93 % Table 7).

Mean number of live larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT)µ Insecticides! 0DBT! ! 2DAT1 9DAT1 11DAT1 13DAT1 (1) 0.36(0)a 0.36(10)a 0.37(13.61)a 0.44(12.66)a 0.34(12.82)a (2) 0.32(0)a 0.2(37.5)a 0.24(29.47)a 0.34(23.52)a 0.34(20.05)a (3) 0.52(0)a 0.24(57.14)a 0.29(45.70)a 0.25(52.93)a 0.23(51.51)a (4) 0.44(0)a 0.48(0)a 0.26(17.91)a 0.20(21.91)a 0.18(27.39)a (5) 0.12(0)a 0.24(0)a 0.22(0)a 0.25(0)a 0.20(0)a

analysis F= 1.37 F=0.90 F= 0.57 F=0.63 F=0.27 ANOVA df =4,116 df =4,116 df =4,116 df =4,116 df =4,116 -GLM P =0.24 P =0.46 P = 0.67 P=0.64 P= 0.89

! :(1):triflumuron(Alystin), (2) plant extract (Tutafort), (3) emamectin benzoate (Proclaim®) (4)

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 7. Mean number of live *T. absoluta* larvae on indicated days before treatments and

At 13 days after the first application, the mean number of live larvae did not significantly vary between treatments and control (Table 7). However, the percentage of mortality significantly varies between treated and control plants (F = 3.53 df = 4, 120; P= 0.009)

At 16DAT1 and just before the second spray, the mean number of live larvae shows no significant difference between treated and control plants (table 7. continued). However, the percentage of mortality did significantly vary between treated and control plants (One way

control plants harboured less live larvae (table 7). There is no larval mortality.

showing the best performance of Proclaim® (57.14 %; Table 7).

mortality is given by Proclaim® (45.70%, table 7).

**3.1.2 Assays in 2010** 

Statistical

diafenthiuron (Pegasus ) and (5) Control.

µ: Data under brackets denote percentage of mortality

days after treatments (laboratory trial, 2010)

(ANOVA-GLM procedure) followed by Tukey multiple comparison

showing the good efficacy of the compound Proclaim® (51.51 %, table 7).

## **3.2.1 Natural products experimented in 2010 under greenhouse**

The first spray was undertaken on March 26, 2010, then on April 8 and on April 19, 2010. At three days following the first application, the mean live larvae (small and old larvae) per leaf did not significantly vary between treated and control plots (GLM-ANOVA Procedure,

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 343

At nine days after the third spray, the mean number of total larvae varied between 0.2 and 2.05. The ANOVA-GLM procedure showed a significant difference between treatments. The products Tracer, Armorex and Deffort were effective in reducing *T. absoluta* larval densities

Mean number of total larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT) Insecticides 11 DAT2! 4DAT3! ! 9DAT3 18DAT3 Armorex(1) 0.525(0)a 0.1(85.18)a 0.3(85.36)b 0.9(12.2)a Deffort(1) 0 .925(0)a 0.3(55.55)a 0.2(90.24)b 0.65(36.85)a Oleargan (1) 0.325(13.33)a 0.075(88.88)ab 0.55(73.17)b 0.375(63.4)a Konflic(1) 0.475(0)a 0(100)ab 0.825(59.75)a 0.325(68.3)a Prev-amTM (2) 0.225(40) a 0.175(74.07)a 1.675(18.30)a 0.7(31.7)a Surround(3) (3)0.1(73.33)a 0.2(70.37)a 0.55(73.17)b 0.375(63.4)a Tracer(4) 0.35(6.66)a 0.1(85.18)a 0.25(87.80)b 0.75(26.8)a Control 0.375a 0.675a 2.05a 1.025a

GLM-ANOVA P=0.201 P=0.017 P=0.000 P=0.220

Mean number of live larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT)

SL\* OL\* SL\* OL\*

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 8. (Continued) Mean number of total live larvae following natural products

Insecticides 3DAT1! ! 10DAT1

Armorex(1) 0.075(40)a 0.125(0)a 0.1(50)a 0(100)a Deffort(1) 0.05(60)a 0.2(0)a 0.225(0)a 0,225(0)a Oleargan (1) 0.2(0)a 0.125(0)a 0.175(12.5)a 0.05**(**60)a Konflic(1) 0.425(0)b 0.15(0)a 0.1(50)a 0.1(20)a Prev-amTM (2) 0.25(0)ab 0.125(0)a 0.275(0)a 0.175(0)a Surround WPTM (3) 0.3(0)a 0.025(80)a 0(100)a 0.25(0)a Tracer(4) O(100)a 0.075(40)a 0.05(75)a 0(100)a Control 0.125(0)ab 0.125(0)a 0.2(0)a 0.125(0)a Statistical Analysis F= 4.03 F= 0.77 F= 1.76 F= 1.53

GLM-ANOVA P= 0.00 P= 0.611 P= 0.096 P=0.157

Table 9. Mean number of live small and old larvae following natural products applications

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

(ANOVA-GLM procedure) followed by Tukey multiple comparison

\*: SL : Small larvae (L1-L2), OL: Old larvae (L3-L4)

under tomato greenhouse (Saheline, Tunisia, 2010).

(ANOVA-GLM procedure) followed by Tukey multiple comparison.

applications under tomato greenhouse (Saheline, Tunisia, 2010).

F=1.41 F=2.49 F=2.49 F=1.36 df= 7,309 df= 7,309 df= 7,309 df= 7,309

df= 3,309 df= 3,309 df= 3,309 df= 3,309

(table 8).

Statistical Analysis

! : third spray

P= 0.09). Although, plots treated with spinosad show the minimum live larvae as demonstrated by 70% efficacy according to Abbott formula (Table 8). The details of larval instars (small larvae: first and second instars and old larvae: three and fourth instars) show a significant difference between insecticides tested. The compounds Tracer, Armorex and Deffort performed well (table 9).


(1): Botanical extracts

(2): Orange extract

(3) : Kaolin

\* Corrected mortality according to Abbott formula

µ = second spray

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 8. Mean number of total live larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).

At 10 days after the first natural products applications, the ANOVA-GLM procedure shows no significant difference between treatments regarding the mean number live larvae (Table 8). The Abbott's percentages of efficacy show the performance of spinosad (84.61 %) and the plant extract (Armorex; 69.23%).

At two days after the second spray, (April 10) there is a significant difference between treated plots (AVOVA-GLM procedure, P= 0.008, table 8). The plots treated with Deffort show the maximum density of mean live larvae per leaf (table 8). However, there is no significant difference between the other products and control. The details of larval stages confirm the low efficacy of Deffort compared with the other products and control (small larvae : P= 0.026; Old larvae P= 0.019; table 9).

Six days following the second application (April 14), the mean number of live larvae shows no significant difference between treated and untreated plots (Table 8).

At eleven days after the second spray, the mean number of live larvae per leaf is relatively similar among treatments and did not significantly vary (ANOVA-GLM procedure P= 0.211) varying from 0.1 to 0.9. Plots treated with Kaolin (Surround) harbour the minimum density.

Four days after the third spray (April 23, 2010), the treated plot differed significantly showing the good performance of the compounds neem extract, Tracer and Konflic (table 8). This is confirmed by the analysis of detailed larval instars (table 9).

At nine days after the third spray, the mean number of total larvae varied between 0.2 and 2.05. The ANOVA-GLM procedure showed a significant difference between treatments. The products Tracer, Armorex and Deffort were effective in reducing *T. absoluta* larval densities (table 8).


! : third spray

342 Insecticides – Pest Engineering

P= 0.09). Although, plots treated with spinosad show the minimum live larvae as demonstrated by 70% efficacy according to Abbott formula (Table 8). The details of larval instars (small larvae: first and second instars and old larvae: three and fourth instars) show a significant difference between insecticides tested. The compounds Tracer, Armorex and

Mean number of total larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT) Insecticides 1DBT! ! 3 DAT1\* 10DAT1(µ) 2DAT2 6DAT2 Armorex(1) 0.30a 0.20(20)a 0.1(69.23)a 0(100)a 0.325(0)a Deffort(1) 0.30a 0.25(0)a 0.45(0)a 0.475(0)b 0.3(0)a Oleargan (1) 0.20a 0.32(0)a 0.225(30.76)a 0.05(33.33)a 0.25(0)a Konflic(1) 050a 0.57(0)a 0.2(38.46)a 0.125(0)a 0.075(0)a Prev-amTM (2) 0.32a 0.37(0)a 0.45(0)a 0.1(0)a 0.2(0)a

WPTM (3) 0.30a 0.32(0)a 0.25(23.07)a 0.075(0)a 0.15(0)a Tracer(4) 0.1a 0.075(70)a 0.05(84.61)a 0(100)a 0.025(0)a Control 0.20a 0.25a 0.325a 0.075a 0.025a

GLM- ANOVA P= 0.120 P= 0.09 P= 0.131 P=0.008 P=0.066

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 8. Mean number of total live larvae following natural products applications under

At 10 days after the first natural products applications, the ANOVA-GLM procedure shows no significant difference between treatments regarding the mean number live larvae (Table 8). The Abbott's percentages of efficacy show the performance of spinosad (84.61 %) and the

At two days after the second spray, (April 10) there is a significant difference between treated plots (AVOVA-GLM procedure, P= 0.008, table 8). The plots treated with Deffort show the maximum density of mean live larvae per leaf (table 8). However, there is no significant difference between the other products and control. The details of larval stages confirm the low efficacy of Deffort compared with the other products and control (small

Six days following the second application (April 14), the mean number of live larvae shows

At eleven days after the second spray, the mean number of live larvae per leaf is relatively similar among treatments and did not significantly vary (ANOVA-GLM procedure P= 0.211) varying from 0.1 to 0.9. Plots treated with Kaolin (Surround) harbour the minimum density. Four days after the third spray (April 23, 2010), the treated plot differed significantly showing the good performance of the compounds neem extract, Tracer and Konflic (table

no significant difference between treated and untreated plots (Table 8).

8). This is confirmed by the analysis of detailed larval instars (table 9).

F= 1.42 F= 1.94 F= 1.61 F= 1.61 F= 1.92 df =3, 309 df =3, 309 df =3, 309 df =3, 309 df =3, 309

Deffort performed well (table 9).

Surround

Statistical Analysis

(1): Botanical extracts (2): Orange extract (3) : Kaolin

µ = second spray

\* Corrected mortality according to Abbott formula

tomato greenhouse (Saheline, Tunisia, 2010).

larvae : P= 0.026; Old larvae P= 0.019; table 9).

plant extract (Armorex; 69.23%).

(ANOVA-GLM procedure) followed by Tukey multiple comparison

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 8. (Continued) Mean number of total live larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).


\*: SL : Small larvae (L1-L2), OL: Old larvae (L3-L4)

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison.

Table 9. Mean number of live small and old larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 345

Mean number of larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT)

df = 3,309 df = 3,309 df = 3,309 df = 3,309

Insecticides 9 DAT3! ! 18DAT3 SL\* OL\* SL\* OL\* Armorex(1) 0.125(72.22)a 0.175(89.06)b 0.75(0)a 0.15(75) a Deffort(1) 0.025(94.44)b 0.175(89.06)b 0.4(5.88)a 0.25(58.33)a Oleargan (1) 0.225(50)a 0.325(79.68)b 0.075(82.35)a 0.3(50)a Konflic(1) 0.275(38.88)a 0.55(65.62)a 0.175(58.82)a 0.15(75) a Prev-amTM (2) 0.375(16.78)a 1.3(18.75)a 0.45(0)a 0.25(58.33)a Surround WPTM (3) 0.1(77.77)a 0.45(71.87)a 0.2a 0.175(70.83)a Tracer(4) 0.125(72.22)a 0.125(92.18)b 0.45(0)a 0.3(50)a Control 0.45a 1.6a 0.425a 0.6a Statistical Analysis F= 2.33 F=5.68 F=1.41 F= 1.97

ANOVA-GLM P= 0.00 P=0.000 P=0.201 P=0.06

Table 9. (Continued) Mean number of live small and old larvae following natural products

Three days following the first insecticide application, the mean number of live larvae (small and large) did not vary significantly between treated and untreated plots (ANOVA-GLM Procedure F= 1.94, df = 3, 309 P= 0.063). However, the plants treated with spinosad (Tracer)

Four days before the first insecticide application, the mean number of live larvae per leaf varied between 0.6 and 0.97 showing no significant difference between treatments and

Two days following the first treatment (March 24), the mean number of live larvae remains relatively low and did not significantly vary between treatment and control (F = 0.34; df = 3, 153; P= 0.79). The corrected mortality according to Abbott formula shows slight efficacy of

At 12 days following the first application, the mean number of live larvae significantly differed between treatments (GLM, F=2.90, df = 3, 156; P= 0.037). The Tukey multiple comparisons showed the good performance of indoxacarb (Avaunt) (Table 10). There is no significant difference between plot treated with triflumuron (Alystin), diafenthiuron

Three days after the second treatment, there is a significant difference between treated plots and control (GLM, F= 16.45 df = 3, 153; P= 0.000). The three compounds performed well

Nine days following the second spray, all insecticides performed well compared with the control (F= 46.7 df =3,153; P=0.000) with the best performance of indoxacarb (Avaunt) (96.87

! !: Means followed by the same letter within a column are not significantly different at P= 0.05

**3.2.2 Insecticides compounds experimented under tomato greenhouse in 2010** 

\* Data in brackets denote percent Abbott mortality (Abbott, 1925)

harbor the minimal larval density (Table 9).

tested products (Table 10).

(Pegasus) and untreated plots.

control (ANOVA. GLM, F= 0.82, df =3, 156; P=0.82).

particularly Avaunt (92.30 % according to Abbott formula).

% efficacy according to Abbott formula, Table 10).

(ANOVA-GLM procedure) followed by Tukey multiple comparison

applications under tomato greenhouse (Saheline, Tunisia, 2010).


µ: undetermined Abbott percentage of efficacy (zero Small larvae in the control plot)

SL: Small larvae (L1-L2), OL: Old larvae (L3-L4).

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 9. (Continued) Mean number of live small and old larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).


\*: SL : Small larvae (L1-L2), OL: Old larvae (L3-L4)

! ! : Means followed by the same letter within a column are not significantly different at P= 0.005 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 9. (Continued) Mean number of live small and old larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).


\* Data in brackets denote percent Abbott mortality (Abbott, 1925)

344 Insecticides – Pest Engineering

Mean number of larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT)

 SL\* OL\* SL\*µ OL\* Armorex(1) 0(100)a 0(100)b 0.1b 0.225(0)a Deffort(1) 0.175(0)b 0.3(0)a 0.025a 0.275(0)a Oleargan (1) 0.025(0)a 0.025(50) b 0.175b 0.075(0)a Konflic(1) 0.05(0)a 0.075(0)b 0a 0.075(0)a Prev-amTM (2) 0(100)a 0.1(0)b 0.125b 0.075(0)a Surround(3) 0.025(0)a 0.05(0)b 0.1b 0.05(0)a Tracer(4) 0(100)a 0(100)b 0a 0.025(0)a Control 0.025a 0.05b 0a 0.025a

> F= 2.31 F= 2.44 F= 2.18 F= 1.34 df= 3,309 df= 3,309 df= 3,309 df= 3,309 P= 0.026 P=0.019 P=0.036 P=0.069

Insecticides 2 DAT2! ! 6DAT2

µ: undetermined Abbott percentage of efficacy (zero Small larvae in the control plot)

(ANOVA-GLM procedure) followed by Tukey multiple comparison

applications under tomato greenhouse (Saheline, Tunisia, 2010).

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

Insecticides 11 DAT2! ! 4DAT3

Table 9. (Continued) Mean number of live small and old larvae following natural products

Mean number of larvae/plant on indicated days before treatment (DBF) and days after treatment (DAT)

 SL\* OL\* SL\* OL\* Armorex(1) 0.375(0)a 0.15(0)a 0(100)a 0.1(84.61)ab Deffort(1) 0.7(0)a 0.225(0)a 0.15(0)b 0.15(76.9)ab Oleargan (1) 0.25(0)a 0.075(50)a 0.025(0) a 0.05(92.30)b Konflic(1) 0.425(0)a 0.05(66.66)a 0(100)a 0(100)b Prev-amTM (2) 0.075(66.66)a 0.15(0)a 0(100)a 0.175(73.0)b Surround WPTM (3) 0.07(66.66)a 0.02(83.33)a 0.125(0)b 0.075(88.4)b Tracer(4) 0.275(0)a 0.075(50)a 0.075(0)ab 0.02 (96.15)b Control 0.225a 0.15a 0.025a 0.65a Statistical Analysis F=1.31 F=1.10 F=2.75 F=2.82

GLM-ANOVA P=0.246 P=0.361 P=0.009 P=0.007

Table 9. (Continued) Mean number of live small and old larvae following natural products

! ! : Means followed by the same letter within a column are not significantly different at P= 0.005

df=3,309 df=3,309 df=3,309 df=3,309

Statistical analysis GLM-ANOVA

SL: Small larvae (L1-L2), OL: Old larvae (L3-L4).

\*: SL : Small larvae (L1-L2), OL: Old larvae (L3-L4)

(ANOVA-GLM procedure) followed by Tukey multiple comparison

applications under tomato greenhouse (Saheline, Tunisia, 2010).

! !: Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 9. (Continued) Mean number of live small and old larvae following natural products applications under tomato greenhouse (Saheline, Tunisia, 2010).

Three days following the first insecticide application, the mean number of live larvae (small and large) did not vary significantly between treated and untreated plots (ANOVA-GLM Procedure F= 1.94, df = 3, 309 P= 0.063). However, the plants treated with spinosad (Tracer) harbor the minimal larval density (Table 9).

## **3.2.2 Insecticides compounds experimented under tomato greenhouse in 2010**

Four days before the first insecticide application, the mean number of live larvae per leaf varied between 0.6 and 0.97 showing no significant difference between treatments and control (ANOVA. GLM, F= 0.82, df =3, 156; P=0.82).

Two days following the first treatment (March 24), the mean number of live larvae remains relatively low and did not significantly vary between treatment and control (F = 0.34; df = 3, 153; P= 0.79). The corrected mortality according to Abbott formula shows slight efficacy of tested products (Table 10).

At 12 days following the first application, the mean number of live larvae significantly differed between treatments (GLM, F=2.90, df = 3, 156; P= 0.037). The Tukey multiple comparisons showed the good performance of indoxacarb (Avaunt) (Table 10). There is no significant difference between plot treated with triflumuron (Alystin), diafenthiuron (Pegasus) and untreated plots.

Three days after the second treatment, there is a significant difference between treated plots and control (GLM, F= 16.45 df = 3, 153; P= 0.000). The three compounds performed well particularly Avaunt (92.30 % according to Abbott formula).

Nine days following the second spray, all insecticides performed well compared with the control (F= 46.7 df =3,153; P=0.000) with the best performance of indoxacarb (Avaunt) (96.87 % efficacy according to Abbott formula, Table 10).

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 347

At 18 days following the second application, the mean number of live larvae significantly varies between treated and control plots (GLM F= 40.88; df = 3, 153; P= 0.000). The efficacy

At 3 and 12 days following the third insecticide application all tested insecticides continue to be effective compared with the control (F= 20.91 df =3, 153; P= 0.00 ; F=10.87; df =3, 153; P= 0.00). Nevertheless, indoxacarb (Avaunt) tend to be a powerful suppressor of *T. absoluta*

In Argentina, the primary *T. absoluta* management tactic was chemical sprays (Lietti *et al*., 2005). Organophosphates were initially used for *T. absoluta* control then were gradually replaced by pyrethroids during the 1970s. During the early 1980s, cartap which alternates with pyrethroids and thiocyclam were sprayed showing the good effectiveness of the former. During the 1990s, insecticides with novel mode of actions were introduced such as abamectin, acylurea, insect growth regulators, tenbufenozide and chlorfenapyr (Lietti *et al*.,

Our laboratory results demonstrate the efficacy of spinosad (Tracer), rotenone (Rotargan), methomyl (Lannate) and abamectin (Vertimec). Methomyl was only tried due to its highly

Spinosad, a mixture of spinosyns A and D, is derived from the naturally occurring actionomycete, *Saccharopolyspora spinosa* (Sparks *et al.,* 1998). Because of its unique mode of action, involving the postsynaptic nicotinic acetylcholine and Gamma-aminobutyric (GABA) receptors, spinosad has strong insecticidal activity against insects (Salgado, 1998) especially Lepidoptera (e.g. *Helicoverpa armigera* (Wang *et a*l., 2009), *Spodoptera frugiperda* (Méndez *et al*., 2002), Diptera (King and Hennesey 1996; Collier and Vanstynwyk , 2003 ; Bond *et al*., 2004),

To date, spinosad is considered a good alternative control of Lepidopteran pests due to its high activity at low rates and its use in integrated pest management programs. The product possesses advantages in term of safety for farm workers and consumers due to its low mammalian toxicity and rapid breakdown in the environment (Sparks et al., 1998). The compound is considered as a standard product for the control of *T. absoluta* in Brazil (Maraus *et al.*, 2008) showing, however low efficacy compared with the insecticide

Rotenone has been reported to be an excellent insecticide against a wide range of insect pests. Davidson (1930) found that rotenone was a toxic and effective contact insecticide against several species of whiteflies, aphids, caterpillars and mites. Also, Turner (1932) reported a high toxicity of rotenone to larvae of the Colorado potato beetle *Leptinotarsa* 

Azadirachtin, a tetranortriterpenoid isolated from the seeds of neem tree, *Azadirachta indica* (Meliaceae), and the fruit of chinaberry, *Melia azaderach* (Meliaceae) acts as an antifeedant and inhibits the growth and the development of several insects (Meisner *et al.*, 1981, Raffa, 1987; McMillian *et al*., 1969). The antifeefant effects of azadirachtin are partly due to sensory

Acetamiprid (Mospilan) is a neonicotinoid insecticide that is formulated for both soil and foliar application. It is a broad-spectrum insecticide effective against several groups of

detection and avoidance by insects (Simmonds and Blaney 1984).

used frequency in tomato production against Noctuid larvae in Tunisia.

some Coleoptera (Elliott et al., 2007) as well as stored grains (Hertlein *et al*, 2011).

of tested insecticide remains high compared with the control.

larvae (table 10).

**4. Discussion** 

2005).

novaluron.

*decemlineata* (Say).


µ : the first treatment was undertaken on March 22, 2010.

! : data in brackets denote percentage of efficacy (Abbott Formula)

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

(ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 10. Mean number of *T. absoluta* larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT) (Saheline tomato greenhouse, 2010).


! ! : Means followed by the same letter within a column are not significantly different at P= 0.05 (ANOVA-GLM procedure) followed by Tukey multiple comparison

Table 10 (continued). Mean number of *T. absoluta* larvae per leaf on indicated days before treatment (DBF) and days after treatment (DAT) (Saheline tomato greenhouse, 2010).

At 18 days following the second application, the mean number of live larvae significantly varies between treated and control plots (GLM F= 40.88; df = 3, 153; P= 0.000). The efficacy of tested insecticide remains high compared with the control.

At 3 and 12 days following the third insecticide application all tested insecticides continue to be effective compared with the control (F= 20.91 df =3, 153; P= 0.00 ; F=10.87; df =3, 153; P= 0.00). Nevertheless, indoxacarb (Avaunt) tend to be a powerful suppressor of *T. absoluta* larvae (table 10).

## **4. Discussion**

346 Insecticides – Pest Engineering

Mean number of larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT)µ Insecticides 4DBT1! ! 2 DAT1 12DAT1 3DAT2 9DAT2 indoxacarb 0.87a 0.7(15. 15)a! 0.2(71.42) a! 0.05(92 .30)a 0.075(96.87)a triflumuron 0.97a 0.6(27.27)a 0.52 (25)ab 0.1(84.61)a 0.4(83.33)a diafenthiuron 0.6a 0. 72 (12.12)a 0.4(42.85)ab 0.125(80.76)a 0.30(87.5)a Control 0.87a 0.85a 0.7 b 0.65b 2.4b

GLM-ANOVA P=0.48 P=0.72 P=0.037 P=0.000 P=0.000

Table 10. Mean number of *T. absoluta* larvae/leaf on indicated days before treatment (DBF)

Mean number of larvae/leaf on indicated days before treatment (DBF) and days after treatment (DAT) Insecticides 18DAT2! ! 3DAT3 12DAT3 indoxacarb (Avaunt) 0.05(95.83)a 0.075(95.45)a 0.35(78.12)a triflumuron (Alystin) 0.05((95.83)a 0.5(69.69)a 0.7(56.25)a

(Pegasus) 0.075(93.75)a 0.325(80.30)a 0.32(87.5)a Control 1.2b 1.65b 1.6b Statistical analysis F= 40.88 F= 20.91 F=10.87

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

Table 10 (continued). Mean number of *T. absoluta* larvae per leaf on indicated days before treatment (DBF) and days after treatment (DAT) (Saheline tomato greenhouse, 2010).

(ANOVA-GLM procedure) followed by Tukey multiple comparison

df =3, 153 df =3, 153 df =3, 153 P = 0.00 P= 0.00 P= 0.000

! ! : Means followed by the same letter within a column are not significantly different at P= 0.05

µ : the first treatment was undertaken on March 22, 2010.

! : data in brackets denote percentage of efficacy (Abbott Formula)

(ANOVA-GLM procedure) followed by Tukey multiple comparison

and days after treatment (DAT) (Saheline tomato greenhouse, 2010).

F=0.82 F= 0.43 F=2.90 F= 16.45 F= 46.7 df =3, 153 df = 3, 153 df = 3, 153 df =3, 153 df =3, 153

Statistical analysis

diafenthiuron

In Argentina, the primary *T. absoluta* management tactic was chemical sprays (Lietti *et al*., 2005). Organophosphates were initially used for *T. absoluta* control then were gradually replaced by pyrethroids during the 1970s. During the early 1980s, cartap which alternates with pyrethroids and thiocyclam were sprayed showing the good effectiveness of the former. During the 1990s, insecticides with novel mode of actions were introduced such as abamectin, acylurea, insect growth regulators, tenbufenozide and chlorfenapyr (Lietti *et al*., 2005).

Our laboratory results demonstrate the efficacy of spinosad (Tracer), rotenone (Rotargan), methomyl (Lannate) and abamectin (Vertimec). Methomyl was only tried due to its highly used frequency in tomato production against Noctuid larvae in Tunisia.

Spinosad, a mixture of spinosyns A and D, is derived from the naturally occurring actionomycete, *Saccharopolyspora spinosa* (Sparks *et al.,* 1998). Because of its unique mode of action, involving the postsynaptic nicotinic acetylcholine and Gamma-aminobutyric (GABA) receptors, spinosad has strong insecticidal activity against insects (Salgado, 1998) especially Lepidoptera (e.g. *Helicoverpa armigera* (Wang *et a*l., 2009), *Spodoptera frugiperda* (Méndez *et al*., 2002), Diptera (King and Hennesey 1996; Collier and Vanstynwyk , 2003 ; Bond *et al*., 2004), some Coleoptera (Elliott et al., 2007) as well as stored grains (Hertlein *et al*, 2011).

To date, spinosad is considered a good alternative control of Lepidopteran pests due to its high activity at low rates and its use in integrated pest management programs. The product possesses advantages in term of safety for farm workers and consumers due to its low mammalian toxicity and rapid breakdown in the environment (Sparks et al., 1998). The compound is considered as a standard product for the control of *T. absoluta* in Brazil (Maraus *et al.*, 2008) showing, however low efficacy compared with the insecticide novaluron.

Rotenone has been reported to be an excellent insecticide against a wide range of insect pests. Davidson (1930) found that rotenone was a toxic and effective contact insecticide against several species of whiteflies, aphids, caterpillars and mites. Also, Turner (1932) reported a high toxicity of rotenone to larvae of the Colorado potato beetle *Leptinotarsa decemlineata* (Say).

Azadirachtin, a tetranortriterpenoid isolated from the seeds of neem tree, *Azadirachta indica* (Meliaceae), and the fruit of chinaberry, *Melia azaderach* (Meliaceae) acts as an antifeedant and inhibits the growth and the development of several insects (Meisner *et al.*, 1981, Raffa, 1987; McMillian *et al*., 1969). The antifeefant effects of azadirachtin are partly due to sensory detection and avoidance by insects (Simmonds and Blaney 1984).

Acetamiprid (Mospilan) is a neonicotinoid insecticide that is formulated for both soil and foliar application. It is a broad-spectrum insecticide effective against several groups of

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 349

Tutafort (plant extract) shows little efficacy after the first application but increases effectiveness after the second application engendering about 80 % of larval mortality (table 7.Cont.). Yet according to manufacturer, (Altinco, 2011), the product has a preventive action and should be applied against eggs and adults. The compound acts by contact penetrating the insect cuticle and dissolves the cell membranes causing the insect dehydrate and its

Management of resistance to prevent or delay the development of resistance to an insecticide and cross resistance to additional insecticides is necessary for increasing the chance of chemical control of *T. absoluta*. Thus, the avoidance of resistance requires the development of pest management programs in which efforts are made to take advantages of natural enemies of pests, plant resistant cultivars, if available, appropriate cultural and

Accordingly, diversification of control tactics should be implemented with the minimum use of chemicals. Insecticides should be applied only as needed basis and only used as the last form of control. When insecticides are applied, the way that they are used should be rationalized and optimized to exploit the full diversity of synthetic chemicals and natural

Development of resistance in *T. absoluta* is an important problem in regions where the insect is established. The expanding international trade of plant material not only spread the pest but also spreads the resistance genes associated with the pest (Denholm and Jespersen, 1998). It is possible that the Mediterranean populations of *T. absoluta* already carried gene resistance from South American counterpart populations and thus, may already express high level of resistance to one or multiple insecticide. Indeed, Cifuentes *et al*., (2011), demonstrated high genetic homogeneity of *T. absoluta* populations came from Mediterranean basin and from South America countries using ribosomal and

Our field results (tomato greenhouse) suggest the good performance of the tested compounds (indoxacarb, triflumuron and diafenthiuron). So far, the product indoxacarb

Indoxacarb is reported by several authors as a powerful insecticide in managing many Lepidopteran pests. Wakil *et al*. (2009) in their study for the management of the pod borer, *Helicoverpa armigera* Hubner (Lepidoptera : Noctuidae) in Pakistan showed the integration of weeding, larvae hand picking and indoxacarb sprays was the most effective in reducing the larval population, pod infestation and maximum grain yield. Also, in Cameroon, Brévault et *al.*, (2008) reported a good efficacy of indoxacarb as a larval insecticide of *H.* 

In the United Kingdom, three insecticides were registered for the control of *T. absoluta* under protected tomato, pepper and aubergine: *Bacillus thuringiensis var. kurstaki*, indoxacarb and

Indoxacarb belongs to a novel class of insecticides, the oxadiazines. It a broad spectrum non-systemic insecticide active especially against Lepidoptera. Indoxacarb affects insect primarily through ingestion but also by contact with treated plant surface. It kills by binding to a site of sodium channels and blocking the flow of sodium ions into nerve cells. The result is impaired nerve function, feeding cessation, paralysis and death (Wing *et al*,

death (Altinco, 2011).

physical methods.

mithochondrial markers.

spinosad (FERA, 2009).

*armigera.*

2000).

products mostly used at rotational basis.

tend to be a powerful suppressor of *T. absoluta* larvae.

insects including Lepidopterans, Coleopterans, Hemipterans and Thysanopterans. The insecticide has an ingestion and stomach action and has a strong osmotic and systemic action (Takahashi *et al*., 1998). The compounds interact with Acetylcholine receptors (AChRs) in a structure-activity relationship, resulting in excitation and paralysis followed by death (Ishaaya *et al.*, 2007).

Abamectin a mixture of avermectins is extracted by the fermentation of the soil bacterium *Streptomyces avermitilis* (Strong & Brown 1987). The insecticide acts on the GABA receptor activating the chloride channel (nerve and muscles) (Aliferis and Jabaji, 2011).

Throughout the assay, the product emamectin benzoate (Proclaim®) showed the best efficacy strongly suppressed *T. absoluta* larval populations. Indeed, several authors reported the performance of this product against several insects, for example, Seal (2005), reported the efficacy of emamectin benzoate at various rates in reducing the densities of the melon thrips, *Thrips palmi* adults and larvae. Stanley *et al.,* (2005) reported the high acute toxicity of emamectin benzoate to *Helicoverpa armigera* under laboratory conditions.

Cook et al., (2004) conducted field and laboratory trials on cotton and soybean for the control of the beet armyworm *Spodoptera exigua* (Hübner) and the fall armyworm *Spodoptera frugiperda* using indoxacarb, pyridalyl, spinosad methoxyfenozide and emamectin benzoate demonstrated the good efficacy of tested products compared with the control. Plots treated with indoxacarb, spinosad and emamectin benzoate had significantly fewer beet armyworm larvae.

Avermectins are a family of 16-membered macrocyclic lactone natural product homologues produced by the soil microorganisms, *Streptomyces avermitilis.* They act as agonists on GABA and glutamate gated chloride channels. The chloride ion flux produced by the direct opening of channels into neuronal cells results in loss cell function and disruption of nerve impulses. Consequently, arthropods are paralyzed irreversibly and stop feeding. Maximum mortality is achieved within four days (Jansson *et al.,* 1997).

Emamectin benzoate (Proclaim) is a novel semi-synthetic derivative of the natural product abamectin in the avermectin family. This insecticide has a high potency against a broad spectrum of lepidopterous pests with an efficacy of about 1,500-fold more potent against certain armyworm species (Jansson *et al.,* 1996)

Insect growth regulators like triflumuron, lufenuron are claimed to be safe and have little impact on beneficial arthropods compared with conventional insecticides and thus attracted considerable attention for their inclusion in IPM programs (Ishaaya *et al*., 2007). In this study, triflumuron showed low efficacy against *T. absoluta* larvae. These results are in accordance with data reported by El-Sheikh and Aamir (2011) suggesting the greater efficiency of lufenuron in controlling *Spodoptera littoralis* Boisd compared with triflumuron or flufenoxuron. Similarly, low effectiveness of triflumuron (Alystin SC48) for the control of *Cactoblastis cactorum* (Lepidoptera: Pyralidae) was reported in Argentina by Labos et al., (2002). Yet the concentration used was lower (30 cc/ hl). Regarding the control of the Mediterranean fruitfly, *Ceratitis capitata*, triflumuron (Alystin 25) failed to give satisfactory results (a concentration of 150 ppm did not kill adults, Zapata *et al.,* (2006)).

Diafenthiuron (Pegasus) is a new type of thiourea derivative that affects respiration in insects. It disrupts oxidative phosphorylation by inhibition of the mitochondrial ATP synthase, an enzyme with essential role in cellular bioenergetics (Ishaaya, 2010). It is an insecticide and acaricide which kills larvae, nymphs and adults by contact and/or stomach action, showing also some ovicidal action (e-pesticide manual, 2005). In our laboratory trial, diafenthiuron (Pegasus) shows little efficacy in *T. absoluta* larval suppression (table 10).

insects including Lepidopterans, Coleopterans, Hemipterans and Thysanopterans. The insecticide has an ingestion and stomach action and has a strong osmotic and systemic action (Takahashi *et al*., 1998). The compounds interact with Acetylcholine receptors (AChRs) in a structure-activity relationship, resulting in excitation and paralysis followed

Abamectin a mixture of avermectins is extracted by the fermentation of the soil bacterium *Streptomyces avermitilis* (Strong & Brown 1987). The insecticide acts on the GABA receptor

Throughout the assay, the product emamectin benzoate (Proclaim®) showed the best efficacy strongly suppressed *T. absoluta* larval populations. Indeed, several authors reported the performance of this product against several insects, for example, Seal (2005), reported the efficacy of emamectin benzoate at various rates in reducing the densities of the melon thrips, *Thrips palmi* adults and larvae. Stanley *et al.,* (2005) reported the high acute toxicity of

Cook et al., (2004) conducted field and laboratory trials on cotton and soybean for the control of the beet armyworm *Spodoptera exigua* (Hübner) and the fall armyworm *Spodoptera frugiperda* using indoxacarb, pyridalyl, spinosad methoxyfenozide and emamectin benzoate demonstrated the good efficacy of tested products compared with the control. Plots treated with indoxacarb, spinosad and emamectin benzoate had significantly fewer beet armyworm

Avermectins are a family of 16-membered macrocyclic lactone natural product homologues produced by the soil microorganisms, *Streptomyces avermitilis.* They act as agonists on GABA and glutamate gated chloride channels. The chloride ion flux produced by the direct opening of channels into neuronal cells results in loss cell function and disruption of nerve impulses. Consequently, arthropods are paralyzed irreversibly and stop feeding. Maximum

Emamectin benzoate (Proclaim) is a novel semi-synthetic derivative of the natural product abamectin in the avermectin family. This insecticide has a high potency against a broad spectrum of lepidopterous pests with an efficacy of about 1,500-fold more potent against

Insect growth regulators like triflumuron, lufenuron are claimed to be safe and have little impact on beneficial arthropods compared with conventional insecticides and thus attracted considerable attention for their inclusion in IPM programs (Ishaaya *et al*., 2007). In this study, triflumuron showed low efficacy against *T. absoluta* larvae. These results are in accordance with data reported by El-Sheikh and Aamir (2011) suggesting the greater efficiency of lufenuron in controlling *Spodoptera littoralis* Boisd compared with triflumuron or flufenoxuron. Similarly, low effectiveness of triflumuron (Alystin SC48) for the control of *Cactoblastis cactorum* (Lepidoptera: Pyralidae) was reported in Argentina by Labos et al., (2002). Yet the concentration used was lower (30 cc/ hl). Regarding the control of the Mediterranean fruitfly, *Ceratitis capitata*, triflumuron (Alystin 25) failed to give satisfactory

Diafenthiuron (Pegasus) is a new type of thiourea derivative that affects respiration in insects. It disrupts oxidative phosphorylation by inhibition of the mitochondrial ATP synthase, an enzyme with essential role in cellular bioenergetics (Ishaaya, 2010). It is an insecticide and acaricide which kills larvae, nymphs and adults by contact and/or stomach action, showing also some ovicidal action (e-pesticide manual, 2005). In our laboratory trial, diafenthiuron (Pegasus) shows little efficacy in *T. absoluta* larval suppression (table 10).

results (a concentration of 150 ppm did not kill adults, Zapata *et al.,* (2006)).

activating the chloride channel (nerve and muscles) (Aliferis and Jabaji, 2011).

emamectin benzoate to *Helicoverpa armigera* under laboratory conditions.

mortality is achieved within four days (Jansson *et al.,* 1997).

certain armyworm species (Jansson *et al.,* 1996)

by death (Ishaaya *et al.*, 2007).

larvae.

Tutafort (plant extract) shows little efficacy after the first application but increases effectiveness after the second application engendering about 80 % of larval mortality (table 7.Cont.). Yet according to manufacturer, (Altinco, 2011), the product has a preventive action and should be applied against eggs and adults. The compound acts by contact penetrating the insect cuticle and dissolves the cell membranes causing the insect dehydrate and its death (Altinco, 2011).

Management of resistance to prevent or delay the development of resistance to an insecticide and cross resistance to additional insecticides is necessary for increasing the chance of chemical control of *T. absoluta*. Thus, the avoidance of resistance requires the development of pest management programs in which efforts are made to take advantages of natural enemies of pests, plant resistant cultivars, if available, appropriate cultural and physical methods.

Accordingly, diversification of control tactics should be implemented with the minimum use of chemicals. Insecticides should be applied only as needed basis and only used as the last form of control. When insecticides are applied, the way that they are used should be rationalized and optimized to exploit the full diversity of synthetic chemicals and natural products mostly used at rotational basis.

Development of resistance in *T. absoluta* is an important problem in regions where the insect is established. The expanding international trade of plant material not only spread the pest but also spreads the resistance genes associated with the pest (Denholm and Jespersen, 1998). It is possible that the Mediterranean populations of *T. absoluta* already carried gene resistance from South American counterpart populations and thus, may already express high level of resistance to one or multiple insecticide. Indeed, Cifuentes *et al*., (2011), demonstrated high genetic homogeneity of *T. absoluta* populations came from Mediterranean basin and from South America countries using ribosomal and mithochondrial markers.

Our field results (tomato greenhouse) suggest the good performance of the tested compounds (indoxacarb, triflumuron and diafenthiuron). So far, the product indoxacarb tend to be a powerful suppressor of *T. absoluta* larvae.

Indoxacarb is reported by several authors as a powerful insecticide in managing many Lepidopteran pests. Wakil *et al*. (2009) in their study for the management of the pod borer, *Helicoverpa armigera* Hubner (Lepidoptera : Noctuidae) in Pakistan showed the integration of weeding, larvae hand picking and indoxacarb sprays was the most effective in reducing the larval population, pod infestation and maximum grain yield. Also, in Cameroon, Brévault et *al.*, (2008) reported a good efficacy of indoxacarb as a larval insecticide of *H. armigera.*

In the United Kingdom, three insecticides were registered for the control of *T. absoluta* under protected tomato, pepper and aubergine: *Bacillus thuringiensis var. kurstaki*, indoxacarb and spinosad (FERA, 2009).

Indoxacarb belongs to a novel class of insecticides, the oxadiazines. It a broad spectrum non-systemic insecticide active especially against Lepidoptera. Indoxacarb affects insect primarily through ingestion but also by contact with treated plant surface. It kills by binding to a site of sodium channels and blocking the flow of sodium ions into nerve cells. The result is impaired nerve function, feeding cessation, paralysis and death (Wing *et al*, 2000).

Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 351

Bielza P., Fernández E., Graválos C., and Albellán J., 2009. Carbamates synergize the toxicity

Bond J.G., Marina C.F., and Williams T., 2004. The naturally derived insecticide Spinosad is

Brévault B., Oumarou Y., Achaleke J., Vaissayre M. & Nebouche S. 2008. Initial activity and

Campbell C.D., Walgenbach J.F., and Kennedy G.C., 1991. Effect of parasitoids on

Cifuentes D., Chynoweth R., and Bielza P., 2011. Genetic study of Mediterranean and South

Cook D.R. Leonard B.R., and Gore J., 2004. Field and laboratory performance of novel

Davidson W.M. 1930. Rotenone as a contact insecticide. J. Econ. Entomol. 23 : 868-

Denholm I., and Jepersen J.B ., 1998. Insecticide resistance management in Europe: recent

DGPA, Direction Générale de la Production Agricole 2009. Tunisian Ministry of Agriculture.

Collier T.R., & Vanstynwyk R., 2003. Olive fruitfly in California: prospects for integrated

http://alerce.inia.cl/docs/informativos/informativo09.pdf. Accessed, 12 May

Elliott R.H. Benjamin M.C., & Gillott G., 2007. Laboratory studies of the toxicity of spinosad

El-sheikh E. A. and Aamir M. M., 2011. Comparative effectiveness and field persistence of

FERA, 2009. The food and environment Research Agency. Plant Pest notice. South American

*littoralis*, Boisd (Lepidoptera : Noctuidae). Crop Protection. 30 : 645-650. E-pesticide manual. Version 3.3. 2005. Edited by British Crop Production Council (BCPC).

and deltamethrin to *Phyllotreta cruciferae* (Coleoptera : Chrysomelidae). Can.

insect growth regulators on a field strain of the cotton leafworm, *Spodoptera* 

of acrinathrin in resistant western flower thrips (Thysanoptera: Thripidae). J. Econ.

highly toxic to *Aedes* and *Anopheles* mosquito larvae. Medical and Veterinary

persistance of insecticides for the control of bollworms (Lepidoptera : Noctuidae) in

Lepidopterous pests in insecticide-treated and untreated tomatoes in western

American populations of tomato leafminer *Tuta absoluta* (Povolny, 1994) (Lepidoptera: Gelechiidae) using ribosomal and mitochondrial markers. Pest.

insecticides against armyworms (Lepidoptera: Noctuidae). Florida Entomologist.

Altinco, 2011. www.altinco.org. Accessed on July 7, 2011.

cotton crops. Crop Protection. 28: 401-406.

control. California Agriculture, 57: 28-32. EstayP. 2000. Polilla del tomate *Tuta absoluta* (Meyrick).

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tomato moth *Tuta absoluta*. N° 56: 1-4.

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Manag. Sci. Wileyonlinelibrary.com. DOI10.1002/ps.2166.

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Annual report.

Entomol. 139: 534-544.

ISBN. 1901396401.

874.

2010.

## **5. Conclusions**

*T. absoluta* has been a serious pest of tomatoes in Tunisia since the autumn 2008. Farmers have gradually come to understand that conventional insecticides such as organophosphates and carbamates are not effective against the insect. Even though more expensive compared with other insecticides, spinosad (Tracer) is now the widely used bioinsecticide to manage the insect.

It is not the intent in this study to advocate one insecticide over another but to enlarge the array of effective insecticide and bio-insecticides with different modes of action. These studies clearly demonstrated the efficacious of several chemicals such as spinosad, abamectin, emamectin benzoate, triflumuron and diafenthiuron. Although, plant extracts such as Armorex and Deffort show mild efficacy in controlling *T. ab*soluta larvae, they can be used in conjunction with chemical products and integrated in a whole program of control.

The efficacies of sprayings using mixtures of natural products and synthetic chemicals for the control of the pest are planned in our laboratory studies. Indeed, insecticides that work in synergy when mixed together are an avenue to explore in *T. absoluta* control. It has been proposed that pesticides mixtures with different modes of action may delay the onset of resistance developing in pest populations (Bielza *et al*., 2009). However, some problems need to be considered when two or more insecticides are mixed together especially phytotoxicity.

The use of insecticides to control *T. absoluta* must not divert attention from the implementation of alternative pest management strategies including cultural, mass-trapping and biological control that can reduce reliance to chemical products.

Chemical pesticides continue to be an important component of insect pest management even with the development of other control methods (mass-trapping, plant resistance…). The use of insecticides based on different chemistries and with varying modes of action is an important component of an integrated pest management strategy. Hence, insecticides will continue to be an integral component of pest management programs due mainly to their effectiveness and simple use. However, the principal factor account for the possible reluctance to shift to the newer insecticides is the high cost.

## **6. Acknowledgements**

The financial support for this work was provided by the Institution of Agricultural Research and Higher Education (IRESA. Ministry of Agriculture and Environment. Tunisia) through the research project "*Tuta absoluta*". We wish to thank the former President Pr Mougou A. and the current President Pr Amamou H. for their help.

We thank Bensalem A., Benmaâti S., Hajjeji F., Ammar A. and Rhouma J. for technical assistance in both laboratory and field.

## **7. References**

Abbott W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18:265-267.

Aliferis K.A., & Jabaji, S., 2011. Metabolomics- A robust bioanalytical approach for the discovery of the modes of action of pesticides: A review. Pesticide Biochemistry and physiology. 100: 105-117.

Altinco, 2011. www.altinco.org. Accessed on July 7, 2011.

350 Insecticides – Pest Engineering

*T. absoluta* has been a serious pest of tomatoes in Tunisia since the autumn 2008. Farmers have gradually come to understand that conventional insecticides such as organophosphates and carbamates are not effective against the insect. Even though more expensive compared with other insecticides, spinosad (Tracer) is now the widely used bio-

It is not the intent in this study to advocate one insecticide over another but to enlarge the array of effective insecticide and bio-insecticides with different modes of action. These studies clearly demonstrated the efficacious of several chemicals such as spinosad, abamectin, emamectin benzoate, triflumuron and diafenthiuron. Although, plant extracts such as Armorex and Deffort show mild efficacy in controlling *T. ab*soluta larvae, they can be used in conjunction with chemical products and integrated in a whole program of

The efficacies of sprayings using mixtures of natural products and synthetic chemicals for the control of the pest are planned in our laboratory studies. Indeed, insecticides that work in synergy when mixed together are an avenue to explore in *T. absoluta* control. It has been proposed that pesticides mixtures with different modes of action may delay the onset of resistance developing in pest populations (Bielza *et al*., 2009). However, some problems need to be considered when two or more insecticides are mixed together especially phyto-

The use of insecticides to control *T. absoluta* must not divert attention from the implementation of alternative pest management strategies including cultural, mass-trapping

Chemical pesticides continue to be an important component of insect pest management even with the development of other control methods (mass-trapping, plant resistance…). The use of insecticides based on different chemistries and with varying modes of action is an important component of an integrated pest management strategy. Hence, insecticides will continue to be an integral component of pest management programs due mainly to their effectiveness and simple use. However, the principal factor account for the possible

The financial support for this work was provided by the Institution of Agricultural Research and Higher Education (IRESA. Ministry of Agriculture and Environment. Tunisia) through the research project "*Tuta absoluta*". We wish to thank the former President Pr Mougou A.

We thank Bensalem A., Benmaâti S., Hajjeji F., Ammar A. and Rhouma J. for technical

Abbott W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ.

Aliferis K.A., & Jabaji, S., 2011. Metabolomics- A robust bioanalytical approach for the

discovery of the modes of action of pesticides: A review. Pesticide Biochemistry

and biological control that can reduce reliance to chemical products.

reluctance to shift to the newer insecticides is the high cost.

and the current President Pr Amamou H. for their help.

assistance in both laboratory and field.

Entomol. 18:265-267.

and physiology. 100: 105-117.

**5. Conclusions** 

control.

toxicity.

**6. Acknowledgements** 

**7. References** 

insecticide to manage the insect.


Management of *Tuta absoluta* (Lepidoptera, Gelechiidae) with Insecticides on Tomatoes 353

Roditakis E., Papachristos D., & Roditakis N.E. 2010. Current status of the tomato leafminer

Salgado V.L., 1998. Studies on the mode of action of Spinosad: insect symptoms and physiological correlates. Pesticides Biochemistry and Physiology. 60: 91-102. Seal D.R., 2005. Management of melon thrips, *Thrips palmi* Karny (Thysanoptera: Thripidae)

Simmonds M.S.J., and Blaney W.M., 1984. Some neurophysiological effects of azadirachtin

Sparks T.C., Thompson G.D., Kirst H.A., Hertlein M.B., Larson L.L. Worden T.N & Thibault

Siqueira H.A.A, Alvaro A., Guedes R.N.C, & Picanço M.C., 2000a. Insecticide resistance in

Siqueira H.A.A, Guedes R.N., & Picanço M.C., 2000b. Cartap resistance and synergism in

Stanley J., Chandrasekaran & Regupathy A. 2005. Basesline toxicity of emamectin and

Strong L., & Brown T.A., 1987. Avermectins in insect control and biology. Bull. Entomol.

Takahashi H., Takakusa N., Suzuki J., & Kishimoto T., 1998. Development of a new

Tomatonews, 2011. http://www.tomatonews.com/resources.html. Accessed March 10,

*Tuta absoluta*. 2011. www.tutaabsoluta.com/agrinewsfull.php?news=3. Accessed 15 March

Urbaneja A., Vercher R., Navarro V., Garcia-Mari F., & Porcuna J.L., 2007. La polilla del

Wang D., Cong P.Y., Li M., Qui X.H., & Wang K.Y. 2009. Sublethal effects of Spinosad on

Wakil W., Ashfaq M., & Ghazanfar M.U., 2009. Integrated management of in chickpea in

Walgenbach J.F., Leidy R.B., & Sheets T.J., 1991. Persistence of insecticides on tomato foliage

rainfed areas of Punjab, Pakistan. Phytoparasitica. 37: 415-420.

survival, growth and reproduction of *Helicoverpa armigera* (Lepidoptera :

and implications for control of tomato fruitworm (Lepidoptera : Noctuidae). J.

Turner N. 1932. Notes on rotenone as an insecticide. J. Econ. Entomol. 25: 1228-1237.

monitoring. Entomological Research. 39: 321-325.

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tomate *Tuta absoluta*. Phytoma España. 194:16-23.

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Econ. Entomol. 84: 978-986.

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M.B 1998. Biological activity of Spinosyns, new fermentain derived insect control agents on tobacco budworm (Lepidoptera: Noctuidae) larvae. J. Econ. Ent. 91: 1277-

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**16** 

*USA* 

**Management Strategies for Western Flower** 

*1United States Department of Agriculture, Agricultural Research Service, Center for* 

*2North Florida Research and Education Center, University of Florida, Quincy, FL,* 

Today, the western flower thrips, *Frankliniella occidentalis* (Pergande) (Thysanoptera: Thripidae) is one of the most significant agricultural pests globally because of the damage it is able to inflict on a wide range of crops. Adults and larvae feed by piercing plant tissues with their needle-shaped mandible and draining the contents of punctured cells (reviewed in Kirk, 1997b). Feeding by adults and larvae produces scarring on foliage, flowers and fruits, which results in aesthetic crop damage and disrupts plant growth and physiology. Also, oviposition can produce a wound response in fruiting structures, which reduces the marketability of certain horticultural produce (Childers, 1997). Most importantly, western flower thrips is able to transmit several species of destructive plant viruses in the genus *Tospovirus* (Bunyaviridae). It is the most important vector of *Tomato spotted wilt virus* and *Impatiens necrotic spot virus* worldwide, and it is also known to vector Chrysanthemum stem necrosis virus, *Groundnut ringspot virus* and *Tomato chlorotic spot virus* (Pappu et al., 2009;

The actual amounts of economic losses attributable to any pest are difficult to determine, but Goldbach and Peters (1994) estimated that *Tomato spotted wilt virus* alone caused over US\$1 billion in losses annually on a global basis. This estimate did not include the direct damage caused by western flower thrips, and it still would further underestimate present day losses, as the western flower thrips has continued to spread throughout the world (Kirk & Terry, 2003; Reitz et al., 2011). The state of Georgia, USA compiles estimates of the economic costs of pests to its crops. These estimates include both crop losses and the costs of control measures. From 2001 – 2006, costs from thrips and TSWV for tomatoes (*Solanum lycopersicum*) and peppers (*Capsicum annuum*) have averaged over 12% of the harvested value of those crops per year (Sparks, 2003, 2004, 2005, 2006; Sparks & Riley, 2001, 2002). Over 60% of the total for economic losses from pests and control costs in tomato and pepper are from thrips and tomato spotted wilt virus (Sparks, 2003). In addition, losses in Georgia

1 The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the

**1. Introduction** 

Webster et al., 2011).

exclusion of others that may be suitable.

**Thrips and the Role of Insecticides<sup>1</sup>**

*Medical, Agricultural and Veterinary Entomology, Tallahassee, FL,* 

Stuart R. Reitz1 and Joe Funderburk2


## **Management Strategies for Western Flower Thrips and the Role of Insecticides<sup>1</sup>**

Stuart R. Reitz1 and Joe Funderburk2

*1United States Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural and Veterinary Entomology, Tallahassee, FL, 2North Florida Research and Education Center, University of Florida, Quincy, FL, USA* 

## **1. Introduction**

354 Insecticides – Pest Engineering

Wing K.D., Sacher M., Kagaya Y., Tsurubuchi Y., Muldirig L.,& Connair M., 2000.

Zapata N., Budia F., Viñuela E., & Pilar M. 2006. Laboratory evaluation of natural pyrehrins,

Protection 19: 537-545.

Phytoparasitica 34(4): 420-427.

Bioactivation and mode of action of the oxidiazine indoxacarb in insects. Crop

pymetrozine and triflumuron as alternatives to control *Ceratitis capitata* adults.

Today, the western flower thrips, *Frankliniella occidentalis* (Pergande) (Thysanoptera: Thripidae) is one of the most significant agricultural pests globally because of the damage it is able to inflict on a wide range of crops. Adults and larvae feed by piercing plant tissues with their needle-shaped mandible and draining the contents of punctured cells (reviewed in Kirk, 1997b). Feeding by adults and larvae produces scarring on foliage, flowers and fruits, which results in aesthetic crop damage and disrupts plant growth and physiology. Also, oviposition can produce a wound response in fruiting structures, which reduces the marketability of certain horticultural produce (Childers, 1997). Most importantly, western flower thrips is able to transmit several species of destructive plant viruses in the genus *Tospovirus* (Bunyaviridae). It is the most important vector of *Tomato spotted wilt virus* and *Impatiens necrotic spot virus* worldwide, and it is also known to vector Chrysanthemum stem necrosis virus, *Groundnut ringspot virus* and *Tomato chlorotic spot virus* (Pappu et al., 2009; Webster et al., 2011).

The actual amounts of economic losses attributable to any pest are difficult to determine, but Goldbach and Peters (1994) estimated that *Tomato spotted wilt virus* alone caused over US\$1 billion in losses annually on a global basis. This estimate did not include the direct damage caused by western flower thrips, and it still would further underestimate present day losses, as the western flower thrips has continued to spread throughout the world (Kirk & Terry, 2003; Reitz et al., 2011). The state of Georgia, USA compiles estimates of the economic costs of pests to its crops. These estimates include both crop losses and the costs of control measures. From 2001 – 2006, costs from thrips and TSWV for tomatoes (*Solanum lycopersicum*) and peppers (*Capsicum annuum*) have averaged over 12% of the harvested value of those crops per year (Sparks, 2003, 2004, 2005, 2006; Sparks & Riley, 2001, 2002). Over 60% of the total for economic losses from pests and control costs in tomato and pepper are from thrips and tomato spotted wilt virus (Sparks, 2003). In addition, losses in Georgia

<sup>1</sup> The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

Management Strategies for Western Flower Thrips and the Role of Insecticides 357

thrips populations did not warrant treatment. Further complicating management issues were the findings by Shorey et al. (1962) that carbamates actually led to rapid resurgence of western flower thrips populations after applications in cotton, which were likely related to

Harding (1961a) observed that western flower thrips was the predominant thrips species infesting onions (*Allium cepa*) grown in southern Texas. It was also observed to be the predominant thrips in California onions (Hale & Shorey, 1965). Multiple applications of insecticides, predominately organophosphates, were found to reduce thrips abundance but

Western flower thrips were noted as a minor pest of other vegetable crops in California. It was confirmed as a vector of *Tomato spotted wilt virus* in the 1930's (see Sakimura, 1962), but viral epidemics caused by western flower thrips apparently were not common at this time, and most damage was a result direct feeding rather than virus transmission (Sakimura, 1961). Similar to the situation with seedling cotton, western flower thrips feeding on young tomato plants was known to reduce photosynthesis and lead to defoliation and plant death (Shorey & Hall, 1963). Thrips feeding on lettuce (Latuca sativa) produces scarred, corky tissue on leaves. Shorey found that organophosphates would reduce populations for a short time after applications, but that populations would rebound soon after applications (Shorey & Hall, 1962). One of the causes identified for the lack of long term control of western flower thrips by insecticides in open field crops was that crops were subject to repeated ongoing dispersal of adults from outside crop fields (Shorey & Hall, 1963). Although tomato spotted wilt epidemics were uncommon in California at this time, Shorey and Hall (1963) also noted the inability of conventional insecticides to reduce Tomato spotted wilt virus transmission within fields because of the repeated dispersal of viruliferous thrips into fields from external

Notably in major review articles concerning floriculture crops, western flower thrips was seldom discussed as a significant pest before the 1970's (Bryan & Smith, 1956; Naegele & Jefferson, 1964; Price et al., 1961), although the damage it caused to greenhouse-grown cut flowers was well recognized in California by the 1930's (Bohart, 1943). At this time, greenhouse growers relied on methods such as intensive sprays of nicotine sulfate or tartar emetic, and fumigation with nicotine, napthalene or calcium cyanide. Fumigation was found to kill adults but was far less effective against immature stages. Therefore, populations could rebound quickly after a fumigation treatment, thus exacerbating overall pest

It is likely that Naegele and Jefferson (1964) did not discuss western flower thrips because of its limited distribution at the time of their review article on floriculture pests. However, the pest status of western flower thrips began to expand rapidly in the late 1970's when growers in California began to experience more extensive damage to cut flower crops from thrips feeding and virus transmission (Robb, 1989). Because of the exceedingly low damage thresholds for these crops, growers responded to the threat with intensive insecticide treatments, leading to the rapid development of resistance to all major classes of insecticides available at the time, including pyrethroids, carbamates, organophosphates and abamectin

adverse effects of the insecticides on beneficial insects.

not to improve yields significantly (Harding, 1961b).

sources.

management concerns for growers.

(Immaraju et al., 1992).

**2. Western flower thrips as an invasive species** 

in 2006 to ornamentals caused primarily by western flower thrips damage exceeded US\$ 15 million. Clearly, these economic assessments show that the western flower thrips is one of the most destructive agricultural pests globally, but its emergence as a major pest has only occurred relatively recently.

The species *"Frankliniella occidentalis"* was first described as a member of the genus *Euthrips* by Theodore Pergande in 1895 from specimens collected in California, where its widespread distribution and abundance across many flowering plants was noted (Pergande, 1895). Subsequently, the species was described under several other names, which have since been synonymized (Mound, 2011). During the early to mid 20th century, the western flower thrips was regularly mentioned as a member of complexes of pestiferous thrips in western North America, but it was generally regarded as a less significant problem than species such as *Taeniothrips inconsequens* (Uzel), *Thrips tabaci* Lindeman, *Scirtothrips citri* (Moulton) and *Heliothrips haemorrhoidalis* (Bouché) (Moulton, 1931). For most of the early 20th century, western flower thrips remained a localized problem in California and other areas of the western USA and Canada, with sporadic problems reported from southern Texas to British Columbia.

One of the first cases of damage attributed to western flower thrips occurred on potatoes grown in the San Gabriel Valley of southern California. Feeding damage from large populations of the western flower thrips was implicated as the cause of curling of new foliage on potato *(Solanum tuberosum)*. The condition, termed potato curly leaf, would lead to significant reductions in tuber size and yield (Crawford, 1915). The recommended control for these thrips infestations was early season applications of Bordeaux mixture combined with extracts of tobacco, which was able to reduce losses when timed appropriately.

Also early in the 20th century, the western flower thrips was recorded as a pest of other vegetable, fruit and nut crops. Riherd (1942) observed oviposition damage on peas grown in the Rio Grande Valley of Texas, which reduced the aesthetic quality of fresh market peas (*Pisum sativum*). Similar spotting patterns, termed "pansy spots", on apples (*Malus domestica*) grown in the northwestern USA were attributed to western flower thrips oviposition (Childs, 1927; Moulton, 1931), as was damage to table grapes (*Vitis vinifera*) grown in California (Jensen, 1973; Yokoyama, 1977). Another significant type of damage to fruit crops resulted from feeding on floral tissues that would cause flower loss before fruit set. Occasionally, larval feeding was known to produce scarring of citrus fruit (Essig, 1926; Woglum & Lewis, 1935). During this period, western flower thrips also was implicated as one of thrips causing damage to alfalfa flowers (*Medicago sativa*), which reduced set seed (Borden, 1915; Seamaxs, 1923).

Later, during the 1950's the western flower thrips began to receive more attention as a pest of seedling cotton (*Gossypium hirsutum*) in the southwestern USA. Extensive use was made of all major classes of insecticides, including organochlorines, organophosphates, and carbamates, during the 1950's – 1960's, to combat this early season threat. Recommendations were made to growers to make multiple applications of insecticides to prevent loss of seedlings from thrips feeding damage. However, control failures with organochlorine insecticides were observed as early as 1960 (Race, 1961). The control failures with organochlorines then led growers to make preventative applications of organophosphates to protect seedlings. These were often made as systemic treatments at planting. However, Race (1965) recognized a significant disadvantage with this approach of intense preventative insecticide use, namely that growers could be making unnecessary applications when the

in 2006 to ornamentals caused primarily by western flower thrips damage exceeded US\$ 15 million. Clearly, these economic assessments show that the western flower thrips is one of the most destructive agricultural pests globally, but its emergence as a major pest has only

The species *"Frankliniella occidentalis"* was first described as a member of the genus *Euthrips* by Theodore Pergande in 1895 from specimens collected in California, where its widespread distribution and abundance across many flowering plants was noted (Pergande, 1895). Subsequently, the species was described under several other names, which have since been synonymized (Mound, 2011). During the early to mid 20th century, the western flower thrips was regularly mentioned as a member of complexes of pestiferous thrips in western North America, but it was generally regarded as a less significant problem than species such as *Taeniothrips inconsequens* (Uzel), *Thrips tabaci* Lindeman, *Scirtothrips citri* (Moulton) and *Heliothrips haemorrhoidalis* (Bouché) (Moulton, 1931). For most of the early 20th century, western flower thrips remained a localized problem in California and other areas of the western USA and Canada, with sporadic problems reported from southern Texas to British

One of the first cases of damage attributed to western flower thrips occurred on potatoes grown in the San Gabriel Valley of southern California. Feeding damage from large populations of the western flower thrips was implicated as the cause of curling of new foliage on potato *(Solanum tuberosum)*. The condition, termed potato curly leaf, would lead to significant reductions in tuber size and yield (Crawford, 1915). The recommended control for these thrips infestations was early season applications of Bordeaux mixture combined with extracts of tobacco, which was able to reduce losses when timed

Also early in the 20th century, the western flower thrips was recorded as a pest of other vegetable, fruit and nut crops. Riherd (1942) observed oviposition damage on peas grown in the Rio Grande Valley of Texas, which reduced the aesthetic quality of fresh market peas (*Pisum sativum*). Similar spotting patterns, termed "pansy spots", on apples (*Malus domestica*) grown in the northwestern USA were attributed to western flower thrips oviposition (Childs, 1927; Moulton, 1931), as was damage to table grapes (*Vitis vinifera*) grown in California (Jensen, 1973; Yokoyama, 1977). Another significant type of damage to fruit crops resulted from feeding on floral tissues that would cause flower loss before fruit set. Occasionally, larval feeding was known to produce scarring of citrus fruit (Essig, 1926; Woglum & Lewis, 1935). During this period, western flower thrips also was implicated as one of thrips causing damage to alfalfa flowers (*Medicago sativa*), which reduced set seed

Later, during the 1950's the western flower thrips began to receive more attention as a pest of seedling cotton (*Gossypium hirsutum*) in the southwestern USA. Extensive use was made of all major classes of insecticides, including organochlorines, organophosphates, and carbamates, during the 1950's – 1960's, to combat this early season threat. Recommendations were made to growers to make multiple applications of insecticides to prevent loss of seedlings from thrips feeding damage. However, control failures with organochlorine insecticides were observed as early as 1960 (Race, 1961). The control failures with organochlorines then led growers to make preventative applications of organophosphates to protect seedlings. These were often made as systemic treatments at planting. However, Race (1965) recognized a significant disadvantage with this approach of intense preventative insecticide use, namely that growers could be making unnecessary applications when the

occurred relatively recently.

Columbia.

appropriately.

(Borden, 1915; Seamaxs, 1923).

thrips populations did not warrant treatment. Further complicating management issues were the findings by Shorey et al. (1962) that carbamates actually led to rapid resurgence of western flower thrips populations after applications in cotton, which were likely related to adverse effects of the insecticides on beneficial insects.

Harding (1961a) observed that western flower thrips was the predominant thrips species infesting onions (*Allium cepa*) grown in southern Texas. It was also observed to be the predominant thrips in California onions (Hale & Shorey, 1965). Multiple applications of insecticides, predominately organophosphates, were found to reduce thrips abundance but not to improve yields significantly (Harding, 1961b).

Western flower thrips were noted as a minor pest of other vegetable crops in California. It was confirmed as a vector of *Tomato spotted wilt virus* in the 1930's (see Sakimura, 1962), but viral epidemics caused by western flower thrips apparently were not common at this time, and most damage was a result direct feeding rather than virus transmission (Sakimura, 1961). Similar to the situation with seedling cotton, western flower thrips feeding on young tomato plants was known to reduce photosynthesis and lead to defoliation and plant death (Shorey & Hall, 1963). Thrips feeding on lettuce (Latuca sativa) produces scarred, corky tissue on leaves. Shorey found that organophosphates would reduce populations for a short time after applications, but that populations would rebound soon after applications (Shorey & Hall, 1962). One of the causes identified for the lack of long term control of western flower thrips by insecticides in open field crops was that crops were subject to repeated ongoing dispersal of adults from outside crop fields (Shorey & Hall, 1963). Although tomato spotted wilt epidemics were uncommon in California at this time, Shorey and Hall (1963) also noted the inability of conventional insecticides to reduce Tomato spotted wilt virus transmission within fields because of the repeated dispersal of viruliferous thrips into fields from external sources.

Notably in major review articles concerning floriculture crops, western flower thrips was seldom discussed as a significant pest before the 1970's (Bryan & Smith, 1956; Naegele & Jefferson, 1964; Price et al., 1961), although the damage it caused to greenhouse-grown cut flowers was well recognized in California by the 1930's (Bohart, 1943). At this time, greenhouse growers relied on methods such as intensive sprays of nicotine sulfate or tartar emetic, and fumigation with nicotine, napthalene or calcium cyanide. Fumigation was found to kill adults but was far less effective against immature stages. Therefore, populations could rebound quickly after a fumigation treatment, thus exacerbating overall pest management concerns for growers.

## **2. Western flower thrips as an invasive species**

It is likely that Naegele and Jefferson (1964) did not discuss western flower thrips because of its limited distribution at the time of their review article on floriculture pests. However, the pest status of western flower thrips began to expand rapidly in the late 1970's when growers in California began to experience more extensive damage to cut flower crops from thrips feeding and virus transmission (Robb, 1989). Because of the exceedingly low damage thresholds for these crops, growers responded to the threat with intensive insecticide treatments, leading to the rapid development of resistance to all major classes of insecticides available at the time, including pyrethroids, carbamates, organophosphates and abamectin (Immaraju et al., 1992).

Management Strategies for Western Flower Thrips and the Role of Insecticides 359

having landed on plants, western flower thrips preferentially reside within flowers or other concealed, protected places on plants (Hansen et al., 2003; Kirk, 1997a). This thigmotactic behavior of thrips limits their exposure to many foliar applied insecticides. Also, the anthophilous nature of western flower thrips limits their exposure to systemic insecticides, which are not readily transported into floral tissues (Cloyd & Sadof, 1998; Daughtrey et al., 1997). Therefore, some of the most effective materials are those with translaminar properties, which increase the probability of thrips concealed in flowers actually ingesting toxins (Kay

While delivering toxins to western flower thrips can be problematic, and the species behavior and ecology can minimize exposure to insecticides, the species is well suited to evolve resistance to multiple classes of insecticides. Since the first reported case of control failures with insecticides (toxaphene, an organochlorine, Race, 1961), there have been numerous incidences of resistance reported to all major classes of insecticides from all regions of the world (Bielza et al., 2007b; Brødsgaard, 1994; Daǧli & Tunç, 2007; Immaraju et al., 1992; Jensen, 2000a; Kay & Herron, 2010; Morishita, 2001; Robb et al., 1995; Weiss et al., 2009; Zhao et al., 1995). Resistance has not only developed against insecticides targeting western flower thrips, but also insecticides used to treat other pest species. Correspondingly, a number of different resistance mechanisms have been characterized to date, including metabolic detoxification, reduced penetration, altered target site resistance,

One class of insecticides where resistance problems have been particularly acute is the pyrethroids. Robb (1989) recorded control failures with pyrethroids in ornamental greenhouses in California. Management became so difficult, with a lack of alternative insecticides that growers returned to using legacy non-synthetic insecticides, such as nicotine sulfate. Despite these early reports regarding difficulties with pyrethroids, they have continued to be used against western flower thrips extensively, with the same outcome of resistance development (Broadbent & Pree, 1997; Espinosa et al., 2002b; Frantz & Mellinger, 2009; Immaraju et al., 1992; Seaton et al., 1997; Thalavaisundaram et al., 2008; Zhao et al., 1995). In these cases, the development of resistance to pyrethroids has tended to occur rapidly. Resistance to pyrethroids led Australia to abandon their use for western flower thrips management less than eight years after the pest was first detected (Herron & Gullick, 2001). Likewise, pyrethroids are no longer recommended for use in Turkish greenhouses because of the rapid development of resistance and cross resistance to other

Resistance to pyrethroids is primarily derived from metabolic detoxification pathways (Broadbent & Pree, 1997; Espinosa et al., 2005; Maymó et al., 2006; Zhao et al., 1995). A broad range of enzymatic detoxification pathways to detoxify pyrethroids have been identified, including cytochrome P450 monooxygenases, glutathione S-transferases and esterases. The predisposition of western flower thrips to evolve resistance based on metabolic detoxification is likely a product of its polyphagous nature. Because individuals move from host to host, they are likely to encounter a variety of plant defensive chemicals. Therefore, it is adaptive to have multiple means to contend with the unpredictable suite of host plant defenses that they may encounter (Rosenheim et al.,

& Herron, 2010).

**3. Insecticide use and insecticide resistance** 

and knockdown resistance (Bielza, 2008).

chemicals (Dağl & Tunç, 2008).

1996).

This insecticide-resistant strain(s) of western flower thrips that originated in California is thought to then have spread around the world in association with the globalization of the cut flower and horticulture industries (Bonarriva, 2003; Huang, 2004). The western flower thrips was established throughout agroecosystems of eastern North America by the mid 1990's, and its spread through Europe was even more rapid (Kirk & Terry, 2003). It was first recorded in the Netherlands in 1983 (Mantel & Van de Vrie, 1988), and large outbreaks were observed in almost all European countries by 1990 (Kirk & Terry, 2003). In northern areas of Europe, western flower thrips is largely restricted to glasshouses2 because climatic conditions prevent persistent populations from establishing (McDonald et al., 1997). In more southerly areas around the Mediterranean basin, populations have become established in open field crops, which facilitates the repeated colonization of protected crops (Brødsgaard, 1993; Kontsedalov et al., 1998). Since the distribution review by Kirk and Terry (2003), western flower thrips has continued to spread to new regions. Significantly, it is now a widely established pest in China (Reitz et al., 2011). As a reflection of its current cosmopolitan distribution and pest status, western flower thrips, a native of North America, is one of the most frequently intercepted insect species at USA ports of entry (Nickle, 2004).

Recently, the taxonomic status of *Frankliniella occidentalis* has been called into question. Molecular evidence indicates that *"Frankliniella occidentalis"* is a complex of two cryptic, sympatric species (Rugman-Jones et al., 2010). Rugman-Jones et al. (2010) have designated the two species as the lupin (L) and glasshouse (G) species. Neither of these genetic species corresponds to the historic morphological descriptions of the species of *F. moultoni* (Hood) or *F. occidentalis* (Pergande)*.* The "lupin" and "glasshouse" designations are based on the similarity of certain individuals with a "western flower thrips" population that has been associated with *Lupinus arboreus* in New Zealand since the 1930's (Martin & Workman, 1994) and other individuals corresponding to pest strains found in European glasshouses (Brødsgaard, 1994). This recognition of species diversity within *"F. occidentalis"* adds uncertainty as to which species is under consideration in earlier literature. However, circumstantial evidence suggests that recent pest problems are largely the result of the highly invasive "glasshouse" type (Martin & Workman, 1994; Rugman-Jones et al., 2010).

Ironically perhaps, responses to these new, expanding problems from invasive western flower thrips populations were similar to historic control efforts attempted in the western USA; namely, there was a heavy reliance on insecticide use for control. Despite pervasive attempts to control western flower thrips with the widespread, intense use of insecticides, there are many factors that limit the efficacy of insecticides. These include ecological, behavioral and physiological factors, and these need to be appreciated and understood to place insecticide use in a proper context. Ecologically, western flower thrips is highly polyphagous and capable of reproducing on numerous host plants (Northfield et al., 2008; Paini et al., 2007). As large populations can develop on non-crop hosts, mass dispersal into crops occurs, whether open field crops (Pearsall & Myers, 2001; Puche et al., 1995; Ramachandran et al., 2001) or crops in protected environments (Antignus et al., 1996). The potential for continual recolonization of crops limits the observed field efficacy of insecticides (Eger et al., 1998; Reitz et al., 2003). This ongoing dispersal means that even repeated insecticide applications have little utility in reducing pest damage, especially in high value crops with low damage thresholds (Bauske, 1998; Kontsedalov et al., 1998). Once

<sup>2</sup> The terms "greenhouse" and "glasshouse" are used interchangeably in this article for convenience, although they are not necessarily structurally equivalent.

having landed on plants, western flower thrips preferentially reside within flowers or other concealed, protected places on plants (Hansen et al., 2003; Kirk, 1997a). This thigmotactic behavior of thrips limits their exposure to many foliar applied insecticides. Also, the anthophilous nature of western flower thrips limits their exposure to systemic insecticides, which are not readily transported into floral tissues (Cloyd & Sadof, 1998; Daughtrey et al., 1997). Therefore, some of the most effective materials are those with translaminar properties, which increase the probability of thrips concealed in flowers actually ingesting toxins (Kay & Herron, 2010).

## **3. Insecticide use and insecticide resistance**

358 Insecticides – Pest Engineering

This insecticide-resistant strain(s) of western flower thrips that originated in California is thought to then have spread around the world in association with the globalization of the cut flower and horticulture industries (Bonarriva, 2003; Huang, 2004). The western flower thrips was established throughout agroecosystems of eastern North America by the mid 1990's, and its spread through Europe was even more rapid (Kirk & Terry, 2003). It was first recorded in the Netherlands in 1983 (Mantel & Van de Vrie, 1988), and large outbreaks were observed in almost all European countries by 1990 (Kirk & Terry, 2003). In northern areas of Europe, western flower thrips is largely restricted to glasshouses2 because climatic conditions prevent persistent populations from establishing (McDonald et al., 1997). In more southerly areas around the Mediterranean basin, populations have become established in open field crops, which facilitates the repeated colonization of protected crops (Brødsgaard, 1993; Kontsedalov et al., 1998). Since the distribution review by Kirk and Terry (2003), western flower thrips has continued to spread to new regions. Significantly, it is now a widely established pest in China (Reitz et al., 2011). As a reflection of its current cosmopolitan distribution and pest status, western flower thrips, a native of North America, is one of the most frequently intercepted insect species at USA ports of entry (Nickle, 2004). Recently, the taxonomic status of *Frankliniella occidentalis* has been called into question. Molecular evidence indicates that *"Frankliniella occidentalis"* is a complex of two cryptic, sympatric species (Rugman-Jones et al., 2010). Rugman-Jones et al. (2010) have designated the two species as the lupin (L) and glasshouse (G) species. Neither of these genetic species corresponds to the historic morphological descriptions of the species of *F. moultoni* (Hood) or *F. occidentalis* (Pergande)*.* The "lupin" and "glasshouse" designations are based on the similarity of certain individuals with a "western flower thrips" population that has been associated with *Lupinus arboreus* in New Zealand since the 1930's (Martin & Workman, 1994) and other individuals corresponding to pest strains found in European glasshouses (Brødsgaard, 1994). This recognition of species diversity within *"F. occidentalis"* adds uncertainty as to which species is under consideration in earlier literature. However, circumstantial evidence suggests that recent pest problems are largely the result of the highly invasive "glasshouse" type (Martin & Workman, 1994; Rugman-Jones et al., 2010). Ironically perhaps, responses to these new, expanding problems from invasive western flower thrips populations were similar to historic control efforts attempted in the western USA; namely, there was a heavy reliance on insecticide use for control. Despite pervasive attempts to control western flower thrips with the widespread, intense use of insecticides, there are many factors that limit the efficacy of insecticides. These include ecological, behavioral and physiological factors, and these need to be appreciated and understood to place insecticide use in a proper context. Ecologically, western flower thrips is highly polyphagous and capable of reproducing on numerous host plants (Northfield et al., 2008; Paini et al., 2007). As large populations can develop on non-crop hosts, mass dispersal into crops occurs, whether open field crops (Pearsall & Myers, 2001; Puche et al., 1995; Ramachandran et al., 2001) or crops in protected environments (Antignus et al., 1996). The potential for continual recolonization of crops limits the observed field efficacy of insecticides (Eger et al., 1998; Reitz et al., 2003). This ongoing dispersal means that even repeated insecticide applications have little utility in reducing pest damage, especially in high value crops with low damage thresholds (Bauske, 1998; Kontsedalov et al., 1998). Once

2 The terms "greenhouse" and "glasshouse" are used interchangeably in this article for convenience,

although they are not necessarily structurally equivalent.

While delivering toxins to western flower thrips can be problematic, and the species behavior and ecology can minimize exposure to insecticides, the species is well suited to evolve resistance to multiple classes of insecticides. Since the first reported case of control failures with insecticides (toxaphene, an organochlorine, Race, 1961), there have been numerous incidences of resistance reported to all major classes of insecticides from all regions of the world (Bielza et al., 2007b; Brødsgaard, 1994; Daǧli & Tunç, 2007; Immaraju et al., 1992; Jensen, 2000a; Kay & Herron, 2010; Morishita, 2001; Robb et al., 1995; Weiss et al., 2009; Zhao et al., 1995). Resistance has not only developed against insecticides targeting western flower thrips, but also insecticides used to treat other pest species. Correspondingly, a number of different resistance mechanisms have been characterized to date, including metabolic detoxification, reduced penetration, altered target site resistance, and knockdown resistance (Bielza, 2008).

One class of insecticides where resistance problems have been particularly acute is the pyrethroids. Robb (1989) recorded control failures with pyrethroids in ornamental greenhouses in California. Management became so difficult, with a lack of alternative insecticides that growers returned to using legacy non-synthetic insecticides, such as nicotine sulfate. Despite these early reports regarding difficulties with pyrethroids, they have continued to be used against western flower thrips extensively, with the same outcome of resistance development (Broadbent & Pree, 1997; Espinosa et al., 2002b; Frantz & Mellinger, 2009; Immaraju et al., 1992; Seaton et al., 1997; Thalavaisundaram et al., 2008; Zhao et al., 1995). In these cases, the development of resistance to pyrethroids has tended to occur rapidly. Resistance to pyrethroids led Australia to abandon their use for western flower thrips management less than eight years after the pest was first detected (Herron & Gullick, 2001). Likewise, pyrethroids are no longer recommended for use in Turkish greenhouses because of the rapid development of resistance and cross resistance to other chemicals (Dağl & Tunç, 2008).

Resistance to pyrethroids is primarily derived from metabolic detoxification pathways (Broadbent & Pree, 1997; Espinosa et al., 2005; Maymó et al., 2006; Zhao et al., 1995). A broad range of enzymatic detoxification pathways to detoxify pyrethroids have been identified, including cytochrome P450 monooxygenases, glutathione S-transferases and esterases. The predisposition of western flower thrips to evolve resistance based on metabolic detoxification is likely a product of its polyphagous nature. Because individuals move from host to host, they are likely to encounter a variety of plant defensive chemicals. Therefore, it is adaptive to have multiple means to contend with the unpredictable suite of host plant defenses that they may encounter (Rosenheim et al., 1996).

Management Strategies for Western Flower Thrips and the Role of Insecticides 361

the most efficacious insecticides against western flower thrips in recent years have been spinosyns, which are metabolites derived from fermentation of the actinomycete bacterium, *Saccharopolyspora spinosa* (Sparks et al., 1999). Spinosyns are in a group of insecticides with a novel mode of action, the nicotinic acetylcholine receptor (nAChR) allosteric activators (Group 5 – Insecticide Resistance Action Committee) (IRAC International MoA Working Group, 2011). The unique mode of action of spinosyn-based insecticides and their translaminar properties have made them highly effective against western flower thrips. The first products with spinosad as the active ingredient (a combination of spinosyns A and D; Dow AgroSciences, Indianapolis, IN) were registered for use in the late 1990's (Thompson et al., 2000). This was followed later by the release of spinetoram in 2008. Given the effectiveness of spinosyns and the lack of effective alternatives, growers tended to place an overreliance on spinosad, making them a victim of their own success. The first evidence of resistance to spinosyns was detected in western flower thrips populations in Australia by 2002 (Herron & James, 2005), Spain by 2003 (Bielza et al., 2007b) and the US by 2006 (Weiss et al., 2009). Spinosyn resistance in western flower thrips appears to be based on altered target site resistance, with spinosad resistance in a Spanish population based a single locus, autosomal recessive trait (Bielza et al., 2007c). The evidence that spinosad resistance is a recessive trait means that this resistance may not be stable, which would facilitate reversion to susceptibility in populations (Weiss et al., 2009). However, as with other resistance cases, mechanisms and genetics of spinosyn resistance likely vary across populations. Zhang et al. (2008) reported that spinosad resistance in a population from Japan is likely polygenic. Perhaps, more troubling is the potential cross resistance to other insecticides that Zhang et al (2008) found in their spinosad resistant strain. This potential will necessitate further caution

In efforts to broaden the range of insecticides available for western flower thrips management, there has been interest in adapting other existing chemistries not previously labeled for use against western flower thrips. Neonicotinoids, which have been used since the 1990's to manage various types of other sucking insect pests, have recently received attention for a potential role in western flower thrips management. In experimental trials in Australia, Broughton and Herron (2009) found that two neonicotinoids, acetamiprid and thiamethoxam, were as effective as spinosad against larvae and adults of western flower thrips in pepper and lettuce. However, none of the tested insecticides, including spinosad, were effective in reducing the abundance of western flower thrips adults in tomato. Coutts and Jones (2005) found that drenching lettuce seedlings with neonicotinoids, in particular imidacloprid, just before transplanting reduced the incidence of tomato spotted wilt significantly in field trials. However, during their field trial, the predominant vector species were *Frankliniella schultzei* (Trybom) and *Thrips tabaci,* with very few western flower thrips found. Interestingly, their results showed that there were minimal effects of the neonicotinoids on the abundance of any of the thrips species. Likewise, western flower thrips populations showed little effect from applications of another neonicotinoid, dinotefuran, in pepper and strawberry (Dripps et al., 2010). It is possible that in these cases, mortality induced by the insecticide was counteracted by the loss of natural enemies of thrips from the insecticide applications. Alternatively, disease reductions observed by Coutts and Jones (2005) may have resulted from sublethal effects of the insecticides on thrips. Imidacloprid was found to actually enhance feeding of western flower thrips on tomato foliage while it reduced feeding by another TSWV vector, *Frankliniella fusca* (Hinds) (Joost & Riley, 2005). Likewise, the anthranilic diamide, cyantraniliprole, which disrupts

in managing the use of spinosyn products.

These inherent metabolic detoxification pathways predispose the western flower thrips to overcome pyrethroids and other classes of insecticides. Adding to the factors that make western flower thrips amenable to developing insecticide resistance are its rapid development rate so that populations may pass through several generations within a single cropping system, its high fecundity, so that resistant females can produce many offspring, and the haplodiploid sex determination characteristic of thrips (Reitz, 2009). In this type of sex determination, females are diploid, but males are haploid. Because males are haploid, their alleles are exposed directly to selection, which enable alleles for resistance to become fixed rapidly in a population (Denholm et al., 1998).

Insecticide resistance in western flower thrips is a complex phenomenon. It is important to note that resistance to a particular insecticide may not derive from a single trait and can involve multiple pathways (Jensen, 1998). For example, resistance to diazinon, an organophosphate, and methiocarb, a carbamate, has been linked to both metabolic detoxification and altered target site sensitivity (Jensen, 2000b; Zhao et al., 1994). In addition, distinct populations can evolve resistance to a particular insecticide through different mechanisms (Jensen, 2000b; Thalavaisundaram et al., 2008). However, a single metabolic mechanism may confer cross resistance between different classes of insecticides. Espinosa et al. (2002a) found that single metabolic pathway appears to confer resistance to pyrethroids and carbamates. In turn, Bielza et al. (2007a) proposed that such a single detoxification mechanism for the pyrethroid acrinathrin and for carbamates could be exploited, by using carbamates as synergists to increase the activity of acrinathrin through competitive substrate inhibition. These variable pathways complicate the prediction of resistance development and the potential for cross resistance to multiple classes of insecticides in any population.

Individuals carrying resistance alleles are often considered to be at a fitness disadvantage in a population when the particular insecticide to which they are resistant is not used (i.e., in the absence of particular selection pressures) (Georghiou & Taylor, 1986). When such a fitness disadvantage is present in resistant individuals, reversion to a susceptible population should occur soon after removal of the specific insecticide (selective pressure). However, this outcome may not always be the case for western flower thrips. There are cases of long term maintenance of resistance in the absence of insecticide exposure. Kontsedalov et al. (1998) reported that resistance to the pyrethroid cypermethrin did not decline after more than seven years of non-exposure for a laboratory colony. Likewise, resistance to organophosphates can be maintained in field and laboratory populations for several years without exposure (Brødsgaard, 1994; Robb, 1989). Bielza et al. (2008) compared fecundity, fertility, longevity, and egg-to-adult developmental time in populations of western flower thrips from Spain that were resistant and susceptible to acrinathrin (pyrethroid) and spinosad (a spinosyn formulation, see below), and found that resistance to either material did not carry significant fitness costs, at least in terms of the parameters they measured. Therefore, resistance may be expected to develop rapidly and be maintained for long periods of time in the absence of the use of an insecticide, and these scenarios must be taken into account in developing insecticide resistance management programs, which are integral to integrated pest management (IPM) programs.

Given the ongoing issues with management failures and resistance development with synthetic insecticides, and the cancellation of registrations for many other synthetic insecticides as a result of the Food Quality and Protection Act of 1996 (US EPA, 1996), there is a limited pool of efficacious insecticides for use against western flower thrips. Consequently, there is intense interest in developing new, alternative insecticides. Some of

These inherent metabolic detoxification pathways predispose the western flower thrips to overcome pyrethroids and other classes of insecticides. Adding to the factors that make western flower thrips amenable to developing insecticide resistance are its rapid development rate so that populations may pass through several generations within a single cropping system, its high fecundity, so that resistant females can produce many offspring, and the haplodiploid sex determination characteristic of thrips (Reitz, 2009). In this type of sex determination, females are diploid, but males are haploid. Because males are haploid, their alleles are exposed directly to selection, which enable alleles for resistance to become

Insecticide resistance in western flower thrips is a complex phenomenon. It is important to note that resistance to a particular insecticide may not derive from a single trait and can involve multiple pathways (Jensen, 1998). For example, resistance to diazinon, an organophosphate, and methiocarb, a carbamate, has been linked to both metabolic detoxification and altered target site sensitivity (Jensen, 2000b; Zhao et al., 1994). In addition, distinct populations can evolve resistance to a particular insecticide through different mechanisms (Jensen, 2000b; Thalavaisundaram et al., 2008). However, a single metabolic mechanism may confer cross resistance between different classes of insecticides. Espinosa et al. (2002a) found that single metabolic pathway appears to confer resistance to pyrethroids and carbamates. In turn, Bielza et al. (2007a) proposed that such a single detoxification mechanism for the pyrethroid acrinathrin and for carbamates could be exploited, by using carbamates as synergists to increase the activity of acrinathrin through competitive substrate inhibition. These variable pathways complicate the prediction of resistance development and the potential for cross resistance to multiple classes of insecticides in any population. Individuals carrying resistance alleles are often considered to be at a fitness disadvantage in a population when the particular insecticide to which they are resistant is not used (i.e., in the absence of particular selection pressures) (Georghiou & Taylor, 1986). When such a fitness disadvantage is present in resistant individuals, reversion to a susceptible population should occur soon after removal of the specific insecticide (selective pressure). However, this outcome may not always be the case for western flower thrips. There are cases of long term maintenance of resistance in the absence of insecticide exposure. Kontsedalov et al. (1998) reported that resistance to the pyrethroid cypermethrin did not decline after more than seven years of non-exposure for a laboratory colony. Likewise, resistance to organophosphates can be maintained in field and laboratory populations for several years without exposure (Brødsgaard, 1994; Robb, 1989). Bielza et al. (2008) compared fecundity, fertility, longevity, and egg-to-adult developmental time in populations of western flower thrips from Spain that were resistant and susceptible to acrinathrin (pyrethroid) and spinosad (a spinosyn formulation, see below), and found that resistance to either material did not carry significant fitness costs, at least in terms of the parameters they measured. Therefore, resistance may be expected to develop rapidly and be maintained for long periods of time in the absence of the use of an insecticide, and these scenarios must be taken into account in developing insecticide resistance management programs, which are integral

Given the ongoing issues with management failures and resistance development with synthetic insecticides, and the cancellation of registrations for many other synthetic insecticides as a result of the Food Quality and Protection Act of 1996 (US EPA, 1996), there is a limited pool of efficacious insecticides for use against western flower thrips. Consequently, there is intense interest in developing new, alternative insecticides. Some of

fixed rapidly in a population (Denholm et al., 1998).

to integrated pest management (IPM) programs.

the most efficacious insecticides against western flower thrips in recent years have been spinosyns, which are metabolites derived from fermentation of the actinomycete bacterium, *Saccharopolyspora spinosa* (Sparks et al., 1999). Spinosyns are in a group of insecticides with a novel mode of action, the nicotinic acetylcholine receptor (nAChR) allosteric activators (Group 5 – Insecticide Resistance Action Committee) (IRAC International MoA Working Group, 2011). The unique mode of action of spinosyn-based insecticides and their translaminar properties have made them highly effective against western flower thrips. The first products with spinosad as the active ingredient (a combination of spinosyns A and D; Dow AgroSciences, Indianapolis, IN) were registered for use in the late 1990's (Thompson et al., 2000). This was followed later by the release of spinetoram in 2008. Given the effectiveness of spinosyns and the lack of effective alternatives, growers tended to place an overreliance on spinosad, making them a victim of their own success. The first evidence of resistance to spinosyns was detected in western flower thrips populations in Australia by 2002 (Herron & James, 2005), Spain by 2003 (Bielza et al., 2007b) and the US by 2006 (Weiss et al., 2009). Spinosyn resistance in western flower thrips appears to be based on altered target site resistance, with spinosad resistance in a Spanish population based a single locus, autosomal recessive trait (Bielza et al., 2007c). The evidence that spinosad resistance is a recessive trait means that this resistance may not be stable, which would facilitate reversion to susceptibility in populations (Weiss et al., 2009). However, as with other resistance cases, mechanisms and genetics of spinosyn resistance likely vary across populations. Zhang et al. (2008) reported that spinosad resistance in a population from Japan is likely polygenic. Perhaps, more troubling is the potential cross resistance to other insecticides that Zhang et al (2008) found in their spinosad resistant strain. This potential will necessitate further caution in managing the use of spinosyn products.

In efforts to broaden the range of insecticides available for western flower thrips management, there has been interest in adapting other existing chemistries not previously labeled for use against western flower thrips. Neonicotinoids, which have been used since the 1990's to manage various types of other sucking insect pests, have recently received attention for a potential role in western flower thrips management. In experimental trials in Australia, Broughton and Herron (2009) found that two neonicotinoids, acetamiprid and thiamethoxam, were as effective as spinosad against larvae and adults of western flower thrips in pepper and lettuce. However, none of the tested insecticides, including spinosad, were effective in reducing the abundance of western flower thrips adults in tomato. Coutts and Jones (2005) found that drenching lettuce seedlings with neonicotinoids, in particular imidacloprid, just before transplanting reduced the incidence of tomato spotted wilt significantly in field trials. However, during their field trial, the predominant vector species were *Frankliniella schultzei* (Trybom) and *Thrips tabaci,* with very few western flower thrips found. Interestingly, their results showed that there were minimal effects of the neonicotinoids on the abundance of any of the thrips species. Likewise, western flower thrips populations showed little effect from applications of another neonicotinoid, dinotefuran, in pepper and strawberry (Dripps et al., 2010). It is possible that in these cases, mortality induced by the insecticide was counteracted by the loss of natural enemies of thrips from the insecticide applications. Alternatively, disease reductions observed by Coutts and Jones (2005) may have resulted from sublethal effects of the insecticides on thrips. Imidacloprid was found to actually enhance feeding of western flower thrips on tomato foliage while it reduced feeding by another TSWV vector, *Frankliniella fusca* (Hinds) (Joost & Riley, 2005). Likewise, the anthranilic diamide, cyantraniliprole, which disrupts

Management Strategies for Western Flower Thrips and the Role of Insecticides 363

have lethal insecticidal properties to pests can be phytotoxic (Cloyd et al., 2009, S. R. Reitz,

There has been considerable interest in the use of microbial insecticides against western flower thrips (Butt & Brownbridge, 1997). Although experimental work has demonstrated the effectiveness of these materials and natural epizootics have been recorded (Vacante et al., 1994), there has been limited commercial success with them. Several products are available for commercial use (Shah & Goettel, 1999), and new formulations are still being

There has been a long history of insecticide use against the western flower thrips. Yet, there has only been a limited number of efficacious available at any given time, and none have been able to serve as a stand-alone management tactic. Although several new insecticides have been reported to have efficacy against western flower thrips, there is still a limited suite of insecticides that are effective. This limitation is likely to be an ongoing constraint because of the cost of developing and registering new insecticides. Therefore, to maintain the utility of efficacious insecticides as a part of IPM programs for western flower thrips, it is

critical to take conservative and judicious approaches to the use of these insecticides.

Given the history of insecticide use against western flower thrips, resistance development is more than likely to occur to any insecticide, regardless of mode of action. Therefore, it is critical to develop strategies to employ them effectively. A key element in this regard is proper insecticide resistance management programs to maintain efficacy for as long as possible. In general, insecticide resistance management programs for western flower thrips do not differ conceptually from those designed for other pests. The basic concept is to rotate among insecticides with different modes of action at appropriate intervals to delay or inhibit

Most current insecticide resistance management plans recommend rotation of chemical classes after every generation of thrips (Broadbent & Pree, 1997; Herron & Cook, 2002; Robb et al., 1995). In Australia, the initial recommendations for insecticide resistance management were for growers to alternate among insecticides from different chemical classes with each application, a practice recommended in other regions (Funderburk, 2009). However, Herron and Cook (2002) proposed that this simple strategy would not be effective because of the long term persistence of resistance in populations to cypermethrin, a pyrethroid. They argued that reversion to a susceptible population would not occur before an insecticide was used again, rendering that material ineffective. It is also possible that alternating chemical classes too frequently (within a generation) could more readily select for individuals with resistance to multiple insecticides. In a subsequent study, Broughton and Herron (2007) advocated a three-consecutive spray program of a particular insecticide before rotating to a different chemical class. A key component to this strategy was that the three-consecutive applications needed to be made within a single thrips generation to gain the maximum

The actual implementation of such an approach is limited by the continuous, overlapping generations present within a crop (Reitz, 2009), and may best be interpreted as rotating chemistries on an appropriate time interval (approximately 3 weeks). Bielza (2008) further cautioned that it is not simply enough to rotate among different chemistries. Rather rotation schedules should be based on known resistance mechanisms to avoid problems with cross

unpublished).

developed (e.g., Zhang et al., 2009).

**4. Insecticide resistance management** 

the evolution of resistance within a pest population.

effectiveness for that treatment.

insect feeding activity, reduced TSWV transmission by *F. fusca,* but had no effect on transmission by western flower thrips (Jacobson & Kennedy, 2011). Therefore, more thorough evaluations should be made of the use of neonicotinoids specifically for western flower thrips management in different cropping systems. Given these results, it is important also not to extrapolate from the effects of insecticides on one thrips species, even congeneric ones, to *F. occidentalis.* 

Despite indications of resistance to abamectin developing (Immaraju et al., 1992), it and related materials in the avermectin class have continued to receive interest as a means to manage western flower thrips in greenhouse and open field crops. Spiers et al. (2006) recently found that abamectin was as effective as spinosad in reducing western flower thrips feeding damage to gerbera daisies (*Gerbera jamesonii*). Overall flower quality was high not only because of the low amount of damage but also because of the lack of phytotoxic effects. The abamectin derivative, emamectin benzoate, has also been found to have efficacy against western flower thrips (Ishaaya et al., 2002). This material was found to be more potent than the parent material abamectin, and it was more effective against larvae than against adults. An advantageous property of abamectin and emamectin benzoate is that they are translaminar, increasing the likelihood that toxins will reach concealed thrips adults and larvae. However, other results have shown that these avermectins are not effective against western flower thrips in field trials (Kay & Herron, 2010). They found that their test population of western flower thrips was highly susceptible to abamectin and emamectin benzoate in direct exposure assays so that the field failures could result from difficulties in applying adequate doses of avermectins to plants in the field (Broughton & Herron, 2007).

Other newly developed insecticides with novel modes of action continue to be evaluated for their potential role in western flower thrips management. Pyridalyl, which has not been classified as yet for its mode of action, is more toxic to larvae than to adults of the western flower thrips, but it is compatible with biological control agents such the predatory bug *Orius strigicollis* (Poppius) (Isayama et al., 2005). This feature makes it an attractive insecticide for rotational use in an overall integrated pest management program. Pyridalyl is registered for greenhouse ornamentals in the USA and certain other crops elsewhere in the world. Likewise, the pyrrrol chlorfenapyr is registered for greenhouse ornamental and vegetables in the USA, but is approved for use in other situations globally. It has shown efficacy comparable to spinosad in greenhouse trials conducted in Australia (Broughton & Herron, 2009). Both pyridalyl and chlorfenapyr have translaminar properties. The systemic insecticide fipronil, a phenylpyrazole, is effective against western flower thrips larvae, and to a lesser extent, adults (Kay & Herron, 2010). In an interesting approach to synergizing insecticides, Cook et al. (2002) found that the addition of dodecyl acetate, a component of the western flower thrips alarm pheromone, increased the efficacy of fipronil in field trials against western flower thrips in strawberry (*Fragaria × ananassa*).

There has also been recent interest in the use botanically derived insecticides for use against the western flower thrips. Certain essential oils can help reduce the incidence of tomato spotted wilt in tomato (Reitz et al., 2008), and other *Chenopodium* based materials have shown efficacy under greenhouse conditions (Chiasson et al., 2004). *Chenopodium* based products, while not highly toxic under open field conditions, provide sufficient suppression of western flower thrips larvae to warrant inclusion in insecticide rotation schemes (Funderburk, 2009). These essential oil products tend to have little negative impact on natural enemies, so they may be compatible in overall IPM programs (Bostanian et al., 2005). One drawback in the use of plant essential oils has been that concentrations of oils needed to have lethal insecticidal properties to pests can be phytotoxic (Cloyd et al., 2009, S. R. Reitz, unpublished).

There has been considerable interest in the use of microbial insecticides against western flower thrips (Butt & Brownbridge, 1997). Although experimental work has demonstrated the effectiveness of these materials and natural epizootics have been recorded (Vacante et al., 1994), there has been limited commercial success with them. Several products are available for commercial use (Shah & Goettel, 1999), and new formulations are still being developed (e.g., Zhang et al., 2009).

There has been a long history of insecticide use against the western flower thrips. Yet, there has only been a limited number of efficacious available at any given time, and none have been able to serve as a stand-alone management tactic. Although several new insecticides have been reported to have efficacy against western flower thrips, there is still a limited suite of insecticides that are effective. This limitation is likely to be an ongoing constraint because of the cost of developing and registering new insecticides. Therefore, to maintain the utility of efficacious insecticides as a part of IPM programs for western flower thrips, it is critical to take conservative and judicious approaches to the use of these insecticides.

## **4. Insecticide resistance management**

362 Insecticides – Pest Engineering

insect feeding activity, reduced TSWV transmission by *F. fusca,* but had no effect on transmission by western flower thrips (Jacobson & Kennedy, 2011). Therefore, more thorough evaluations should be made of the use of neonicotinoids specifically for western flower thrips management in different cropping systems. Given these results, it is important also not to extrapolate from the effects of insecticides on one thrips species, even congeneric

Despite indications of resistance to abamectin developing (Immaraju et al., 1992), it and related materials in the avermectin class have continued to receive interest as a means to manage western flower thrips in greenhouse and open field crops. Spiers et al. (2006) recently found that abamectin was as effective as spinosad in reducing western flower thrips feeding damage to gerbera daisies (*Gerbera jamesonii*). Overall flower quality was high not only because of the low amount of damage but also because of the lack of phytotoxic effects. The abamectin derivative, emamectin benzoate, has also been found to have efficacy against western flower thrips (Ishaaya et al., 2002). This material was found to be more potent than the parent material abamectin, and it was more effective against larvae than against adults. An advantageous property of abamectin and emamectin benzoate is that they are translaminar, increasing the likelihood that toxins will reach concealed thrips adults and larvae. However, other results have shown that these avermectins are not effective against western flower thrips in field trials (Kay & Herron, 2010). They found that their test population of western flower thrips was highly susceptible to abamectin and emamectin benzoate in direct exposure assays so that the field failures could result from difficulties in applying adequate doses of avermectins to plants in the field (Broughton & Herron, 2007). Other newly developed insecticides with novel modes of action continue to be evaluated for their potential role in western flower thrips management. Pyridalyl, which has not been classified as yet for its mode of action, is more toxic to larvae than to adults of the western flower thrips, but it is compatible with biological control agents such the predatory bug *Orius strigicollis* (Poppius) (Isayama et al., 2005). This feature makes it an attractive insecticide for rotational use in an overall integrated pest management program. Pyridalyl is registered for greenhouse ornamentals in the USA and certain other crops elsewhere in the world. Likewise, the pyrrrol chlorfenapyr is registered for greenhouse ornamental and vegetables in the USA, but is approved for use in other situations globally. It has shown efficacy comparable to spinosad in greenhouse trials conducted in Australia (Broughton & Herron, 2009). Both pyridalyl and chlorfenapyr have translaminar properties. The systemic insecticide fipronil, a phenylpyrazole, is effective against western flower thrips larvae, and to a lesser extent, adults (Kay & Herron, 2010). In an interesting approach to synergizing insecticides, Cook et al. (2002) found that the addition of dodecyl acetate, a component of the western flower thrips alarm pheromone, increased the efficacy of fipronil in field trials

against western flower thrips in strawberry (*Fragaria × ananassa*).

There has also been recent interest in the use botanically derived insecticides for use against the western flower thrips. Certain essential oils can help reduce the incidence of tomato spotted wilt in tomato (Reitz et al., 2008), and other *Chenopodium* based materials have shown efficacy under greenhouse conditions (Chiasson et al., 2004). *Chenopodium* based products, while not highly toxic under open field conditions, provide sufficient suppression of western flower thrips larvae to warrant inclusion in insecticide rotation schemes (Funderburk, 2009). These essential oil products tend to have little negative impact on natural enemies, so they may be compatible in overall IPM programs (Bostanian et al., 2005). One drawback in the use of plant essential oils has been that concentrations of oils needed to

ones, to *F. occidentalis.* 

Given the history of insecticide use against western flower thrips, resistance development is more than likely to occur to any insecticide, regardless of mode of action. Therefore, it is critical to develop strategies to employ them effectively. A key element in this regard is proper insecticide resistance management programs to maintain efficacy for as long as possible. In general, insecticide resistance management programs for western flower thrips do not differ conceptually from those designed for other pests. The basic concept is to rotate among insecticides with different modes of action at appropriate intervals to delay or inhibit the evolution of resistance within a pest population.

Most current insecticide resistance management plans recommend rotation of chemical classes after every generation of thrips (Broadbent & Pree, 1997; Herron & Cook, 2002; Robb et al., 1995). In Australia, the initial recommendations for insecticide resistance management were for growers to alternate among insecticides from different chemical classes with each application, a practice recommended in other regions (Funderburk, 2009). However, Herron and Cook (2002) proposed that this simple strategy would not be effective because of the long term persistence of resistance in populations to cypermethrin, a pyrethroid. They argued that reversion to a susceptible population would not occur before an insecticide was used again, rendering that material ineffective. It is also possible that alternating chemical classes too frequently (within a generation) could more readily select for individuals with resistance to multiple insecticides. In a subsequent study, Broughton and Herron (2007) advocated a three-consecutive spray program of a particular insecticide before rotating to a different chemical class. A key component to this strategy was that the three-consecutive applications needed to be made within a single thrips generation to gain the maximum effectiveness for that treatment.

The actual implementation of such an approach is limited by the continuous, overlapping generations present within a crop (Reitz, 2009), and may best be interpreted as rotating chemistries on an appropriate time interval (approximately 3 weeks). Bielza (2008) further cautioned that it is not simply enough to rotate among different chemistries. Rather rotation schedules should be based on known resistance mechanisms to avoid problems with cross

Management Strategies for Western Flower Thrips and the Role of Insecticides 365

larvae on developing tomato fruits, which reduce their aesthetic quality and marketability (Ghidiu et al., 2006; Salguero-Navas et al., 1991). Similar scarring damage from feeding can occur on pepper fruit (Funderburk et al., 2009). However, soon after the invasion of the western flower thrips, epidemics of tomato spotted wilt began to occur throughout the southeast (Csinos et al., 2009) and crops remain at risk if proper management is not employed (Reitz et al., 2008). It is important to note that the most prevalent *Frankliniella*  species in Florida and the southeastern USA are *F. tritici* (Fitch) and *F. bispinosa* (Morgan) (in southern Florida, Hansen et al., 2003), but these species do not cause the damage that

The extensive crop losses caused by western flower thrips and *Tomato spotted wilt virus* have spurred considerable research to develop effective management programs. Understandably though, when western flower thrips and tomato spotted wilt first emerged as problems, tomato and pepper growers in the region responded with intensive insecticide treatments in attempts to prevent disease spread. By the 1990's growers in northern Florida were making an average of 16 separate applications of insecticides, with each application often being a mixture of multiple insecticides (Bauske, 1998). Despite such intense insecticide treatments, attempts at vector control through insecticides did not substantially reduce the problems. This lack of success results from the fact that most virus transmission in these crops is a result of primary spread of the pathogen – that is, infection comes from viruliferous individuals that disperse into the crop from external sources (Gitaitis et al., 1998; Puche et al., 1995). Furthermore, *Tomato spotted wilt virus* transmission occurs in as little as 5 minutes of an adult thrips feeding on a plant (Wijkamp et al., 1996). The following is a description of key pest management tactics that have been developed to successfully manage thrips and *Tomato spotted wilt virus* in open field

Because thrips species vary in their pest status, and because insecticides can differentially impact populations of native flower thrips and the invasive western flower thrips, it is necessary to accurately identify the species in order to make and evaluate management decisions. Thrips can be easily sampled by collecting flower samples into containers with alcohol. These samples can then be examined under a microscope with at least 40X magnification to determine the species. Various identification keys are available to assist with species identifications at this level (e.g., Frantz & Fasulo, n.d.). Periodic sampling can be used to assess shifts in the relative abundance of species of thrips throughout the growing season. When done systematically, this sampling is invaluable for determining the

Sampling for crop management scouting purposes can be accomplished by counting the thrips from samples of ten flowers collected from each of several locations throughout a field. The number of samples needed to collect depends, in part, on field size. Because of the potential for scarring damage to fruit, it is also important to examine small, medium, and large fruits for thrips, with care taken to look under the calyx because of the thigmotactic behavior of thrips. Small fruits especially need to be inspected frequently as the eggs generally are laid during the flower stage, and larvae on the small fruit are the first indication of a developing problem. Again, it is important to sample fruit from several

need for any insecticide application and the effects of such applications.

western flower thrips do in vegetable crops.

fruiting vegetables.

locations in each field.

**5.1 Scouting** 

resistance. An example would be to rotate from chemistries in which metabolic resistance is likely to develop to chemistries in which target site resistance is likely to develop. The more types of resistance modes that can be built into a rotation plan, the more effective each material would be expected to be. Unfortunately in some cropping systems, growers may be faced with having only one or two efficacious classes of insecticides, which increases the risk of resistance development (Broughton & Herron, 2007). Many growers will make applications that are mixtures of more than one insecticides (Cloyd, 2009a). This is done either to combat more than one pest at a particular time, or in the belief that better control of a particular pest can be achieved with mixtures. However, Bielza (2008) also cautioned against using mixtures of insecticides because the structure of western flower thrips populations and resistance mechanisms may actually increase rates of resistance development when mixtures are used.

Bielza (2008) outlined a general resistance management protocol that also serves as a foundation for a sound IPM program. The four recommendations are to: 1) apply insecticides only when required; 2) make accurate and precise insecticide applications; 3) diversify the types of management methods that are used in a crop; and 4) conserve natural enemies. In addition, resistance monitoring needs to be conducted on an ongoing basis so that insecticides can be quickly removed from use before complete failures occur, and so that susceptibility to those materials can be restored. The proper stewardship of insecticide use will help to forestall the development of resistance in western flower thrips populations, as well as populations of other pest species inhabiting particular crops. Even with sound insecticide resistance management programs in effect, it is clear that insecticides cannot function as a stand-alone control method for western flower thrips, and most authors have advocated that insecticides not be used as a stand-alone management tactic. In fact, there cannot be a reliance on any single tactic, and truly integrated management approaches need to be employed.

## **5. Western flower thrips IPM in open field vegetables in the Southeastern USA**

IPM programs developed in Florida for open field vegetable crops are an example of the evolution management programs for the western flower thrips and tomato spotted wilt virus. The development of these IPM programs has relied on a thorough understanding of western flower thrips biology and ecology. Perhaps, the most important aspect for successful management is the recognition that complete control of western flower thrips and elimination of damage is not attainable. Rather, the goal should be to manage thrips within acceptable limits that do not result in economically significant damage, and this goal has become the focus of current western flower thrips management programs (Funderburk, 2009).

Northern Florida, and the rest of the southeastern USA, is a major producer of fresh market tomatoes and peppers, although farms in this region tend to be relatively small (10 – 100 ha) and dispersed throughout the landscape (Bauske, 1998). Vegetable crops in the region are grown on beds covered with plastic mulches (Castro et al., 1993). Crops are started from transplants, with the typical crop growing in the field for 12 – 14 weeks. Tomato and pepper crops in the region did not experience pest problems from thrips until the western flower thrips invaded in the 1980's (Beshear, 1983; Olson & Funderburk, 1986). Initially, damage from western flower thrips was observed from oviposition and direct feeding of adults and larvae on developing tomato fruits, which reduce their aesthetic quality and marketability (Ghidiu et al., 2006; Salguero-Navas et al., 1991). Similar scarring damage from feeding can occur on pepper fruit (Funderburk et al., 2009). However, soon after the invasion of the western flower thrips, epidemics of tomato spotted wilt began to occur throughout the southeast (Csinos et al., 2009) and crops remain at risk if proper management is not employed (Reitz et al., 2008). It is important to note that the most prevalent *Frankliniella*  species in Florida and the southeastern USA are *F. tritici* (Fitch) and *F. bispinosa* (Morgan) (in southern Florida, Hansen et al., 2003), but these species do not cause the damage that western flower thrips do in vegetable crops.

The extensive crop losses caused by western flower thrips and *Tomato spotted wilt virus* have spurred considerable research to develop effective management programs. Understandably though, when western flower thrips and tomato spotted wilt first emerged as problems, tomato and pepper growers in the region responded with intensive insecticide treatments in attempts to prevent disease spread. By the 1990's growers in northern Florida were making an average of 16 separate applications of insecticides, with each application often being a mixture of multiple insecticides (Bauske, 1998). Despite such intense insecticide treatments, attempts at vector control through insecticides did not substantially reduce the problems. This lack of success results from the fact that most virus transmission in these crops is a result of primary spread of the pathogen – that is, infection comes from viruliferous individuals that disperse into the crop from external sources (Gitaitis et al., 1998; Puche et al., 1995). Furthermore, *Tomato spotted wilt virus* transmission occurs in as little as 5 minutes of an adult thrips feeding on a plant (Wijkamp et al., 1996). The following is a description of key pest management tactics that have been developed to successfully manage thrips and *Tomato spotted wilt virus* in open field fruiting vegetables.

#### **5.1 Scouting**

364 Insecticides – Pest Engineering

resistance. An example would be to rotate from chemistries in which metabolic resistance is likely to develop to chemistries in which target site resistance is likely to develop. The more types of resistance modes that can be built into a rotation plan, the more effective each material would be expected to be. Unfortunately in some cropping systems, growers may be faced with having only one or two efficacious classes of insecticides, which increases the risk of resistance development (Broughton & Herron, 2007). Many growers will make applications that are mixtures of more than one insecticides (Cloyd, 2009a). This is done either to combat more than one pest at a particular time, or in the belief that better control of a particular pest can be achieved with mixtures. However, Bielza (2008) also cautioned against using mixtures of insecticides because the structure of western flower thrips populations and resistance mechanisms may actually increase rates of resistance

Bielza (2008) outlined a general resistance management protocol that also serves as a foundation for a sound IPM program. The four recommendations are to: 1) apply insecticides only when required; 2) make accurate and precise insecticide applications; 3) diversify the types of management methods that are used in a crop; and 4) conserve natural enemies. In addition, resistance monitoring needs to be conducted on an ongoing basis so that insecticides can be quickly removed from use before complete failures occur, and so that susceptibility to those materials can be restored. The proper stewardship of insecticide use will help to forestall the development of resistance in western flower thrips populations, as well as populations of other pest species inhabiting particular crops. Even with sound insecticide resistance management programs in effect, it is clear that insecticides cannot function as a stand-alone control method for western flower thrips, and most authors have advocated that insecticides not be used as a stand-alone management tactic. In fact, there cannot be a reliance on any single tactic, and truly integrated management approaches need

**5. Western flower thrips IPM in open field vegetables in the Southeastern** 

IPM programs developed in Florida for open field vegetable crops are an example of the evolution management programs for the western flower thrips and tomato spotted wilt virus. The development of these IPM programs has relied on a thorough understanding of western flower thrips biology and ecology. Perhaps, the most important aspect for successful management is the recognition that complete control of western flower thrips and elimination of damage is not attainable. Rather, the goal should be to manage thrips within acceptable limits that do not result in economically significant damage, and this goal has become the focus of current western flower thrips management programs (Funderburk,

Northern Florida, and the rest of the southeastern USA, is a major producer of fresh market tomatoes and peppers, although farms in this region tend to be relatively small (10 – 100 ha) and dispersed throughout the landscape (Bauske, 1998). Vegetable crops in the region are grown on beds covered with plastic mulches (Castro et al., 1993). Crops are started from transplants, with the typical crop growing in the field for 12 – 14 weeks. Tomato and pepper crops in the region did not experience pest problems from thrips until the western flower thrips invaded in the 1980's (Beshear, 1983; Olson & Funderburk, 1986). Initially, damage from western flower thrips was observed from oviposition and direct feeding of adults and

development when mixtures are used.

to be employed.

**USA** 

2009).

Because thrips species vary in their pest status, and because insecticides can differentially impact populations of native flower thrips and the invasive western flower thrips, it is necessary to accurately identify the species in order to make and evaluate management decisions. Thrips can be easily sampled by collecting flower samples into containers with alcohol. These samples can then be examined under a microscope with at least 40X magnification to determine the species. Various identification keys are available to assist with species identifications at this level (e.g., Frantz & Fasulo, n.d.). Periodic sampling can be used to assess shifts in the relative abundance of species of thrips throughout the growing season. When done systematically, this sampling is invaluable for determining the need for any insecticide application and the effects of such applications.

Sampling for crop management scouting purposes can be accomplished by counting the thrips from samples of ten flowers collected from each of several locations throughout a field. The number of samples needed to collect depends, in part, on field size. Because of the potential for scarring damage to fruit, it is also important to examine small, medium, and large fruits for thrips, with care taken to look under the calyx because of the thigmotactic behavior of thrips. Small fruits especially need to be inspected frequently as the eggs generally are laid during the flower stage, and larvae on the small fruit are the first indication of a developing problem. Again, it is important to sample fruit from several locations in each field.

Management Strategies for Western Flower Thrips and the Role of Insecticides 367

*Orius insidiosus* (Say) colonizes peppers and can effectively suppress thrips populations in the crop (Funderburk et al., 2000; Ramachandran et al., 2001). In particular, *O. insidiosus*  preferentially preys on western flower thrips over the native species *F. tritici* and *F. bispinosa* (Baez et al., 2004; Reitz et al., 2006). Therefore, conservation of these valuable naturally occurring biological control agents can significantly reduce pest problems in pepper and related crops (e.g., eggplant, *Solanum melongena*) and has become a cornerstone of IPM for pepper production (Funderburk et al., 2009). One of the keys to conservation of *Orius*  species is to use insecticides that are minimally toxic to *Orius* spp., whether for thrips management or for management of other pests (Reitz et al., 2003). In contrast to pepper, *Orius* species do not have an affinity for tomato (Baez et al., 2011; Pfannenstiel & Yeargan,

1998), and so this naturally occurring biological control is not available for tomato.

Biotic limitations on western flower thrips populations can come from other species of thrips as well as predators, such as *O. insidiosus*. Recent studies have shown that interspecific competition from native thrips limits the larval survivorship of western flower thrips (Paini et al., 2008). In a survey conducted in northern Florida, Northfield et al. (2008) observed that over 75% of thrips collected from a range of uncultivated hosts were the native species *F. tritici,* with only 1% being western flower thrips. Similar results have been observed in crop fields where two-thirds or more of thrips in untreated or spinosad-treated pepper are the native species. Yet, the demographics differ in pyrethroid treated plots, where western flower thrips predominant (Hansen et al., 2003; Reitz et al., 2003). Because the native species *F. tritici* and *F. bispinosa* do not cause the economic damage that western flower thrips do, and because they outcompete western flower thrips, their conservation contributes to overall pest management. This difference in pest status among the species is why species identifications are an essential component of

Thrips locate host plants primarily through a combination of visual cues, with anthophilous thrips tending to be attracted to colors of flowers. Western flower thrips are attuned to spectral radiation in the ultraviolet range (~365 nm) and in the yellow-green range (~540 nm) (Matteson et al., 1992). The yellow-green sensitivity is thought to play a role in long distance orientation to plants, and the ultraviolet sensitivity is part of the visual system to distinguish flowers. Anthophilous thrips, such as western flower thrips, are attracted to colors of flowers, especially white, blue and yellow flowers with low ultraviolet reflectance (Antignus, 2000). Therefore, increasing the reflectivity in ultraviolet range of the spectrum

The ultraviolet reflective mulches available for the raised-bed plastic mulch production system of Florida are effective in repelling migrating adults of the western flower thrips, and this repellency reduces the primary and secondary spread of tomato spotted wilt. The use of ultraviolet reflective mulch also reduces the influx of the native thrips, *F. tritici* and *F. bispinosa,*  but not disproportionately to reductions in western flower thrips (Momol et al., 2004; Reitz et al., 2003). Ultraviolet reflective mulches are most effective early in the crop season before the plant canopy begins to cover the mulch and reduce the surface area available for reflectance. Application of certain bactericides/fungicides and other pesticides also reduces the ultraviolet reflectance and hence the efficacy of the mulch. A single application of copper and mancozeb

**5.4 Interspecific competition** 

scouting in IPM programs.

can repel thrips.

**5.5 Host plant location and ultraviolet reflective mulches** 

#### **5.2 Economic thresholds**

Economic thresholds have been developed for thrips management in fruiting vegetable crops, including tomato, pepper and eggplant (Funderburk, 2009). These thresholds primarily apply to oviposition damage by female western flower thrips and to the feeding damage caused by adult western flower thrips and larvae of this and other thrips. Consequently, species identification in scouting is critical for the use of thresholds. Thresholds can guide growers for making therapeutic insecticide applications to mitigate these types of damage. However, growers must be aware that therapeutic treatments can do little to mitigate virus transmission. Therefore, preventative tactics must be employed to manage the primary spread of disease in crop fields. Secondary spread may be managed by scouting for larvae and treating, as appropriate.

*Tomato:* Although adults of all *Frankliniella* species that occur in Florida feed on pollen, petals and other floral structures in tomato, this feeding injury does not result in economic damage. However, once feeding by adults of western flower thrips and larvae of all species commences on immature fruits, it can produce "flecking" damage, which becomes apparent as the tomatoes ripen (Ghidiu et al., 2006). Oviposition in developing tomato fruit from western flower thrips also causes aesthetic damage (Salguero-Navas et al., 1991). Whereas 25 adults of native thrips (*F. tritici* and/or *F. bispinosa*) per bloom do not cause damage, one western flower thrips adult per flower is the threshold at which growers need to take action. An average of up to two larvae per small, medium or large fruit can be tolerated, but growers should take action at these thresholds (Funderburk, 2009; Funderburk et al., 2011).

*Pepper and eggplant:* As with tomato, adults of *F. tritici* and *F. bispinosa* cause little, if any, damage to pepper and eggplant, even with densities of 25 per bloom, and they beneficially outcompete western flower thrips and melon thrips, *Thrips palmi* Karny (Funderburk, 2009). Direct feeding damage from adults of western flower thrips and melon thrips is less severe than in tomato, and oviposition in immature pepper and eggplant fruit by western flower thrips does not cause the damage that is typical in tomato and some other crops. Therefore, higher thresholds can be tolerated in pepper and eggplant than in tomato. Up to six (6) western flower thrips and/or melon thrips adults per flower can be tolerated without damage. Once fruits begin to develop, growers need to be aware of scarring damage that adults of the western flower thrips and the melon thrips and larvae of all species may cause. Up to two larvae per small, medium, or large fruit on average in a field are tolerable. Growers should be prepared to take action if larval populations exceed two per fruit (Funderburk 2009). Because of the critical role that *Orius* spp. play in suppressing thrips populations and secondary virus spread, their populations should be monitored in scouting programs, and considered when assessing the need for insecticide treatments.

#### **5.3 Biological control**

Despite certain similarities between crops of tomato and pepper, there are fundamental differences in interactions between thrips and these two plant species. These differences mean that management programs must be designed for each crop. Adult western flower thrips readily colonize pepper and tomato (Baez et al., 2011). Tomato, though, is not a significant reproductive host for western flower thrips (Funderburk, 2009), but pepper can be a good reproductive host for these thrips (van den Meiracker & Ramakers, 1991). Consequently, there is the potential for secondary virus spread from within the crop (Gitaitis et al., 1998). However, Funderburk et al. (2000) demonstrated that the predator *Orius insidiosus* (Say) colonizes peppers and can effectively suppress thrips populations in the crop (Funderburk et al., 2000; Ramachandran et al., 2001). In particular, *O. insidiosus*  preferentially preys on western flower thrips over the native species *F. tritici* and *F. bispinosa* (Baez et al., 2004; Reitz et al., 2006). Therefore, conservation of these valuable naturally occurring biological control agents can significantly reduce pest problems in pepper and related crops (e.g., eggplant, *Solanum melongena*) and has become a cornerstone of IPM for pepper production (Funderburk et al., 2009). One of the keys to conservation of *Orius*  species is to use insecticides that are minimally toxic to *Orius* spp., whether for thrips management or for management of other pests (Reitz et al., 2003). In contrast to pepper, *Orius* species do not have an affinity for tomato (Baez et al., 2011; Pfannenstiel & Yeargan, 1998), and so this naturally occurring biological control is not available for tomato.

#### **5.4 Interspecific competition**

366 Insecticides – Pest Engineering

Economic thresholds have been developed for thrips management in fruiting vegetable crops, including tomato, pepper and eggplant (Funderburk, 2009). These thresholds primarily apply to oviposition damage by female western flower thrips and to the feeding damage caused by adult western flower thrips and larvae of this and other thrips. Consequently, species identification in scouting is critical for the use of thresholds. Thresholds can guide growers for making therapeutic insecticide applications to mitigate these types of damage. However, growers must be aware that therapeutic treatments can do little to mitigate virus transmission. Therefore, preventative tactics must be employed to manage the primary spread of disease in crop fields. Secondary spread may be managed by

*Tomato:* Although adults of all *Frankliniella* species that occur in Florida feed on pollen, petals and other floral structures in tomato, this feeding injury does not result in economic damage. However, once feeding by adults of western flower thrips and larvae of all species commences on immature fruits, it can produce "flecking" damage, which becomes apparent as the tomatoes ripen (Ghidiu et al., 2006). Oviposition in developing tomato fruit from western flower thrips also causes aesthetic damage (Salguero-Navas et al., 1991). Whereas 25 adults of native thrips (*F. tritici* and/or *F. bispinosa*) per bloom do not cause damage, one western flower thrips adult per flower is the threshold at which growers need to take action. An average of up to two larvae per small, medium or large fruit can be tolerated, but growers should take action at these thresholds (Funderburk, 2009; Funderburk et al., 2011). *Pepper and eggplant:* As with tomato, adults of *F. tritici* and *F. bispinosa* cause little, if any, damage to pepper and eggplant, even with densities of 25 per bloom, and they beneficially outcompete western flower thrips and melon thrips, *Thrips palmi* Karny (Funderburk, 2009). Direct feeding damage from adults of western flower thrips and melon thrips is less severe than in tomato, and oviposition in immature pepper and eggplant fruit by western flower thrips does not cause the damage that is typical in tomato and some other crops. Therefore, higher thresholds can be tolerated in pepper and eggplant than in tomato. Up to six (6) western flower thrips and/or melon thrips adults per flower can be tolerated without damage. Once fruits begin to develop, growers need to be aware of scarring damage that adults of the western flower thrips and the melon thrips and larvae of all species may cause. Up to two larvae per small, medium, or large fruit on average in a field are tolerable. Growers should be prepared to take action if larval populations exceed two per fruit (Funderburk 2009). Because of the critical role that *Orius* spp. play in suppressing thrips populations and secondary virus spread, their populations should be monitored in scouting

programs, and considered when assessing the need for insecticide treatments.

Despite certain similarities between crops of tomato and pepper, there are fundamental differences in interactions between thrips and these two plant species. These differences mean that management programs must be designed for each crop. Adult western flower thrips readily colonize pepper and tomato (Baez et al., 2011). Tomato, though, is not a significant reproductive host for western flower thrips (Funderburk, 2009), but pepper can be a good reproductive host for these thrips (van den Meiracker & Ramakers, 1991). Consequently, there is the potential for secondary virus spread from within the crop (Gitaitis et al., 1998). However, Funderburk et al. (2000) demonstrated that the predator

**5.2 Economic thresholds** 

**5.3 Biological control** 

scouting for larvae and treating, as appropriate.

Biotic limitations on western flower thrips populations can come from other species of thrips as well as predators, such as *O. insidiosus*. Recent studies have shown that interspecific competition from native thrips limits the larval survivorship of western flower thrips (Paini et al., 2008). In a survey conducted in northern Florida, Northfield et al. (2008) observed that over 75% of thrips collected from a range of uncultivated hosts were the native species *F. tritici,* with only 1% being western flower thrips. Similar results have been observed in crop fields where two-thirds or more of thrips in untreated or spinosad-treated pepper are the native species. Yet, the demographics differ in pyrethroid treated plots, where western flower thrips predominant (Hansen et al., 2003; Reitz et al., 2003). Because the native species *F. tritici* and *F. bispinosa* do not cause the economic damage that western flower thrips do, and because they outcompete western flower thrips, their conservation contributes to overall pest management. This difference in pest status among the species is why species identifications are an essential component of scouting in IPM programs.

#### **5.5 Host plant location and ultraviolet reflective mulches**

Thrips locate host plants primarily through a combination of visual cues, with anthophilous thrips tending to be attracted to colors of flowers. Western flower thrips are attuned to spectral radiation in the ultraviolet range (~365 nm) and in the yellow-green range (~540 nm) (Matteson et al., 1992). The yellow-green sensitivity is thought to play a role in long distance orientation to plants, and the ultraviolet sensitivity is part of the visual system to distinguish flowers. Anthophilous thrips, such as western flower thrips, are attracted to colors of flowers, especially white, blue and yellow flowers with low ultraviolet reflectance (Antignus, 2000). Therefore, increasing the reflectivity in ultraviolet range of the spectrum can repel thrips.

The ultraviolet reflective mulches available for the raised-bed plastic mulch production system of Florida are effective in repelling migrating adults of the western flower thrips, and this repellency reduces the primary and secondary spread of tomato spotted wilt. The use of ultraviolet reflective mulch also reduces the influx of the native thrips, *F. tritici* and *F. bispinosa,*  but not disproportionately to reductions in western flower thrips (Momol et al., 2004; Reitz et al., 2003). Ultraviolet reflective mulches are most effective early in the crop season before the plant canopy begins to cover the mulch and reduce the surface area available for reflectance. Application of certain bactericides/fungicides and other pesticides also reduces the ultraviolet reflectance and hence the efficacy of the mulch. A single application of copper and mancozeb

Management Strategies for Western Flower Thrips and the Role of Insecticides 369

The single best defense against insect-vectored pathogens is host plant resistance. Numerous cultivars of tomato and pepper are resistant or tolerant to *Tomato spotted wilt virus* are currently commercially available (for a partial listing, see Funderburk et al., 2011). These cultivars have resistance to the virus, but not to thrips feeding or oviposition. All of the commercially available cultivars of tomato share a single source of resistance from the *Sw-5* gene. In pepper, all resistance is conferred by the *Tsw* gene. Both of the *Sw-5* and *Tsw*  genes appear to be single dominant genes (Boiteux & de Avila, 1994; Stevens et al., 1992), and thus susceptible to being compromised by resistance breaking strains of the virus. In fact, such resistance breaking strains have commonly developed around the world (Roselló et al., 1996; Sharman & Persley, 2006). Presently in Florida, tomato spotted wilt resistant cultivars can maintain tomato spotted wilt incidences at economically acceptable levels. However, the threat of epidemics from resistance breaking strains is real. Coupled with the potential damage from western flower thrips feeding and oviposition, growers must

maintain an integrated approach to thrips and tomato spotted wilt management.

production season to prevent scarring damage to fruit.

Insecticides continue to have an important role to play in western flower thrips management. However, the use of insecticides must be done judiciously. Decisions regarding which insecticides to use and when need to be made in the context of both shortterm and long-term management goals. Minimizing resistance development and avoiding the flaring of western flower thrips populations by their release from natural enemies need to be critical factors in insecticide use decisions. Populations of the invasive western flower thrips likely arrived in Florida with resistance to most classes of broad-spectrum insecticides (Immaraju et al., 1992). Further, flaring of the populations of the western flower thrips and other pests is possible when any broad-spectrum synthetic insecticide is used (Funderburk et al., 2000; Reitz et al., 2003). For this reason, growers are encouraged to move to newer, safer, and more selective insecticides in different chemical classes that are becoming available. Although growers are encouraged to use more selective materials when needed, the use of certain organophosphate and carbamate insecticides against western flower thrips may be warranted in certain circumstances. These should only be used in particular instances when nontarget effects would be minimal, for instance near the end of the

As discussed above, the most efficacious insecticides for western flower thrips, at present, are in the spinosyn class. No other insecticide class provides a similar level of effectiveness against western flower thrips. However, as resistance to spinosyns has been documented in Florida and elsewhere, limits are being placed on the number of applications that can be made in each crop to forestall further resistance development. A number of other insecticides are registered or in the process of being registered by the United States Environmental Protection Agency (EPA) that are able to suppress western flower thrips adults and larvae. Lists of currently available insecticides for western flower thrips management and their role in overall IPM programs for fruiting vegetable crops are

The fact that these materials are not as efficacious as spinosyns should not deter their inclusion in IPM programs. The focus of management should not be placed on killing the maximum number of thrips. Rather, the focus of management should be in minimizing damage below economically injurious levels. Because economic damage from oviposition

available (Funderburk, 2009; Funderburk et al., 2011; Funderburk et al., 2009).

**5.8 Host plant resistance** 

**5.9 Insecticides** 

for bacterial or fungal control can reduce the reflectance by nearly 50%. Repeated applications can consequently lead to higher incidences of tomato spotted wilt (S. R. Reitz, unpublished data). Therefore, using alternatives to copper and mancozeb early in the season for foliar pathogen management is advisable.

Ultraviolet reflective mulches also deter other pests, especially whiteflies and aphids, which can vector other plant viruses (Fanigliulo et al., 2009; Stapleton & Summers, 2002; Summers et al., 2010). Consequently, these materials are a good overall IPM tactic to employ where insect vectors are of concern. However, growers need to balance these benefits with the potential delay in plant growth in the spring because these mulches do not warm the soil as readily as standard black plastic mulches (Harpaz, 1982; Maynard & Olson, 2000). Newly developed mulches have helped to mitigate this effect by excluding the reflective metalized layer where transplants are placed.

### **5.6 Host plant fertilization**

Soils in the southeastern USA tend to be nutrient deficient, so that growers need to add up to 200 kg of nitrogen per hectare (the recommended rate for Florida, Olson & Simmonne, 2009). However, growers have often overfertilized crops by up to 70% (Castro et al., 1993). This extra nitrogen fertilization can actually increase densities of western flower thrips. Female western flower thrips, in particular, preferentially settle on plants with higher nitrogen content (Baez et al., 2011; Brodbeck et al., 2001). This association seems to be most closely related to the phenylalanine content of tomato. From a pest management perspective, as vector populations increase with increasing fertilization there is an increase in the incidence of tomato spotted wilt. In north Florida tomatoes, the incidence of tomato spotted wilt was 50% lower for plants grown at recommended nitrogen levels compared with plants grown with supraoptimal nitrogen (Stavisky et al., 2002). Interestingly, *F. tritici*  and *F. bispinosa* do not respond in the same manner to nitrogen fertilization as western flower thrips (Baez et al., 2011; Stavisky et al., 2002). Besides the increasing pest problems, excess nitrogen fertilization does not increase per plant yield. Therefore, growers can improve overall crop production of tomatoes and pepper by maintaining optimal fertilization levels.

#### **5.7 Systemic acquired resistance**

Many plants possess traits for systemic acquired resistance, which are induced defensive mechanisms against pathogens (Sticher et al., 1997). Certain chemicals have been found to stimulate these natural plant defenses against pathogens when applied to plants before infection occurs. Acibenzolar-*S*-methyl is a systemic acquired resistance inducer that stimulates the salicylic acid pathway for disease resistance in tomato and other crops. Commercial formulations of acibenzolar-*S*-methyl have been shown to reduce the incidence of tomato spotted wilt (Momol et al., 2004). Its use has minimal impacts on populations of the flower thrips. When tomatoes are grown on ultraviolet reflective mulches, Momol et al. (2004) concluded that acibenzolar-*S*-methyl provided little additional disease protection because of the large effect of the mulch. Nevertheless, acibenzolar-*S*-methyl is highly effective against bacterial pathogens that afflict tomatoes (Obradovic et al., 2005; Pradhanang et al., 2005), making it an excellent replacement for copper and mancozeb sprays on ultraviolet reflective mulches as well as standard black plastic mulch.

#### **5.8 Host plant resistance**

368 Insecticides – Pest Engineering

for bacterial or fungal control can reduce the reflectance by nearly 50%. Repeated applications can consequently lead to higher incidences of tomato spotted wilt (S. R. Reitz, unpublished data). Therefore, using alternatives to copper and mancozeb early in the season for foliar

Ultraviolet reflective mulches also deter other pests, especially whiteflies and aphids, which can vector other plant viruses (Fanigliulo et al., 2009; Stapleton & Summers, 2002; Summers et al., 2010). Consequently, these materials are a good overall IPM tactic to employ where insect vectors are of concern. However, growers need to balance these benefits with the potential delay in plant growth in the spring because these mulches do not warm the soil as readily as standard black plastic mulches (Harpaz, 1982; Maynard & Olson, 2000). Newly developed mulches have helped to mitigate this effect by excluding the reflective metalized

Soils in the southeastern USA tend to be nutrient deficient, so that growers need to add up to 200 kg of nitrogen per hectare (the recommended rate for Florida, Olson & Simmonne, 2009). However, growers have often overfertilized crops by up to 70% (Castro et al., 1993). This extra nitrogen fertilization can actually increase densities of western flower thrips. Female western flower thrips, in particular, preferentially settle on plants with higher nitrogen content (Baez et al., 2011; Brodbeck et al., 2001). This association seems to be most closely related to the phenylalanine content of tomato. From a pest management perspective, as vector populations increase with increasing fertilization there is an increase in the incidence of tomato spotted wilt. In north Florida tomatoes, the incidence of tomato spotted wilt was 50% lower for plants grown at recommended nitrogen levels compared with plants grown with supraoptimal nitrogen (Stavisky et al., 2002). Interestingly, *F. tritici*  and *F. bispinosa* do not respond in the same manner to nitrogen fertilization as western flower thrips (Baez et al., 2011; Stavisky et al., 2002). Besides the increasing pest problems, excess nitrogen fertilization does not increase per plant yield. Therefore, growers can improve overall crop production of tomatoes and pepper by maintaining optimal

Many plants possess traits for systemic acquired resistance, which are induced defensive mechanisms against pathogens (Sticher et al., 1997). Certain chemicals have been found to stimulate these natural plant defenses against pathogens when applied to plants before infection occurs. Acibenzolar-*S*-methyl is a systemic acquired resistance inducer that stimulates the salicylic acid pathway for disease resistance in tomato and other crops. Commercial formulations of acibenzolar-*S*-methyl have been shown to reduce the incidence of tomato spotted wilt (Momol et al., 2004). Its use has minimal impacts on populations of the flower thrips. When tomatoes are grown on ultraviolet reflective mulches, Momol et al. (2004) concluded that acibenzolar-*S*-methyl provided little additional disease protection because of the large effect of the mulch. Nevertheless, acibenzolar-*S*-methyl is highly effective against bacterial pathogens that afflict tomatoes (Obradovic et al., 2005; Pradhanang et al., 2005), making it an excellent replacement for copper and mancozeb sprays on ultraviolet reflective mulches as well as standard black

pathogen management is advisable.

layer where transplants are placed.

**5.6 Host plant fertilization** 

fertilization levels.

plastic mulch.

**5.7 Systemic acquired resistance** 

The single best defense against insect-vectored pathogens is host plant resistance. Numerous cultivars of tomato and pepper are resistant or tolerant to *Tomato spotted wilt virus* are currently commercially available (for a partial listing, see Funderburk et al., 2011). These cultivars have resistance to the virus, but not to thrips feeding or oviposition. All of the commercially available cultivars of tomato share a single source of resistance from the *Sw-5* gene. In pepper, all resistance is conferred by the *Tsw* gene. Both of the *Sw-5* and *Tsw*  genes appear to be single dominant genes (Boiteux & de Avila, 1994; Stevens et al., 1992), and thus susceptible to being compromised by resistance breaking strains of the virus. In fact, such resistance breaking strains have commonly developed around the world (Roselló et al., 1996; Sharman & Persley, 2006). Presently in Florida, tomato spotted wilt resistant cultivars can maintain tomato spotted wilt incidences at economically acceptable levels. However, the threat of epidemics from resistance breaking strains is real. Coupled with the potential damage from western flower thrips feeding and oviposition, growers must maintain an integrated approach to thrips and tomato spotted wilt management.

#### **5.9 Insecticides**

Insecticides continue to have an important role to play in western flower thrips management. However, the use of insecticides must be done judiciously. Decisions regarding which insecticides to use and when need to be made in the context of both shortterm and long-term management goals. Minimizing resistance development and avoiding the flaring of western flower thrips populations by their release from natural enemies need to be critical factors in insecticide use decisions. Populations of the invasive western flower thrips likely arrived in Florida with resistance to most classes of broad-spectrum insecticides (Immaraju et al., 1992). Further, flaring of the populations of the western flower thrips and other pests is possible when any broad-spectrum synthetic insecticide is used (Funderburk et al., 2000; Reitz et al., 2003). For this reason, growers are encouraged to move to newer, safer, and more selective insecticides in different chemical classes that are becoming available. Although growers are encouraged to use more selective materials when needed, the use of certain organophosphate and carbamate insecticides against western flower thrips may be warranted in certain circumstances. These should only be used in particular instances when nontarget effects would be minimal, for instance near the end of the production season to prevent scarring damage to fruit.

As discussed above, the most efficacious insecticides for western flower thrips, at present, are in the spinosyn class. No other insecticide class provides a similar level of effectiveness against western flower thrips. However, as resistance to spinosyns has been documented in Florida and elsewhere, limits are being placed on the number of applications that can be made in each crop to forestall further resistance development. A number of other insecticides are registered or in the process of being registered by the United States Environmental Protection Agency (EPA) that are able to suppress western flower thrips adults and larvae. Lists of currently available insecticides for western flower thrips management and their role in overall IPM programs for fruiting vegetable crops are available (Funderburk, 2009; Funderburk et al., 2011; Funderburk et al., 2009).

The fact that these materials are not as efficacious as spinosyns should not deter their inclusion in IPM programs. The focus of management should not be placed on killing the maximum number of thrips. Rather, the focus of management should be in minimizing damage below economically injurious levels. Because economic damage from oviposition

Management Strategies for Western Flower Thrips and the Role of Insecticides 371

To facilitate overall crop management, growers are advised to anticipate key pests such as whiteflies or western flower thrips and to employ preventive tactics to minimize their impact. For tomato, one such preventative tactic would ultraviolet reflective mulches. It is also important from an areawide management perspective to maintain crop free periods and remove crops immediately after harvest so that crop residues do not serve as reservoirs for later infestations. In pepper grown in southern Florida, pepper weevil, *Anthonomus eugenii* Cano, is another significant pest. Its management also can be facilitated by crop free periods, and the destruction of crops immediately after harvest. Further sanitation, including the control of solanaceous weeds that serve as alternative hosts helps to reduce future populations. Using such preventative measures would minimize the need for insecticide applications for this pest. Consequently, there would be an overall benefit crop management because many of the available insecticides for pepper weevil management are pyrethroids or other disruptive broad spectrum synthetic insecticides. These are just some of the many pest problems that growers must contend with. Consequently, there is a clear need to integrate management programs for the diverse pests attacking crops. It is also critical to provide growers with realistic economic thresholds for different pests, and proper scouting techniques to assess pest abundances

Only recently has the reliance on insecticides for western flower thrips management been challenged. Yet, failures to control western flower thrips with insecticides have become so severe that Cloyd (2009b) suggested we have reached an impasse in the use of insecticides against western flower thrips in ornamental greenhouse production and diverse management tactics must be employed. The successful management programs for western flower thrips and thrips-vectored viruses developed for solanaceous crops in Florida have been based on an understanding of thrips ecology and how different species interact with different crops (Funderburk, 2009). These strategies involve: an emphasis on scouting and the identification of thrips species present in a crop; the optimal nitrogen fertilization inputs to reduce the attractiveness of crops to western flower thrips without adversely affecting yields; the use of ultraviolet reflective mulches to deter thrips entrance into crop fields; the use of acibenzolar-*S*-methyl to suppress development of tomato spotted wilt symptoms in fruit of susceptible varieties; and the use of economic thresholds to determine the need and timing of insecticide applications; and the use of select insecticides to help suppress reproduction of thrips in the field, and thus manage secondary spread of tomato spotted wilt from within fields. The understanding of the importance of biotic resistance against western flower thrips provided by competing species of native thrips and natural enemies, such as *O. insidiosus,* has been of fundamental importance to improving western flower thrips management. This low input approach helps to avoid other pest problems as well. For example, the conservation biological control program for western flower thrips and *Tomato spotted wilt virus* in peppers has been used by growers in north Florida since the late 1990's, and growers have experienced far fewer problems from whiteflies, aphids, and other pests than when they were following a prophylactic, calendar-based spray program that included

If western flower thrips were the only pest concern for growers, they would, perhaps, feel relieved. However, there are many other insect and pathogen threats to crops. We have now

and the need, if any, to apply pesticides.

**6. Conclusions and future directions** 

frequent use of broad-spectrum insecticides to control pests.

and scarring from feeding only occur at high levels (see above), even limited suppression of western flower thrips adults and larvae can maintain these types of damage well within tolerable limits. Secondary spread of tomato spotted wilt in tomato can also be limited by suppressing populations rather than attempting complete control (Momol et al., 2004). In pepper, conservation of *O. insidiosus* significantly reduces both primary and secondary spread of tomato spotted wilt (Funderburk et al., 2000; Reitz et al., 2003). We have found repeatedly that avoiding treatments that induce outbreaks of western flower thrips populations by killing natural enemies and competing species of native thrips within crop fields is an effective approach to minimizing losses to western flower thrips. Most broadspectrum synthetic insecticides, including pyrethroids, organophosphates, and carbamates kill the native species of thrips that outcompete western flower thrips (Hansen et al., 2003; Reitz et al., 2003; Srivistava et al., 2008), leading to dramatic large scale shifts in thrips demographics (Frantz & Mellinger, 2009). These synthetic broad-spectrum insecticides not only can disrupt western flower thrips management, they also can disrupt management of other pests including spider mites, whiteflies, and leafminers, by eliminating natural enemies of those pests.

#### **5.10 Vertical integration of the management program**

One of the most important keys to successful crop production is not to consider problems in isolation. It is critical to understand how one management tactic may affect other production aspects. For example, in northern Florida, western flower thrips and *Tomato spotted wilt virus* are clearly the most important pest-complex facing tomato and pepper production. The use of ultraviolet reflective mulches has been effective in reducing populations of western flower thrips and the incidence of tomato spotted wilt (e.g., Momol et al., 2004; Reitz et al., 2003). Still, sweetpotato whitefly (*Bemisia tabaci* [Gennadius]) and whitefly-vectored viruses are occasionally important pests in northern Florida tomatoes (Momol et al., 1999). Ultraviolet reflective mulches used to manage thrips and tomato spotted wilt are also efficacious in reducing whitefly-caused damage (Antignus, 2000; Csizinszky et al., 1999).

In contrast, in southern Florida, western flower thrips and tospoviruses have only recently emerged as damaging problems requiring management consideration. Whiteflies and whitefly-vectored viruses have historically been the key insect pest and disease problems in tomato there. Growers use an wide range of insecticides to manage whitefly vectored viruses, particularly *Tomato yellow leaf curl virus*, which can devastate entire crops (Moriones & Navas-Castillo, 2000). Most tomatoes in southern Florida are treated with neonicotinoid insecticides at planting for management of immature whiteflies, with imidacloprid, thiamethoxam, or dinotefuran being most commonly used (Schuster et al., 2010). As the season progresses, growers may rotate "soft" insecticides, such as azadirachtin-based products, microbial insecticides such as *Beauveria bassiana* and insect growth regulators, into management programs against whitefly nymphs. While such materials are compatible with thrips management, they have little effect in suppressing primary virus transmission by whitefly adults that disperse into fields. As a result, growers still place a heavy reliance on broad spectrum insecticides, such as organophosphates and pyrethroids, for management of primary virus spread by whiteflies. The unintended consequence of this approach to whitefly management has been to release populations of western flower thrips from their natural controls, which greatly complicates overall crop management (Frantz & Mellinger, 2009; Weiss et al., 2009).

To facilitate overall crop management, growers are advised to anticipate key pests such as whiteflies or western flower thrips and to employ preventive tactics to minimize their impact. For tomato, one such preventative tactic would ultraviolet reflective mulches. It is also important from an areawide management perspective to maintain crop free periods and remove crops immediately after harvest so that crop residues do not serve as reservoirs for later infestations. In pepper grown in southern Florida, pepper weevil, *Anthonomus eugenii* Cano, is another significant pest. Its management also can be facilitated by crop free periods, and the destruction of crops immediately after harvest. Further sanitation, including the control of solanaceous weeds that serve as alternative hosts helps to reduce future populations. Using such preventative measures would minimize the need for insecticide applications for this pest. Consequently, there would be an overall benefit crop management because many of the available insecticides for pepper weevil management are pyrethroids or other disruptive broad spectrum synthetic insecticides. These are just some of the many pest problems that growers must contend with. Consequently, there is a clear need to integrate management programs for the diverse pests attacking crops. It is also critical to provide growers with realistic economic thresholds for different pests, and proper scouting techniques to assess pest abundances and the need, if any, to apply pesticides.

## **6. Conclusions and future directions**

370 Insecticides – Pest Engineering

and scarring from feeding only occur at high levels (see above), even limited suppression of western flower thrips adults and larvae can maintain these types of damage well within tolerable limits. Secondary spread of tomato spotted wilt in tomato can also be limited by suppressing populations rather than attempting complete control (Momol et al., 2004). In pepper, conservation of *O. insidiosus* significantly reduces both primary and secondary spread of tomato spotted wilt (Funderburk et al., 2000; Reitz et al., 2003). We have found repeatedly that avoiding treatments that induce outbreaks of western flower thrips populations by killing natural enemies and competing species of native thrips within crop fields is an effective approach to minimizing losses to western flower thrips. Most broadspectrum synthetic insecticides, including pyrethroids, organophosphates, and carbamates kill the native species of thrips that outcompete western flower thrips (Hansen et al., 2003; Reitz et al., 2003; Srivistava et al., 2008), leading to dramatic large scale shifts in thrips demographics (Frantz & Mellinger, 2009). These synthetic broad-spectrum insecticides not only can disrupt western flower thrips management, they also can disrupt management of other pests including spider mites, whiteflies, and leafminers, by eliminating natural

One of the most important keys to successful crop production is not to consider problems in isolation. It is critical to understand how one management tactic may affect other production aspects. For example, in northern Florida, western flower thrips and *Tomato spotted wilt virus* are clearly the most important pest-complex facing tomato and pepper production. The use of ultraviolet reflective mulches has been effective in reducing populations of western flower thrips and the incidence of tomato spotted wilt (e.g., Momol et al., 2004; Reitz et al., 2003). Still, sweetpotato whitefly (*Bemisia tabaci* [Gennadius]) and whitefly-vectored viruses are occasionally important pests in northern Florida tomatoes (Momol et al., 1999). Ultraviolet reflective mulches used to manage thrips and tomato spotted wilt are also efficacious in reducing whitefly-caused damage (Antignus, 2000; Csizinszky et al., 1999). In contrast, in southern Florida, western flower thrips and tospoviruses have only recently emerged as damaging problems requiring management consideration. Whiteflies and whitefly-vectored viruses have historically been the key insect pest and disease problems in tomato there. Growers use an wide range of insecticides to manage whitefly vectored viruses, particularly *Tomato yellow leaf curl virus*, which can devastate entire crops (Moriones & Navas-Castillo, 2000). Most tomatoes in southern Florida are treated with neonicotinoid insecticides at planting for management of immature whiteflies, with imidacloprid, thiamethoxam, or dinotefuran being most commonly used (Schuster et al., 2010). As the season progresses, growers may rotate "soft" insecticides, such as azadirachtin-based products, microbial insecticides such as *Beauveria bassiana* and insect growth regulators, into management programs against whitefly nymphs. While such materials are compatible with thrips management, they have little effect in suppressing primary virus transmission by whitefly adults that disperse into fields. As a result, growers still place a heavy reliance on broad spectrum insecticides, such as organophosphates and pyrethroids, for management of primary virus spread by whiteflies. The unintended consequence of this approach to whitefly management has been to release populations of western flower thrips from their natural controls, which greatly complicates overall crop management (Frantz & Mellinger,

enemies of those pests.

2009; Weiss et al., 2009).

**5.10 Vertical integration of the management program** 

Only recently has the reliance on insecticides for western flower thrips management been challenged. Yet, failures to control western flower thrips with insecticides have become so severe that Cloyd (2009b) suggested we have reached an impasse in the use of insecticides against western flower thrips in ornamental greenhouse production and diverse management tactics must be employed. The successful management programs for western flower thrips and thrips-vectored viruses developed for solanaceous crops in Florida have been based on an understanding of thrips ecology and how different species interact with different crops (Funderburk, 2009). These strategies involve: an emphasis on scouting and the identification of thrips species present in a crop; the optimal nitrogen fertilization inputs to reduce the attractiveness of crops to western flower thrips without adversely affecting yields; the use of ultraviolet reflective mulches to deter thrips entrance into crop fields; the use of acibenzolar-*S*-methyl to suppress development of tomato spotted wilt symptoms in fruit of susceptible varieties; and the use of economic thresholds to determine the need and timing of insecticide applications; and the use of select insecticides to help suppress reproduction of thrips in the field, and thus manage secondary spread of tomato spotted wilt from within fields. The understanding of the importance of biotic resistance against western flower thrips provided by competing species of native thrips and natural enemies, such as *O. insidiosus,* has been of fundamental importance to improving western flower thrips management. This low input approach helps to avoid other pest problems as well. For example, the conservation biological control program for western flower thrips and *Tomato spotted wilt virus* in peppers has been used by growers in north Florida since the late 1990's, and growers have experienced far fewer problems from whiteflies, aphids, and other pests than when they were following a prophylactic, calendar-based spray program that included frequent use of broad-spectrum insecticides to control pests.

If western flower thrips were the only pest concern for growers, they would, perhaps, feel relieved. However, there are many other insect and pathogen threats to crops. We have now

Management Strategies for Western Flower Thrips and the Role of Insecticides 373

have improved this manuscript. We are especially indebted to Laurence Mound (CSIRO) for many invaluable discussions regarding thrips taxonomy, biology and ecology. We recognize the contributions of Larry Pedigo (Professor Emeritus, Iowa State University) to the theory and practice of integrated pest management generally and to stressing the importance of

Antignus, Y. (2000) Manipulation of wavelength-dependent behaviour of insects: An IPM

Antignus, Y., Mor, N., Joseph, R.B., Lapidot, M., & Cohen, S. (1996) Ultraviolet-absorbing

Aramburu, J., & Marti, M. (2003) The occurrence in north-east Spain of a variant of Tomato

Baez, I., Reitz, S.R., & Funderburk, J.E. (2004) Predation by *Orius insidiosus* (Heteroptera:

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Tubulifera). *Journal of Georgia Entomological Society* 18: 342-344.

insects. *Environmental Entomology* 25: 919-924.

containing the Sw-5 gene. *Plant Pathology* 52: 407.

tool to impede insects and restrict epidemics of insect-borne viruses. *Virus Research*

plastic sheets protect crops from insect pests and from virus diseases vectored by

spotted wilt virus (TSWV) that breaks resistance in tomato *(Lycopersicon esculentum)* 

Anthocoridae) on species and life stages of *Frankliniella* flower thrips (Thysanoptera: Thripidae) in pepper flowers. *Environmental Entomology* 33: 662-670.

understanding pest status in particular.

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**8. References** 

71: 213-220.

begun to appreciate how management of one pest can interact with and affect management of other pests. Therefore, the need for sustainable, truly integrated pest management programs that do not consider individual pests in isolation is clear. There must be a focus on pest complexes rather than considering individual pest species. For example, insecticide resistance can be exacerbated when western flower thrips are exposed to insecticide treatments aimed at other pests (Immaraju et al., 1992).

The development of more selective insecticides that do not adversely affect natural enemies will improve overall crop management. These materials would reduce the risk of releasing non-target pests from control when treatments are necessary for another pest. Additional management tools that would reduce the need for insecticide applications will continue to be beneficial. Improved host plant resistance is one of those areas. The recent development of cultivars of tomatoes and peppers that show some degree of resistance to *Tomato spotted wilt virus* has eased concerns among growers. However, because the resistance conferred by the *Sw-5* gene in tomato and the *Tsw* gene in pepper is not durable, resistance-breaking strains can readily develop (Aramburu & Marti, 2003; Ciuffo et al., 2005; Latham & Jones, 1998; Margaria et al., 2004; Roggero et al., 2002; Thomas-Carroll & Jones, 2003; Thompson & van Zijl, 1995). In addition, virus resistance does not protect against physical damage. Therefore, it is still important for growers to maintain a multifaceted integrated management program.

Currently there are major research efforts underway to identify new germplasm sources of resistance to *Tomato spotted wilt virus*. One promising source of resistance appears to be the *Sw-7* gene from *Solanum chilense* (Price et al., 2007). Although this resistance is another single gene dominant trait, it is not linked to the *Sw-5* gene. Another exciting prospect for the future of host plant resistance is the identification of germplasm that is resistant to western flower thrips. Several accessions of different *Capsicum* species have been identified that have significant resistance against western flower thrips feeding (Maharijaya et al., 2011). If these traits can be incorporated into commercial cultivars, they may offer some degree of protection against virus transmission and aesthetic damage.

The management of western flower thrips will continue to be an ongoing challenge. Management programs cannot be static as they will need to be continually refined and updated with the advent of new invasive pests or as other conditions change. For example, *Scirtothrips dorsalis* Hood invaded Florida within the past five years and poses a threat to crops including pepper and eggplant. Recently, a new form of *Tospovirus,* a genetic reassortant of *Groundnut ringspot virus* and *Tomato chlorotic spot virus* has been found in Florida (Webster et al., 2010). Although western flower thrips can transmit this virus (Webster et al., 2011), it is not yet known if it or another thrips species is the most important vector. Despite these challenges, we believe that western flower thrips can be successfully managed, given a thorough understanding of its ecology and pest status.

## **7. Acknowledgments**

We appreciate the collaborative efforts of Steve Olson, Pete Andersen, Brent Brodbeck, Julianne Stavisky, Mrittunjai Srivistava, M. Timur Momol, and Norm Leppla (University of Florida), Anthony Weiss and Jim Dripps (Dow AgroSciences), Charles Mellinger and Galen Frantz (Glades Crop Care), and Scott Adkins (USDA-ARS) for their insights and contributions to these research projects and to the management programs that have developed from this research. We appreciate the comments by Katherine Luhring, which have improved this manuscript. We are especially indebted to Laurence Mound (CSIRO) for many invaluable discussions regarding thrips taxonomy, biology and ecology. We recognize the contributions of Larry Pedigo (Professor Emeritus, Iowa State University) to the theory and practice of integrated pest management generally and to stressing the importance of understanding pest status in particular.

## **8. References**

372 Insecticides – Pest Engineering

begun to appreciate how management of one pest can interact with and affect management of other pests. Therefore, the need for sustainable, truly integrated pest management programs that do not consider individual pests in isolation is clear. There must be a focus on pest complexes rather than considering individual pest species. For example, insecticide resistance can be exacerbated when western flower thrips are exposed to insecticide

The development of more selective insecticides that do not adversely affect natural enemies will improve overall crop management. These materials would reduce the risk of releasing non-target pests from control when treatments are necessary for another pest. Additional management tools that would reduce the need for insecticide applications will continue to be beneficial. Improved host plant resistance is one of those areas. The recent development of cultivars of tomatoes and peppers that show some degree of resistance to *Tomato spotted wilt virus* has eased concerns among growers. However, because the resistance conferred by the *Sw-5* gene in tomato and the *Tsw* gene in pepper is not durable, resistance-breaking strains can readily develop (Aramburu & Marti, 2003; Ciuffo et al., 2005; Latham & Jones, 1998; Margaria et al., 2004; Roggero et al., 2002; Thomas-Carroll & Jones, 2003; Thompson & van Zijl, 1995). In addition, virus resistance does not protect against physical damage. Therefore, it is still important for growers to maintain a multifaceted integrated

Currently there are major research efforts underway to identify new germplasm sources of resistance to *Tomato spotted wilt virus*. One promising source of resistance appears to be the *Sw-7* gene from *Solanum chilense* (Price et al., 2007). Although this resistance is another single gene dominant trait, it is not linked to the *Sw-5* gene. Another exciting prospect for the future of host plant resistance is the identification of germplasm that is resistant to western flower thrips. Several accessions of different *Capsicum* species have been identified that have significant resistance against western flower thrips feeding (Maharijaya et al., 2011). If these traits can be incorporated into commercial cultivars, they may offer some degree of

The management of western flower thrips will continue to be an ongoing challenge. Management programs cannot be static as they will need to be continually refined and updated with the advent of new invasive pests or as other conditions change. For example, *Scirtothrips dorsalis* Hood invaded Florida within the past five years and poses a threat to crops including pepper and eggplant. Recently, a new form of *Tospovirus,* a genetic reassortant of *Groundnut ringspot virus* and *Tomato chlorotic spot virus* has been found in Florida (Webster et al., 2010). Although western flower thrips can transmit this virus (Webster et al., 2011), it is not yet known if it or another thrips species is the most important vector. Despite these challenges, we believe that western flower thrips can be successfully

We appreciate the collaborative efforts of Steve Olson, Pete Andersen, Brent Brodbeck, Julianne Stavisky, Mrittunjai Srivistava, M. Timur Momol, and Norm Leppla (University of Florida), Anthony Weiss and Jim Dripps (Dow AgroSciences), Charles Mellinger and Galen Frantz (Glades Crop Care), and Scott Adkins (USDA-ARS) for their insights and contributions to these research projects and to the management programs that have developed from this research. We appreciate the comments by Katherine Luhring, which

treatments aimed at other pests (Immaraju et al., 1992).

protection against virus transmission and aesthetic damage.

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management program.

**7. Acknowledgments** 


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**17** 

*Italy* 

Stefano Civolani

**The Past and Present of Pear Protection** 

*Department of Biology and Evolution, University of Ferrara, Ferrara,* 

**Against the Pear Psylla,** *Cacopsylla pyri* **L.** 

*Agricultural Foundation "Fratelli Navarra", Malborghetto di Boara (Ferrara),* 

The pear psylla, *Cacopsylla pyri* L. (Hemiptera Psyllidae), is known in Europe for its extended infestations which may cause heavy economical losses to most pear growing regions. Pear (*Pyrus sp.* L.) is the second most relevant fruit species of temperate regions: the first one is apple (*Malus domestica* L.) and the third one is peach (*Prunus persica* L.). The top three world regions for pear production are China, Europe and North America. Total European pear production is presently stable around 2.6 million tones, according to 2010 data from the Fruit and Vegetable Service Center (CSO, Ferrara, Italy). In Europe, Italy and Spain are the largest producers, respectively with 35% and 20% of the total production. Pear production in France is decreasing (8%), mainly as a result of fireblight on "Passe Crassane", a highly susceptible variety. Until 2007, in the Netherlands and Belgium there was an increase in production with extensive planting of the "Conference" cultivar, but the first signs of saturation of the "Conference" market started to appear in 2007. The total pear

All commercial varieties in Europe belong to the species *Pyrus communis* L. and they are all susceptible to *C. pyri*. The susceptibility increases when orchard techniques are aimed to maximize fruit production, such as the high density of plants per hectare, the large use of

The damages that *C. pyri* may induce to pear trees are classified in two main types: 1) direct damages, weakening the plant by subtraction of nutrients; when the pest attack is intense, the plant wastes away with reduced production; 2) indirect damages, due to the production of a large amount of honeydew on which sooty molds develop (russetting fruits, Fig. 2), and

In the first case the most damaging stages are the nymphs of all instars because of the high amount of honeydew (produced especially in spring and summer) dripping on everything including fruits. Besides lowering the fruit market value, honeydew favours the growth of sooty molds caused by saprophytic fungi, in turn causing indirect injury to the plant. Sooty molds actually induce alterations of photosynthesis, disruption of metabolism, leaf curling and premature loss, together with lower production. Prolonged attacks and intense

production of the Netherlands and Belgium presently amounts to 9%.

fertilizers and the intense irrigation (Fig. 1).

also to the possible transmission of phytoplasms (Fig. 3).

weakening by *C. pyri* may lead to plant death.

**2. Damages caused by** *C. pyri*

**1. Introduction** 


## **The Past and Present of Pear Protection Against the Pear Psylla,** *Cacopsylla pyri* **L.**

## Stefano Civolani

*Department of Biology and Evolution, University of Ferrara, Ferrara, Agricultural Foundation "Fratelli Navarra", Malborghetto di Boara (Ferrara), Italy* 

## **1. Introduction**

384 Insecticides – Pest Engineering

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laboratory strains of western flower thrips (Thysanoptera: Thripidae). *Journal of* 

The pear psylla, *Cacopsylla pyri* L. (Hemiptera Psyllidae), is known in Europe for its extended infestations which may cause heavy economical losses to most pear growing regions. Pear (*Pyrus sp.* L.) is the second most relevant fruit species of temperate regions: the first one is apple (*Malus domestica* L.) and the third one is peach (*Prunus persica* L.). The top three world regions for pear production are China, Europe and North America. Total European pear production is presently stable around 2.6 million tones, according to 2010 data from the Fruit and Vegetable Service Center (CSO, Ferrara, Italy). In Europe, Italy and Spain are the largest producers, respectively with 35% and 20% of the total production. Pear production in France is decreasing (8%), mainly as a result of fireblight on "Passe Crassane", a highly susceptible variety. Until 2007, in the Netherlands and Belgium there was an increase in production with extensive planting of the "Conference" cultivar, but the first signs of saturation of the "Conference" market started to appear in 2007. The total pear production of the Netherlands and Belgium presently amounts to 9%.

All commercial varieties in Europe belong to the species *Pyrus communis* L. and they are all susceptible to *C. pyri*. The susceptibility increases when orchard techniques are aimed to maximize fruit production, such as the high density of plants per hectare, the large use of fertilizers and the intense irrigation (Fig. 1).

## **2. Damages caused by** *C. pyri*

The damages that *C. pyri* may induce to pear trees are classified in two main types: 1) direct damages, weakening the plant by subtraction of nutrients; when the pest attack is intense, the plant wastes away with reduced production; 2) indirect damages, due to the production of a large amount of honeydew on which sooty molds develop (russetting fruits, Fig. 2), and also to the possible transmission of phytoplasms (Fig. 3).

In the first case the most damaging stages are the nymphs of all instars because of the high amount of honeydew (produced especially in spring and summer) dripping on everything including fruits. Besides lowering the fruit market value, honeydew favours the growth of sooty molds caused by saprophytic fungi, in turn causing indirect injury to the plant. Sooty molds actually induce alterations of photosynthesis, disruption of metabolism, leaf curling and premature loss, together with lower production. Prolonged attacks and intense weakening by *C. pyri* may lead to plant death.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 387

**3. The key to improve** *C. pyri* **control is the detailed knowledge of its life cycle**  In Europe *C. pyri* shows 4-5 generations per year and overwinters as adult in reproductive diapause. The adult winter forms appear at the beginning of September (Fig. 4) (Civolani & Pasqualini, 2003) and overwinter individually or in small groups sheltered in cracks of the

Fig. 2. Damages to fruits due to sooty molds.

Fig. 3. Damages caused by phytoplasmas.

Fig. 1. Pear orchard with high plant density.

Unlike nymphs, adults of *C. pyri* (similarly to those of the congener species *C. pyricola*) are responsible for the transmission of the phytoplasma of "pear decline". The phytoplasma is acquired by the psylla upon feeding on an infected plant and transmitted to an healthy one by salivation during feeding on phloem (Carraro *et al*., 1998). The transmission mechanism is persistent-propagative because the phytoplasm reproduces in the insect body. The acquisition and inoculation of the pathogen requires for the vector insect to feed for 1-2 hours on phloem of infected plants. A latency period in the vector (about 1-2 weeks) follows the acquisition of phytoplasma in which the pathogen circulates and multiplies until it reaches the salivary glands. The first symptom of the disease appears during the summerautumn period, when the leaves exhibit a red-purple hue contrasting with the yellow-green hue of senescent leaves of healthy plants. The leaves also have a stiff lamina with the edges curled upside and the apex folded downwards, and they fall prematurely beginning from the apical ones. In the next spring the infected trees show smaller, light green leaves with upward edges ("transparent canopy"). In some cases a sudden wilting is observed: although still on shoots, leaves become brown and dry. The tree may die within few days or weeks. Some authors (Giunchedi & Refatti, 1997; Davies *et al*., 1998) showed that some plants undergo recovering during the winter dormancy because of degradation of aboveground phloem elements (Schaper & Seemüller, 1982). Moreover, the quinces *Cydonia oblonga* Mill used as rootstocks rarely allow the phyoplasma survival between vegetative cycles, unlike *P. communis* rootstocks which allow phytoplasma reproduction within roots in winter and in the next spring, and the following spreading of the pathogen in aboveground phloem elements. Therefore in pear trees grafted to quince rootstocks the phytoplasma population, in absence of reinfection from overwintering psylla adults, may progressively decrease, disappearing within some years.

Fig. 2. Damages to fruits due to sooty molds.

Unlike nymphs, adults of *C. pyri* (similarly to those of the congener species *C. pyricola*) are responsible for the transmission of the phytoplasma of "pear decline". The phytoplasma is acquired by the psylla upon feeding on an infected plant and transmitted to an healthy one by salivation during feeding on phloem (Carraro *et al*., 1998). The transmission mechanism is persistent-propagative because the phytoplasm reproduces in the insect body. The acquisition and inoculation of the pathogen requires for the vector insect to feed for 1-2 hours on phloem of infected plants. A latency period in the vector (about 1-2 weeks) follows the acquisition of phytoplasma in which the pathogen circulates and multiplies until it reaches the salivary glands. The first symptom of the disease appears during the summerautumn period, when the leaves exhibit a red-purple hue contrasting with the yellow-green hue of senescent leaves of healthy plants. The leaves also have a stiff lamina with the edges curled upside and the apex folded downwards, and they fall prematurely beginning from the apical ones. In the next spring the infected trees show smaller, light green leaves with upward edges ("transparent canopy"). In some cases a sudden wilting is observed: although still on shoots, leaves become brown and dry. The tree may die within few days or weeks. Some authors (Giunchedi & Refatti, 1997; Davies *et al*., 1998) showed that some plants undergo recovering during the winter dormancy because of degradation of aboveground phloem elements (Schaper & Seemüller, 1982). Moreover, the quinces *Cydonia oblonga* Mill used as rootstocks rarely allow the phyoplasma survival between vegetative cycles, unlike *P. communis* rootstocks which allow phytoplasma reproduction within roots in winter and in the next spring, and the following spreading of the pathogen in aboveground phloem elements. Therefore in pear trees grafted to quince rootstocks the phytoplasma population, in absence of reinfection from overwintering psylla adults, may progressively decrease,

Fig. 1. Pear orchard with high plant density.

disappearing within some years.

Fig. 3. Damages caused by phytoplasmas.

## **3. The key to improve** *C. pyri* **control is the detailed knowledge of its life cycle**

In Europe *C. pyri* shows 4-5 generations per year and overwinters as adult in reproductive diapause. The adult winter forms appear at the beginning of September (Fig. 4) (Civolani & Pasqualini, 2003) and overwinter individually or in small groups sheltered in cracks of the

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 389

From eggs laid in winter months, simultaneously to bud opening, the first instar nymphs emerge, infesting the new vegetation. Later, in April-May, the adult summer forms appear (Fig. 5) whose females lay a large number of eggs (Fig. 6) hatching in the second half of May. The first instar (Fig. 7) and late instar nymphs (Fig. 8) live on the shoots, excreting a large amount of honeydew responsible for heavy damages to the plant. The next generations overlap with all developmental stages until autumn. Aestivation is also observed during the

summer months.

Fig. 6. *C. pyri* eggs.

Fig. 7. Newly hatched *C. pyri* nymphs

tree bark, at branch crossing or at the base of buds. As soon as the weather conditions improve, the winter forms leave their shelter and reach the new apical leaves. Here they puncture the buds with their stylets, sucking and excreting fecal droplets which accumulate near the proctiger. In winter the adult male produces active sperms stored in the spermatheca and is therefore ready to mate and inseminate the female. However, the eggs can be fertilized only when oocytes reach maturity (Bonnemaison *et al*., 1956). In January all females are mature, ready to mate and lay eggs. The main factor affecting the physiology of ovopository apparatus is temperature, which must be higher than 10° C for two consecutive days (thermal quiescence) (Nguyen, 1975).

Fig. 4. *C. pyri* adult winter form.

Fig. 5. *C. pyri* adult summer form.

From eggs laid in winter months, simultaneously to bud opening, the first instar nymphs emerge, infesting the new vegetation. Later, in April-May, the adult summer forms appear (Fig. 5) whose females lay a large number of eggs (Fig. 6) hatching in the second half of May. The first instar (Fig. 7) and late instar nymphs (Fig. 8) live on the shoots, excreting a large amount of honeydew responsible for heavy damages to the plant. The next generations overlap with all developmental stages until autumn. Aestivation is also observed during the summer months.

Fig. 6. *C. pyri* eggs.

388 Insecticides – Pest Engineering

tree bark, at branch crossing or at the base of buds. As soon as the weather conditions improve, the winter forms leave their shelter and reach the new apical leaves. Here they puncture the buds with their stylets, sucking and excreting fecal droplets which accumulate near the proctiger. In winter the adult male produces active sperms stored in the spermatheca and is therefore ready to mate and inseminate the female. However, the eggs can be fertilized only when oocytes reach maturity (Bonnemaison *et al*., 1956). In January all females are mature, ready to mate and lay eggs. The main factor affecting the physiology of ovopository apparatus is temperature, which must be higher than 10° C for two consecutive

days (thermal quiescence) (Nguyen, 1975).

Fig. 4. *C. pyri* adult winter form.

Fig. 5. *C. pyri* adult summer form.

Fig. 7. Newly hatched *C. pyri* nymphs

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 391

This treatment, performed at leaf fall, requires pesticides active against adult winter forms such as those belonging to the pyrethroid family (the same principles are also used in late winter against the same generation). These pesticides are completely non-selective, therefore dangerous for the beneficial insects: for this reason the treatment must be performed not too early (in October) when most individuals of *Anthocoris nemoralis* F. (Hemiptera Anthocoridae) are still active on the pear trees, but only at complete leaf fall (late November or early December), when *A. nemoralis* populations have already found shelter in bark crevices while *C. pyri* adult winter forms are still active on plants (Fig. 4) (Civolani, 2000;

Synthetic pyrethroids exhibit a very high abatement-contacticide action on psylla wintering adults. Table 1 shows the results of tests with the active ingredient ciflutrin: the abatement activity on adults is such that no egg deposition occurs in the next spring. Notwithstanding the good results that may be obtained by pyrethroid treatment in autumn (and also in late winter, as shown below) these strategies, common in France on *C. pyri* and in North America on *C. pyricola*, in the Italian pear growing regions are discouraged: here the pest population, after an initial sharp decline, soon recovers and increases again in May, reaching the economic threshold for spring-summer treatments. This event could be explained by the faster recovery of the pest in spring because the natural control by its predator *A. nemoralis* is limited. Moreover, considering only the pesticide efficiency, the results are strictly dependent on seasonal conditions. Indeed, if an early frost occurs at the beginning of autumn, most psylla adults take shelter early and survive to the late autumn treatments. Given the need to preserve auxiliary insect populations, an untimely autumn treatment with broad spectrum pyrethroids could be very dangerous for them and therefore is strongly discouraged in relevant pear growing areas such as Emilia-Romagna (Northern Italy). Experimental tests have been performed to evaluate other active principles such as mineral oils and neonicotinoids. According to tests performed in 2001-2002 in the province of Ferrara (Emilia-Romagna), good results were obtained with mineral oil alone or associated to imidacloprid (Table 1) (Civolani, 2000), although for auxiliary insect preservation the use of neonicotinoids is not suggested against *C. pyri* in European pear growing areas, unlike

North America where is found in field control guidelines against *C. pyricola*.

untreated control - 170 mineral oil 29 November 1999 30

ciflutrin 29 November 1999 0 mineral oil 3 March 2000 53

ciflutrin 3 March 2000 69

mineral oil + imidacloprid 29 November 1999 12

mineral oil + imidacloprid 3 March 2000 65

This treatment is presently based on broad spectrum pyrethroids whose purpose is to break down the population of females emerging from winter shelters and about to lay eggs.

Table 1. Results of a field test with a pyrethroid and mineral oil with or without a

Treatment date Eggs per 100 bubs

**4.1 Autumn treatment** 

Civolani & Pasqualini, 2003).

neonicotinoid (Civolani, 2000).

**4.2 Late winter treatment** 

Fig. 8. *C. pyri* late instar nymphs.

## **4.** *C. pyri* **Integrated pest management**

In Europe this pest was considered of secondary importance until 1960, but its population rapidly increased simultaneously to heavy use of pesticides in pear orchards and intensive agriculture. The need to control pear infestations required the repeated use of different pesticides and several control strategies, but in most cases the efficiency of control progressively decreased, first because of the great adaptability and survival of the insect to the active ingredients, and second because of the negative effects on beneficial insects caused by excessive use of non-selective toxic ingredients. In this way the psylla population freely increased, leading to conditions in which the pest was hard or impossible to control.

Presently the defence against *C. pyri* is mainly based on integrated pest management (IPM), supported by natural control aimed to equilibrate the complex biological relationships of the field community. First of all, *C. pyri* control must follow IPM guidelines to optimize the activity of natural control agents and to reduce the chances of developing pesticide resistance by insect populations.

Among the basic strategies there are the "good agricultural practice" (GAP) techniques, such as reducing excessive plant growth and avoiding overstimulation by fertilizers or incorrect pruning. Also the general strategies to control other pest species may play a relevant role in psylla development and population increase. For example, the technique of mating disruption and the use of granulosis virus (CpGV) employed to control *Cydia pomonella* L. (Lepidoptera Tortricidae), favours the activity of entomophagous fauna by reducing the impact of synthetic products on the orchard, and may also help to reduce the treatments against *C. pyri.* However, in the last decade the chemical control has been the key defence strategy against the pear psylla in intensive pear orchards.

The main strategies of chemical control against *C. pyri* performed in the last 20 years in integrated and conventional farms are listed below. Each strategy has advantages and disadvantages, therefore its efficiency depends both on the active ingredients employed and the weather conditions at the time of treatment.

## **4.1 Autumn treatment**

390 Insecticides – Pest Engineering

In Europe this pest was considered of secondary importance until 1960, but its population rapidly increased simultaneously to heavy use of pesticides in pear orchards and intensive agriculture. The need to control pear infestations required the repeated use of different pesticides and several control strategies, but in most cases the efficiency of control progressively decreased, first because of the great adaptability and survival of the insect to the active ingredients, and second because of the negative effects on beneficial insects caused by excessive use of non-selective toxic ingredients. In this way the psylla population freely increased, leading to conditions in which the pest was hard or impossible to control. Presently the defence against *C. pyri* is mainly based on integrated pest management (IPM), supported by natural control aimed to equilibrate the complex biological relationships of the field community. First of all, *C. pyri* control must follow IPM guidelines to optimize the activity of natural control agents and to reduce the chances of developing pesticide

Among the basic strategies there are the "good agricultural practice" (GAP) techniques, such as reducing excessive plant growth and avoiding overstimulation by fertilizers or incorrect pruning. Also the general strategies to control other pest species may play a relevant role in psylla development and population increase. For example, the technique of mating disruption and the use of granulosis virus (CpGV) employed to control *Cydia pomonella* L. (Lepidoptera Tortricidae), favours the activity of entomophagous fauna by reducing the impact of synthetic products on the orchard, and may also help to reduce the treatments against *C. pyri.* However, in the last decade the chemical control has been the key

The main strategies of chemical control against *C. pyri* performed in the last 20 years in integrated and conventional farms are listed below. Each strategy has advantages and disadvantages, therefore its efficiency depends both on the active ingredients employed and

defence strategy against the pear psylla in intensive pear orchards.

the weather conditions at the time of treatment.

Fig. 8. *C. pyri* late instar nymphs.

resistance by insect populations.

**4.** *C. pyri* **Integrated pest management** 

This treatment, performed at leaf fall, requires pesticides active against adult winter forms such as those belonging to the pyrethroid family (the same principles are also used in late winter against the same generation). These pesticides are completely non-selective, therefore dangerous for the beneficial insects: for this reason the treatment must be performed not too early (in October) when most individuals of *Anthocoris nemoralis* F. (Hemiptera Anthocoridae) are still active on the pear trees, but only at complete leaf fall (late November or early December), when *A. nemoralis* populations have already found shelter in bark crevices while *C. pyri* adult winter forms are still active on plants (Fig. 4) (Civolani, 2000; Civolani & Pasqualini, 2003).

Synthetic pyrethroids exhibit a very high abatement-contacticide action on psylla wintering adults. Table 1 shows the results of tests with the active ingredient ciflutrin: the abatement activity on adults is such that no egg deposition occurs in the next spring. Notwithstanding the good results that may be obtained by pyrethroid treatment in autumn (and also in late winter, as shown below) these strategies, common in France on *C. pyri* and in North America on *C. pyricola*, in the Italian pear growing regions are discouraged: here the pest population, after an initial sharp decline, soon recovers and increases again in May, reaching the economic threshold for spring-summer treatments. This event could be explained by the faster recovery of the pest in spring because the natural control by its predator *A. nemoralis* is limited. Moreover, considering only the pesticide efficiency, the results are strictly dependent on seasonal conditions. Indeed, if an early frost occurs at the beginning of autumn, most psylla adults take shelter early and survive to the late autumn treatments. Given the need to preserve auxiliary insect populations, an untimely autumn treatment with broad spectrum pyrethroids could be very dangerous for them and therefore is strongly discouraged in relevant pear growing areas such as Emilia-Romagna (Northern Italy).

Experimental tests have been performed to evaluate other active principles such as mineral oils and neonicotinoids. According to tests performed in 2001-2002 in the province of Ferrara (Emilia-Romagna), good results were obtained with mineral oil alone or associated to imidacloprid (Table 1) (Civolani, 2000), although for auxiliary insect preservation the use of neonicotinoids is not suggested against *C. pyri* in European pear growing areas, unlike North America where is found in field control guidelines against *C. pyricola*.


Table 1. Results of a field test with a pyrethroid and mineral oil with or without a neonicotinoid (Civolani, 2000).

#### **4.2 Late winter treatment**

This treatment is presently based on broad spectrum pyrethroids whose purpose is to break down the population of females emerging from winter shelters and about to lay eggs.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 393

Fig. 9. Pear trees treated with kaolin in late winter.

Fig. 10. *C. pyri* adult with body soiled by kaolin.

Formerly the highly toxic 4,6-dinitro-*o*-cresol (DNOC) was used, until finally banned in 1999. With a biological activity extended up to 15 days, DNOC was characterized by a strong contact activity especially towards eggs newly laid or about to be laid (Barbieri et al., 1986; Pollini et al., 1992). On pear psylla, the pyrethroids are exclusively active on adults through contact associated to an anti-feeding effect. The pyrethroid treatment is generally performed at pear bud swelling stage or at the latest when they break. The eggs are laid by *C. pyri* at the end of the thermal quiescent period, corresponding to a maximum temperature above 10° C for two consecutive days. The late winter strategy considerably lowers the amount of initial psylla population on pear trees, therefore the first generation is extremely reduced.

Concerning the pesticide activity, the best results of this strategy are obtained after a mild winter, because in these conditions almost all adults leave their shelters at the time of treatment. On the contrary, frost waves at the end of winter interrupt and delay the emergence of adults, reducing the efficiency of the treatment.

The control strategy based on late winter treatments with pyrethroids was recently disputed first because of the toxicity of the active ingredients involved, but mostly because the sharp reduction of the psylla first generation could starve the anthocorids, interfering with their settlement during early plant growth in spring. To avoid the side effects of pyrethroids, in the last ten years alternative solutions to synthetic pesticides have been repeatedly tested against the overwintering generations, and among them kaolin and some oily compounds.

Kaolin, a white, non-abrasive, fine-grained allumosilicate mineral that is purified and sized so that it can be easily dispersed in water, creates a mineral barrier on plants that prevents oviposition and insect feeding (Fig. 9 and Fig. 10) (Puterka et al., 2000). The efficiency of kaolin is shown by the results obtained in two experimental trials in 2001 and 2002 (Pasqualini et al., 2003). These authors found that a double treatment with kaolin affected egg laying of overwintering *C. pyri* by hindering their anchorage on the leaf surface and inhibiting host-plant acceptance (Table 2). It was also found that the body and wings of some adults became soiled, making insects less mobile and preventing them from reaching the laying site (host location) on plants. Indeed, the kaolin-treated plants were almost completely free of nymphs (Table 2). Due to the high mobility of *C. pyri* adults, the effects of kaolin treatment on the summer population were not assessed in this small plot trial. A larger plot trial was therefore performed in 2004 and again kaolin showed a very high control efficiency (Daniel et al., 2006). At the end of June 2004 some *C. pyri* individuals were observed in the kaolin treated plot, but the population density tended to be lower than that in the plot treated by a standard spirodiclofen strategy. In kaolin treated plants *C. pyri* was kept under the economic threshold until harvest (Daniel et al., 2006), therefore it could be an interesting alternative control strategy for this pest in organic and IPM orchards.

Some oily compounds could also be used in this period to interfere with egg deposition by *C. pyri* adults. It has been known from a long time that mineral oils and oily compounds could have negative effects on psylla egg deposition (Zwick & Westigard, 1978). A Turkish researcher (Erler, 2004) tested four types of oils, namely cotton seed oil, neem oil, fish-liver oil and summer oil, observing a delay in egg laying of about four weeks for fish-liver oil and summer oil, but of only one or two weeks for cotton seed oil and neem oil, probably depending on the stability of the oily material in open field. Other field trials aimed to interfere with egg laying by *C. pyri* adult winter forms were performed in 2001 and 2002 in Italy with pure mineral oil ("dormant oil"), obtaining a good reduction of the number of eggs laid (Pasqualini et al., 2003) (Table 2).

Fig. 9. Pear trees treated with kaolin in late winter.

Formerly the highly toxic 4,6-dinitro-*o*-cresol (DNOC) was used, until finally banned in 1999. With a biological activity extended up to 15 days, DNOC was characterized by a strong contact activity especially towards eggs newly laid or about to be laid (Barbieri et al., 1986; Pollini et al., 1992). On pear psylla, the pyrethroids are exclusively active on adults through contact associated to an anti-feeding effect. The pyrethroid treatment is generally performed at pear bud swelling stage or at the latest when they break. The eggs are laid by *C. pyri* at the end of the thermal quiescent period, corresponding to a maximum temperature above 10° C for two consecutive days. The late winter strategy considerably lowers the amount of initial psylla population on pear trees, therefore the first generation is extremely

Concerning the pesticide activity, the best results of this strategy are obtained after a mild winter, because in these conditions almost all adults leave their shelters at the time of treatment. On the contrary, frost waves at the end of winter interrupt and delay the

The control strategy based on late winter treatments with pyrethroids was recently disputed first because of the toxicity of the active ingredients involved, but mostly because the sharp reduction of the psylla first generation could starve the anthocorids, interfering with their settlement during early plant growth in spring. To avoid the side effects of pyrethroids, in the last ten years alternative solutions to synthetic pesticides have been repeatedly tested against the overwintering generations, and among them kaolin and some oily compounds. Kaolin, a white, non-abrasive, fine-grained allumosilicate mineral that is purified and sized so that it can be easily dispersed in water, creates a mineral barrier on plants that prevents oviposition and insect feeding (Fig. 9 and Fig. 10) (Puterka et al., 2000). The efficiency of kaolin is shown by the results obtained in two experimental trials in 2001 and 2002 (Pasqualini et al., 2003). These authors found that a double treatment with kaolin affected egg laying of overwintering *C. pyri* by hindering their anchorage on the leaf surface and inhibiting host-plant acceptance (Table 2). It was also found that the body and wings of some adults became soiled, making insects less mobile and preventing them from reaching the laying site (host location) on plants. Indeed, the kaolin-treated plants were almost completely free of nymphs (Table 2). Due to the high mobility of *C. pyri* adults, the effects of kaolin treatment on the summer population were not assessed in this small plot trial. A larger plot trial was therefore performed in 2004 and again kaolin showed a very high control efficiency (Daniel et al., 2006). At the end of June 2004 some *C. pyri* individuals were observed in the kaolin treated plot, but the population density tended to be lower than that in the plot treated by a standard spirodiclofen strategy. In kaolin treated plants *C. pyri* was kept under the economic threshold until harvest (Daniel et al., 2006), therefore it could be an

interesting alternative control strategy for this pest in organic and IPM orchards.

eggs laid (Pasqualini et al., 2003) (Table 2).

Some oily compounds could also be used in this period to interfere with egg deposition by *C. pyri* adults. It has been known from a long time that mineral oils and oily compounds could have negative effects on psylla egg deposition (Zwick & Westigard, 1978). A Turkish researcher (Erler, 2004) tested four types of oils, namely cotton seed oil, neem oil, fish-liver oil and summer oil, observing a delay in egg laying of about four weeks for fish-liver oil and summer oil, but of only one or two weeks for cotton seed oil and neem oil, probably depending on the stability of the oily material in open field. Other field trials aimed to interfere with egg laying by *C. pyri* adult winter forms were performed in 2001 and 2002 in Italy with pure mineral oil ("dormant oil"), obtaining a good reduction of the number of

emergence of adults, reducing the efficiency of the treatment.

reduced.

Fig. 10. *C. pyri* adult with body soiled by kaolin.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 395

mechanism of action which interferes with biosynthesis of lipids in the target arthropods. The original mechanism of action of spirodiclofen plays a key practical role by allowing successful rotation strategies with abamectin to limit the risks of occurrence of resistance in *C. pyri* control. The best activity of spirodiclofen occurs when it is targeted on yellow eggs some days before the hatching of first instar nymphs: the active ingredient shows instead a decreasing activity with the advancing of the psylla developmental stages (Table 4). At the stage of yellow eggs the activity of spirodiclofen is improved by addition of mineral oil (Table 4) and the use of spirodiclofen followed by treatment with mineral oil (1000 ml/hl) may represent a good alternative to the treatment with abamectin in presence of high and prolonged infestations. In conditions of low-medium pressure by *C. pyri*, one treatment with spirodiclofen may be enough: the few individuals escaping the treatment may be easily captured by anthocorids, given the good selectivity of the product towards these valuable auxiliary insects (Pasqualini & Civolani, 2007). However, the efficiency of spirodiclofen is often lower than that of abamectin

(Table 4) (Pasqualini & Civolani 2007; Boselli & Cristiani 2008; Marčić et al., 2009).

moth *C. pomonella*.

occurrence of resistance in *C. pyri* control.

of sugars with fatty acids. (Puterka et al., 2003).

of populations of natural psylla antagonists.

Other active ingredients have been employed on both *C. pyri* in Europe and *C. pyricola* in North America. For example, in *C. pyricola* the differences of abamectin efficiency observed in the field suggested to employ and recommend neonicotinoids in pear IPM programs, among which imidacloprid, introduced in 1995, thiametoxan in 2001, acetamiprid in 2002 and thiacloprid in 2004. Besides *C. pyricola*, the last ingredient is used mainly for codling

Presently a new active ingredient, spirotetramat, a lipid biosynthesis inhibitor similar to the tetronic acid derivate spirodiclofen, is under investigation in Europe (Nauen et al., 2008) but already commercially available in North America. Due to its mode of action spirotetramat is especially effective against juvenile stages of sucking pests, psyllid included. In the case of female adults the compound significantly reduces fertility and consequently insect populations. Spirotetramat also exhibits unique translocation properties: after foliar uptake the insecticidal activity is translocated within the entire vascular system. This property allows the protection of new shoots or leaves appearing after foliar application (Nauen et al., 2008): given the high efficiency on *C. pyri* (unpublished data) this active principle could represent a future valuable alternative to abamectin in order to manage the risks of

It is also possible to control nymphs by simply washing the trees with high amounts of water to which insecticidal soaps (fatty acids salts) are added to remove the honeydew (Briolini et al., 1989). Recently some other products have been used, similar to liquid glue and able to trap by a physical mechanism small and scarcely active insects such as almost all juvenile instars of *C. pyri.* These products are synthetic sugar esters (sucrose octanoate) and represent a relatively new class of insecticidal compounds that are produced by the reaction

After discussing the active ingredients that could be used against *C. pyri* and the different strategies that could be employed, once again it must be emphasized that an efficient control of *C. pyri* infestations could be obtained by an integrated pest management of the pear orchard which allows a balanced growth of plants and simultaneously favours the growth

During the spring growth period a key point is to protect the useful psylla predators, first of all the most important one, *A. nemoralis*. The populations of this anthocorid are low in early spring but increase in the second half of June, insuring the protection of pear trees until

harvesting and providing the most relevant contribution against *C. pyri*.


Table 2. Results of two late winter field trials with kaolin and mineral oil (Pasqualini et al., 2003).

## **4.3 Spring-summer treatments**

The treatments against summer generations can be performed towards eggs or nymphs. In the first case chitin inhibitors such as hexaflumuron (banned in 2004), triflumuron (banned in 2009), diflubenzuron and teflubenzuron have been employed. These active ingredients are used against second generation eggs, usually laid in the first decade of May. The treatment is usually performed against *C. pomonella* but shows a secondary effect on psylla. The chitin inhibitors provide the best results on *C. pyri* when they are applied on newly laid eggs (white eggs) or on eggs laid in a short time after the treatment. However, no activity is clearly exerted on eggs laid on the new shoots unreached by the treatment. Some authors observed that chitin-inhibitors show control effects similar to those of specific psyllicides described below: this could be explained by the absence of side effects on *C. pyri* natural predator, *A. nemoralis* (Souliotis & Moschos, 2008). However, the most relevant treatment employed against *C. pyri* in past and present times is the one against juvenile stages. This control strategy is base on specific synthetic active ingredients which are often acaricides, such as amitraz (commercially released in 1975 and banned in 2005), abamectin (commercially released in Italy in 1996) and spirodiclofen (commercially released in Italy in 2007), although in the past generic organophosphorates have been used such as monocrotofos and azinphos methyl.

Abamectin is presently the basic pesticide employed against *C. pyri*. Is should be briefly recalled that abamectin belongs to the chemical family of avermectins, compounds produced by the soil bacterium *Streptomyces avermitilis* (Lasota & Dybas, 1991). The activity of abamectin is mainly directed against young nymphs and secondarily against adults. The best results are therefore obtained when yellow eggs are mostly present and when the hatching peak, that could interfere with the pesticide activity because of honeydew, has not yet achieved (Pasqualini & Civolani, 2006). The product is not systemic but translaminary: the addition of mineral oil improves its penetration and after 24 hours no traces of the compound are found on leaf surface. One treatment timely performed against the second generation often represents the final solution, considering the high activity of the principle in comparison to amitraz (Table 3).

Spirodiclofen, commercially available since 2007, is the first member of a new chemical family, that of the tetronic acids (Nauen et al., 2000; Nauen, 2005), characterized by a new and original

Untreated control - 136.75 6

mineral oil (dormant oil) 18 February. 30 2 

Untreated control - 77.75 7.5 kaolin 11 and 19 February 0 0 mineral oil 11 February 12 3.5 Table 2. Results of two late winter field trials with kaolin and mineral oil (Pasqualini et al.,

The treatments against summer generations can be performed towards eggs or nymphs. In the first case chitin inhibitors such as hexaflumuron (banned in 2004), triflumuron (banned in 2009), diflubenzuron and teflubenzuron have been employed. These active ingredients are used against second generation eggs, usually laid in the first decade of May. The treatment is usually performed against *C. pomonella* but shows a secondary effect on psylla. The chitin inhibitors provide the best results on *C. pyri* when they are applied on newly laid eggs (white eggs) or on eggs laid in a short time after the treatment. However, no activity is clearly exerted on eggs laid on the new shoots unreached by the treatment. Some authors observed that chitin-inhibitors show control effects similar to those of specific psyllicides described below: this could be explained by the absence of side effects on *C. pyri* natural predator, *A. nemoralis* (Souliotis & Moschos, 2008). However, the most relevant treatment employed against *C. pyri* in past and present times is the one against juvenile stages. This control strategy is base on specific synthetic active ingredients which are often acaricides, such as amitraz (commercially released in 1975 and banned in 2005), abamectin (commercially released in Italy in 1996) and spirodiclofen (commercially released in Italy in 2007), although in the past generic organophosphorates have been used such as

Abamectin is presently the basic pesticide employed against *C. pyri*. Is should be briefly recalled that abamectin belongs to the chemical family of avermectins, compounds produced by the soil bacterium *Streptomyces avermitilis* (Lasota & Dybas, 1991). The activity of abamectin is mainly directed against young nymphs and secondarily against adults. The best results are therefore obtained when yellow eggs are mostly present and when the hatching peak, that could interfere with the pesticide activity because of honeydew, has not yet achieved (Pasqualini & Civolani, 2006). The product is not systemic but translaminary: the addition of mineral oil improves its penetration and after 24 hours no traces of the compound are found on leaf surface. One treatment timely performed against the second generation often represents the final solution, considering the high activity of the principle

Spirodiclofen, commercially available since 2007, is the first member of a new chemical family, that of the tetronic acids (Nauen et al., 2000; Nauen, 2005), characterized by a new and original

kaolin 18 February and 10

First trial 2001

Second trial 2002

**4.3 Spring-summer treatments** 

monocrotofos and azinphos methyl.

in comparison to amitraz (Table 3).

2003).

Treatment date Eggs per 100

bubs

March 1 0.25

Nimphs per 100 flowers

mechanism of action which interferes with biosynthesis of lipids in the target arthropods. The original mechanism of action of spirodiclofen plays a key practical role by allowing successful rotation strategies with abamectin to limit the risks of occurrence of resistance in *C. pyri* control. The best activity of spirodiclofen occurs when it is targeted on yellow eggs some days before the hatching of first instar nymphs: the active ingredient shows instead a decreasing activity with the advancing of the psylla developmental stages (Table 4). At the stage of yellow eggs the activity of spirodiclofen is improved by addition of mineral oil (Table 4) and the use of spirodiclofen followed by treatment with mineral oil (1000 ml/hl) may represent a good alternative to the treatment with abamectin in presence of high and prolonged infestations. In conditions of low-medium pressure by *C. pyri*, one treatment with spirodiclofen may be enough: the few individuals escaping the treatment may be easily captured by anthocorids, given the good selectivity of the product towards these valuable auxiliary insects (Pasqualini & Civolani, 2007). However, the efficiency of spirodiclofen is often lower than that of abamectin (Table 4) (Pasqualini & Civolani 2007; Boselli & Cristiani 2008; Marčić et al., 2009).

Other active ingredients have been employed on both *C. pyri* in Europe and *C. pyricola* in North America. For example, in *C. pyricola* the differences of abamectin efficiency observed in the field suggested to employ and recommend neonicotinoids in pear IPM programs, among which imidacloprid, introduced in 1995, thiametoxan in 2001, acetamiprid in 2002 and thiacloprid in 2004. Besides *C. pyricola*, the last ingredient is used mainly for codling moth *C. pomonella*.

Presently a new active ingredient, spirotetramat, a lipid biosynthesis inhibitor similar to the tetronic acid derivate spirodiclofen, is under investigation in Europe (Nauen et al., 2008) but already commercially available in North America. Due to its mode of action spirotetramat is especially effective against juvenile stages of sucking pests, psyllid included. In the case of female adults the compound significantly reduces fertility and consequently insect populations. Spirotetramat also exhibits unique translocation properties: after foliar uptake the insecticidal activity is translocated within the entire vascular system. This property allows the protection of new shoots or leaves appearing after foliar application (Nauen et al., 2008): given the high efficiency on *C. pyri* (unpublished data) this active principle could represent a future valuable alternative to abamectin in order to manage the risks of occurrence of resistance in *C. pyri* control.

It is also possible to control nymphs by simply washing the trees with high amounts of water to which insecticidal soaps (fatty acids salts) are added to remove the honeydew (Briolini et al., 1989). Recently some other products have been used, similar to liquid glue and able to trap by a physical mechanism small and scarcely active insects such as almost all juvenile instars of *C. pyri.* These products are synthetic sugar esters (sucrose octanoate) and represent a relatively new class of insecticidal compounds that are produced by the reaction of sugars with fatty acids. (Puterka et al., 2003).

After discussing the active ingredients that could be used against *C. pyri* and the different strategies that could be employed, once again it must be emphasized that an efficient control of *C. pyri* infestations could be obtained by an integrated pest management of the pear orchard which allows a balanced growth of plants and simultaneously favours the growth of populations of natural psylla antagonists.

During the spring growth period a key point is to protect the useful psylla predators, first of all the most important one, *A. nemoralis*. The populations of this anthocorid are low in early spring but increase in the second half of June, insuring the protection of pear trees until harvesting and providing the most relevant contribution against *C. pyri*.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 397

untreated control - 83.25 spirodiclofen 19 May (yellow eggs) 36.25 spirodiclofen + mineral oil 19 May (yellow eggs) 15.50 abamectin + mineral oil 19 May (yellow eggs) 0.75

untreated control - 25 spirodiclofen 30 April (white eggs) 6 spirodiclofen 14 May (yellow eggs) 11 spirodiclofen 17 May (first hatching) 19

amitraz 14 May (yellow eggs) 2

untreated control - 66.5 spirodiclofen 27 April 1.5

abamectin 27 April 0.5

spirodiclofen and abamectin 27 April and 9 May 0 spirodiclofen and mineral oil 27 April and 9 May 0.6

Abamectin and spirodiclofen 27 April and 9 May 0 Table 4. Results of three field trials with spirodiclofen on three pear tree varieties (Pasqualini

ingredient were the enhanced activity of cytochrome P450 monooxygenase (MFO) and also the changes in acetyl cholinesterase, since the susceptibility to the pesticide could not be fully recovered by pretreating *in vivo* the adults with piperonylbutoxide (PBO), a specific

In 1994 in the Avignon region (France) the survey for *C. pyri* resistance was extended to 16 active ingredients belonging to five pesticide families, by topical laboratory tests on adults (Fig. 11). The tests showed that the resistance rates (RR) were extremely low for the family of carbamates (one- to 2.4-fold), relatively low for the family of pyrethroids (4.7- to 6.2-fold). For the family of organophosphorates insecticides, the resistance rates among the active ingredients were very different: lower than one (0.2-fold) for parathion-methyl, low for mevinphos and malathion (3.5- and 2.5-fold, respectively), and higher for chlorpyriphosethyl (10.2-fold), monocrotophos (26-fold), azinphos-methyl (62.2-fold) and phosmet (179.7 fold). In the same Avignon area, tests on resistance selection were performed in laboratory with the organophosphorate azinphos-methyl, which was frequently used in high amounts against *C. pomonella* and could indirectly cause selection also in *C. pyri*. The RR observed ranged from 10 to 40-fold in comparison to wild populations: these values were

spirodiclofen 22 May (20-30 %

First trial (2005, on the variety

Second trial (2002, on the variety "Abbé

Third trial (2007, on the variety "Beurré

& Civolani, 2007; Boselli & Cristiani, 2008).

inhibitor of MFO (Berrada *et al*., 1994).

"Conference")

Fétel")

Bosc")

Treatment date Nymphs per

hatching) <sup>18</sup>

shoot

Another aspect that should not be overlooked in *C. pyri* control is the relevant effect of weather conditions on pest populations. A late winter season with mild temperatures favours an early and fast emergence of adults from their winter shelters and a regular egg laying, while the late winter frosts interrupt the adult emergence and egg laying, producing a gradual development of first and second pest generation which interferes with the precise timing of treatments. Cold and rainy periods during blossoming and petal fall interfere with nymph spreading on plants: in this case the nymphs often crowded in the flower calyx, sometimes causing with their feeding activity russet blotches or young fruit drop. On the contrary, high summer temperatures tend to block psylla development because of high egg mortality and slowing of juvenile growth for a long period.


Table 3. Results of three field trials with abamectin and amitraz on two pear varieties (Pasqualini & Civolani, 2006).

## **5. Evolution of resistance of** *C. pyri* **to pesticides**

As for all phytophagous pests, also for *C. pyri* the repeated use of chemical active ingredients causes the development of resistance. However, in Europe there are less resistance cases documented for *C. pyri* in comparison to those known since 1960 for *C. pyricola* in North America (Harries & Burts, 1965). Among the *C. pyri* resistance events in Europe the best known involve organophosphorates, pyrethroids and chitin inhibitors: in all documented cases a sharp decrease in pesticide activity was observed even after a few years of use. The active ingredient monocrotofos represents the best known case (Berrada *et al*., 1995). The selection induced by this pesticide around the end of 1980 on some *C. pyri* populations near Toulouse (France) caused an increased resistance up to 140 fold in comparison to the susceptible strain in 30 generations, as shown by laboratory tests. Further tests showed that the mechanisms involved in the onset of resistance to this active

Another aspect that should not be overlooked in *C. pyri* control is the relevant effect of weather conditions on pest populations. A late winter season with mild temperatures favours an early and fast emergence of adults from their winter shelters and a regular egg laying, while the late winter frosts interrupt the adult emergence and egg laying, producing a gradual development of first and second pest generation which interferes with the precise timing of treatments. Cold and rainy periods during blossoming and petal fall interfere with nymph spreading on plants: in this case the nymphs often crowded in the flower calyx, sometimes causing with their feeding activity russet blotches or young fruit drop. On the contrary, high summer temperatures tend to block psylla development because of high egg

> untreated control - 16.68 abamectin 6 May 0.15 amitraz 10 May 1.08

> untreated control - 46.88 abamectin 6 May 2.23 amitraz 10 May 8.18

> untreated control - 8.29 abamectin 6 May 0.98 amitraz 10 May 1.39

Table 3. Results of three field trials with abamectin and amitraz on two pear varieties

As for all phytophagous pests, also for *C. pyri* the repeated use of chemical active ingredients causes the development of resistance. However, in Europe there are less resistance cases documented for *C. pyri* in comparison to those known since 1960 for *C. pyricola* in North America (Harries & Burts, 1965). Among the *C. pyri* resistance events in Europe the best known involve organophosphorates, pyrethroids and chitin inhibitors: in all documented cases a sharp decrease in pesticide activity was observed even after a few years of use. The active ingredient monocrotofos represents the best known case (Berrada *et al*., 1995). The selection induced by this pesticide around the end of 1980 on some *C. pyri* populations near Toulouse (France) caused an increased resistance up to 140 fold in comparison to the susceptible strain in 30 generations, as shown by laboratory tests. Further tests showed that the mechanisms involved in the onset of resistance to this active

Treatment date Nymphs per

shoot

mortality and slowing of juvenile growth for a long period.

**5. Evolution of resistance of** *C. pyri* **to pesticides** 

First trial (2000, on the variety

Second trial (2000, on the variety

Third trial (2004, on the variety

(Pasqualini & Civolani, 2006).

"Conference")

"William")

"Conference")


Table 4. Results of three field trials with spirodiclofen on three pear tree varieties (Pasqualini & Civolani, 2007; Boselli & Cristiani, 2008).

ingredient were the enhanced activity of cytochrome P450 monooxygenase (MFO) and also the changes in acetyl cholinesterase, since the susceptibility to the pesticide could not be fully recovered by pretreating *in vivo* the adults with piperonylbutoxide (PBO), a specific inhibitor of MFO (Berrada *et al*., 1994).

In 1994 in the Avignon region (France) the survey for *C. pyri* resistance was extended to 16 active ingredients belonging to five pesticide families, by topical laboratory tests on adults (Fig. 11). The tests showed that the resistance rates (RR) were extremely low for the family of carbamates (one- to 2.4-fold), relatively low for the family of pyrethroids (4.7- to 6.2-fold). For the family of organophosphorates insecticides, the resistance rates among the active ingredients were very different: lower than one (0.2-fold) for parathion-methyl, low for mevinphos and malathion (3.5- and 2.5-fold, respectively), and higher for chlorpyriphosethyl (10.2-fold), monocrotophos (26-fold), azinphos-methyl (62.2-fold) and phosmet (179.7 fold). In the same Avignon area, tests on resistance selection were performed in laboratory with the organophosphorate azinphos-methyl, which was frequently used in high amounts against *C. pomonella* and could indirectly cause selection also in *C. pyri*. The RR observed ranged from 10 to 40-fold in comparison to wild populations: these values were

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 399

Following the sudden outbreak of *C. pyri* populations in some fruit growing area in Emilia-Romagna in 2005, abamectin tests for resistance were performed for the first time only on overwintering adults (Fig. 11), but no resistance was detected, although LC50 and LC90

Besides adults (Table 5) (Fig. 11), in 2007 and 2008 the abamectin tests in Emilia-Romagna were extended also to eggs and nymphs (Fig. 12 and Fig. 13), the stage target in *C. pyri* field treatments (Civolani et al., 2010). On adults, the abamectin topical tests performed in autumn 2007 and 2008 did not show significant differences among all populations tested, but the LC50 values were apparently related to the adult collection dates, as previously

Fig. 11. Topical application of the insecticide solution on *C. pyri* adult winter form with

The egg spray test (LC50 and LC90) did not show relevant differences, although LC50 and LC90 values were always lower in the organic farm than in all others (Table 6). The results of leaf dip tests performed on young and old larvae were generally similar to the egg spray

Overall, the tests data indicate that no apparent resistance to abamectin has been developed

In 2009 and 2010 new cases of loss of efficiency of abamectin reappeared in some orchards in the province of Modena (Emilia-Romagna). In 2010 other tests were again performed on a *C. pyri* population which underwent 7 abamectin field treatment in 2009. However, in this case also the results of LC50 and LC90 did not show significant differences and the RR was just

From 2004 to 2006 tests were carried out also in Spain to monitor the evolution of susceptibility to abamectin in orchards where the number of treatments changed from less than two until 2005 to an average of three after the ban on amitraz, with some orchards

values appeared related to the time of adult field collection (Civolani et al., 2007).

reported (Civolani et al., 2010).

hand-held manual micro-applicator.

up to now in *C. pyri* populations of Emilia-Romagna.

ones (Civolani et al., 2010).

above 2 (unpublished data).

considerably lower than those reached by monocrotofos (Bues *et al*., 2000). Further tests showed that the selection by azinphos-methyl produced a strong cross resistance with phosmet and monocrotofos (155 fold). By the same tests no cross resistance was shown for azinphos-methyl with amitraz, pyrethroids, carbamates and other organophosphorate pesticides such as chlorpyriphos and mevinphos (Bues *et al*., 2000). The same authors started genetic studies by crossing the resistant and susceptible strains and showed that the resistance was autosomically inherited and semi-dominant in expression (Bues *et al*., 2000). They also advanced by backcross the hypothesis that the resistance factor was monogenic. However, the result that the resistance to azinphos-methyl is semi-dominant is different from what previously obtained in Oregon by Van de Baan (1988) on pyrethroids: this author crossed two populations of *C. pyricola*, one susceptible and the other 240-fold resistant to the active ingredient fenvalerate (a pyrethroid), showing that the resistance was in this case semi-recessive (Van de Baan, 1988). Probably there are different mechanisms involved in the resistance to different pesticide families.

As mentioned before, the pyrethroids are another pesticide family largely employed in France against *C. pyri* and in North America against *C. pyricola* during leaf fall and late winter, before egg laying by overwintering adults. In laboratory topical tests on overwintering adults, some of these active ingredients showed very variable RR: for example, in tests performed in Avignon in 1994 the observed RR were 4 or 6-fold higher in comparison to the susceptible laboratory population (Buès *et al*., 1999), but further studies in 1996 in the same southern Rhone valley, on a field population collected in Pont Saint Esprit, showed that the RR was 42.9-fold higher (Buès *et al*., 1999). Later, the same authors confirmed similarly high resistance values to the active ingredient deltametrin in some Southern France populations in which the RR was 30-fold, always with the adult winter forms less susceptible in comparison to the same stage of the adult summer forms (Buès et al., 2003).

Concerning the mechanism of action producing resistance to pyrethroids, laboratory tests showed that the addition of PBO caused an recovery of pyrethroid efficiency almost complete: this shows that other mechanisms of induction of resistance could be secondarily involved.

Therefore the detoxifying enzyme MFO is involved in the mechanism of action of both organophosphorate and pyrethroid active ingredients.

Around 1995, in a region of West Switzerland a lower susceptibility was observed to teflubenzuron, active ingredient belonging to the family of chitin inhibitor (Schaub *et al*., 1996). More recently this resistance to teflubenzuron was also observed in the Czech Republic, according to tests performed in 2004 and 2005 (Kocourek & Stará, 2006). The mechanisms of resistance to teflubenzuron have not yet been completely investigated.

As previously mentioned, abamectin is the most efficient and most used active ingredient against *C. pyri*: it was used against this pest for the first time in 1996, with only one spring treatment and only in some orchards, also because amitraz was an alternative until 2005, the year in which the ingredient was banned. Probably after the ban on this active ingredient the number of treatments per orchard and the area of employment were increased: this trend was also observed in the fruit growing area of Lleida, Girona and Huesca in Spain (Miarnau et al., 2010), and Ferrara and Modena in Italy. The current high selection pressure with this active ingredient, repeatedly applied over both geographical areas could induce selection for resistance.

considerably lower than those reached by monocrotofos (Bues *et al*., 2000). Further tests showed that the selection by azinphos-methyl produced a strong cross resistance with phosmet and monocrotofos (155 fold). By the same tests no cross resistance was shown for azinphos-methyl with amitraz, pyrethroids, carbamates and other organophosphorate pesticides such as chlorpyriphos and mevinphos (Bues *et al*., 2000). The same authors started genetic studies by crossing the resistant and susceptible strains and showed that the resistance was autosomically inherited and semi-dominant in expression (Bues *et al*., 2000). They also advanced by backcross the hypothesis that the resistance factor was monogenic. However, the result that the resistance to azinphos-methyl is semi-dominant is different from what previously obtained in Oregon by Van de Baan (1988) on pyrethroids: this author crossed two populations of *C. pyricola*, one susceptible and the other 240-fold resistant to the active ingredient fenvalerate (a pyrethroid), showing that the resistance was in this case semi-recessive (Van de Baan, 1988). Probably there are different mechanisms involved in the

As mentioned before, the pyrethroids are another pesticide family largely employed in France against *C. pyri* and in North America against *C. pyricola* during leaf fall and late winter, before egg laying by overwintering adults. In laboratory topical tests on overwintering adults, some of these active ingredients showed very variable RR: for example, in tests performed in Avignon in 1994 the observed RR were 4 or 6-fold higher in comparison to the susceptible laboratory population (Buès *et al*., 1999), but further studies in 1996 in the same southern Rhone valley, on a field population collected in Pont Saint Esprit, showed that the RR was 42.9-fold higher (Buès *et al*., 1999). Later, the same authors confirmed similarly high resistance values to the active ingredient deltametrin in some Southern France populations in which the RR was 30-fold, always with the adult winter forms less susceptible in comparison to the same stage of the adult summer forms (Buès et

Concerning the mechanism of action producing resistance to pyrethroids, laboratory tests showed that the addition of PBO caused an recovery of pyrethroid efficiency almost complete: this shows that other mechanisms of induction of resistance could be secondarily

Therefore the detoxifying enzyme MFO is involved in the mechanism of action of both

Around 1995, in a region of West Switzerland a lower susceptibility was observed to teflubenzuron, active ingredient belonging to the family of chitin inhibitor (Schaub *et al*., 1996). More recently this resistance to teflubenzuron was also observed in the Czech Republic, according to tests performed in 2004 and 2005 (Kocourek & Stará, 2006). The mechanisms of resistance to teflubenzuron have not yet been completely investigated. As previously mentioned, abamectin is the most efficient and most used active ingredient against *C. pyri*: it was used against this pest for the first time in 1996, with only one spring treatment and only in some orchards, also because amitraz was an alternative until 2005, the year in which the ingredient was banned. Probably after the ban on this active ingredient the number of treatments per orchard and the area of employment were increased: this trend was also observed in the fruit growing area of Lleida, Girona and Huesca in Spain (Miarnau et al., 2010), and Ferrara and Modena in Italy. The current high selection pressure with this active ingredient, repeatedly applied over both geographical areas could induce

resistance to different pesticide families.

organophosphorate and pyrethroid active ingredients.

al., 2003).

involved.

selection for resistance.

Following the sudden outbreak of *C. pyri* populations in some fruit growing area in Emilia-Romagna in 2005, abamectin tests for resistance were performed for the first time only on overwintering adults (Fig. 11), but no resistance was detected, although LC50 and LC90 values appeared related to the time of adult field collection (Civolani et al., 2007).

Besides adults (Table 5) (Fig. 11), in 2007 and 2008 the abamectin tests in Emilia-Romagna were extended also to eggs and nymphs (Fig. 12 and Fig. 13), the stage target in *C. pyri* field treatments (Civolani et al., 2010). On adults, the abamectin topical tests performed in autumn 2007 and 2008 did not show significant differences among all populations tested, but the LC50 values were apparently related to the adult collection dates, as previously reported (Civolani et al., 2010).

Fig. 11. Topical application of the insecticide solution on *C. pyri* adult winter form with hand-held manual micro-applicator.

The egg spray test (LC50 and LC90) did not show relevant differences, although LC50 and LC90 values were always lower in the organic farm than in all others (Table 6). The results of leaf dip tests performed on young and old larvae were generally similar to the egg spray ones (Civolani et al., 2010).

Overall, the tests data indicate that no apparent resistance to abamectin has been developed up to now in *C. pyri* populations of Emilia-Romagna.

In 2009 and 2010 new cases of loss of efficiency of abamectin reappeared in some orchards in the province of Modena (Emilia-Romagna). In 2010 other tests were again performed on a *C. pyri* population which underwent 7 abamectin field treatment in 2009. However, in this case also the results of LC50 and LC90 did not show significant differences and the RR was just above 2 (unpublished data).

From 2004 to 2006 tests were carried out also in Spain to monitor the evolution of susceptibility to abamectin in orchards where the number of treatments changed from less than two until 2005 to an average of three after the ban on amitraz, with some orchards

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 401

Population (locality) Field strategy <sup>2007</sup> <sup>2008</sup>

Minotti (S. Martino) integrated 4.98 1.59 3.24 0.61 Scagnolaro (Francolino) integrated 2.80 0.89 4.16 0.78 Bonora (Boara) integrated - - 5.95 1.12 Celati (Francolino) integrated - - 4.23 0.80 Marchetti (Diamantina) conventional - - 5.31 1

Buondì-Vezzani (Diamantina) organic 3.13 5.27

Table 5. Response of *C. pyri* overwintering adult populations to topical applications of abamectin (2007 and 2008) in the province of Ferrara (Civolani et al., 2010). The values of

Population (locality) Field strategy <sup>2007</sup> <sup>2008</sup>

(Diamantina) organic 0.20 0.15 Minotti (S. Martino) integrated 0.43 2.15 0.29 1.93 Scagnolaro (Francolino) integrated - - 0.15 1 Bonora (Boara) integrated - - 0.27 1.80 Celati (Francolino) integrated - - 0.34 2.26 Marchetti (Diamantina) conventional - - 0.44 2.93

Table 6. Response of *C. pyri* to egg spray applications (2007 and 2008) in the province of Ferrara (Civolani et al., 2010). The values of LC50 are expressed in mg l-1 of abamectin.

Spirodiclofen is a recently introduced active ingredient in *C. pyri* control. Different LC50 values were observed for spirodiclofen in tests again performed in Emilia-Romagna, regardless of its limited use as an alternative to abamectin. This active ingredient was tested on three populations of the province of Ferrara in 2006 and 2007 by laboratory assays on

The topical tests on overwintering adults showed high susceptibility differences for this active ingredient between the adult population collected in the organic farm and those

collected in the integrated (Minotti) and conventional (Marchetti) farms (Table 7).

Population (locality) Field strategy 2006 2007

Table 7. Results obtained in the topical test with spirodiclofen on *C. pyri* overwintering adults (2006 and 2007). The values of LC50 are expressed in mg l-1 of spirodiclofen.

(Diamantina) organic 19.09 - 89.01 - Minotti (S. Martino) integrated 2582.60 135.28 1931.70 21.,69

(Diamantina) conventional 25.93 1.358 943.72 10.60

LC50 are expressed in mg l-1 of abamectin.

Buondì-Vezzani

adults and eggs.

Buondì-Vezzani

Marchetti

LC50 RR LC50 RR

LC50 RR LC50 RR

LC50 RR LC50 RR

undergoing 6-7 treatments. The tests were performed both on adults and nymphs and no evidence of a high RR was found (Miarnau et al., 2010). However, there are some Spanish populations of *C. pyri* that show low susceptibility in adults as well as in nymphs: these populations come from the fields with the highest number of abamectin treatments per year. As in Emilia-Romagna, these cases also indicate a high risk of selection for resistance to abamectin, especially if the number of treatments per year is high and there are no alternative products to use in a resistance management program.

Fig. 12. Cages for potted pear plants to keep adults of different population separated.

Fig. 13. Potted young pear plants on which psylla eggs were laid and then treated with abamectin.

undergoing 6-7 treatments. The tests were performed both on adults and nymphs and no evidence of a high RR was found (Miarnau et al., 2010). However, there are some Spanish populations of *C. pyri* that show low susceptibility in adults as well as in nymphs: these populations come from the fields with the highest number of abamectin treatments per year. As in Emilia-Romagna, these cases also indicate a high risk of selection for resistance to abamectin, especially if the number of treatments per year is high and there are no

Fig. 12. Cages for potted pear plants to keep adults of different population separated.

Fig. 13. Potted young pear plants on which psylla eggs were laid and then treated with

abamectin.

alternative products to use in a resistance management program.


Table 5. Response of *C. pyri* overwintering adult populations to topical applications of abamectin (2007 and 2008) in the province of Ferrara (Civolani et al., 2010). The values of LC50 are expressed in mg l-1 of abamectin.


Table 6. Response of *C. pyri* to egg spray applications (2007 and 2008) in the province of Ferrara (Civolani et al., 2010). The values of LC50 are expressed in mg l-1 of abamectin.

Spirodiclofen is a recently introduced active ingredient in *C. pyri* control. Different LC50 values were observed for spirodiclofen in tests again performed in Emilia-Romagna, regardless of its limited use as an alternative to abamectin. This active ingredient was tested on three populations of the province of Ferrara in 2006 and 2007 by laboratory assays on adults and eggs.

The topical tests on overwintering adults showed high susceptibility differences for this active ingredient between the adult population collected in the organic farm and those collected in the integrated (Minotti) and conventional (Marchetti) farms (Table 7).


Table 7. Results obtained in the topical test with spirodiclofen on *C. pyri* overwintering adults (2006 and 2007). The values of LC50 are expressed in mg l-1 of spirodiclofen.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 403

that when the presence of *C. pyri* rapidly increases in the third decade of May, usually we can expect a rapid increase of the predator population about two-three weeks later. This requires for the fruit grower to tolerate a relatively high presence of the pest and "resist" to the temptation to perform specific treatments. Since 1990, other field conditions added to these problems of the natural control, such as the increased chemical treatments against the codling moth, *C. pomonella*. These treatments caused a general weakening of the wild *A. nemoralis* populations, disrupting a very fluctuating natural equilibrium. Another problem recently emerged is that even in equilibrated pear orchards, not undergoing any heavy chemical treatment, for some reason the presence of predators remains low or undergoes

The previously mentioned limits of natural control led to artificially introduce anthocorids in the pear orchard, buying them from biofactories and thus performing a true biological control technique. The aim is to obtain a more numerous presence of the predator in the critical periods, anticipating the reproduction mechanism of the population which could occur naturally but with some delay. The introduction of predators is performed at the end of winter, between the end of March and the beginning of April. About 1000 adult individuals of *A. nemoralis* are placed per hectare of the pear orchard, in three consecutive weekly introductions. This biological control technique was common around 2000 in integrated pear orchards where the active ingredient amitraz was employed for the springsummer control of *C. pyri*, then it was slowly neglected because of the limited results and

high fluctuations over the years or even according to the seasons.

the relatively high costs.

Fig. 14. Adult of *A. nemoralis.*

The results obtained by the spray test on yellow eggs before hatching, using the field doses of spirodiclofen, show low activity on the population collected in the Minotti integrated farm (both on first and second psylla generation). The activity levels in the Minotti farm, expressed as percentage of nymph mortality, are very different from those detected in the other two farms. However, the values detected in the conventional Marchetti farm are unexpectedly similar to those obtained in the organic farm (Table 8): these results were surprising because spirodiclofen was never employed before in none of these farms (unpublished data).


Table 8. Results obtained in the spray test with spirodiclofen on *C. pyri* eggs laid by overwintering and summer adults in 2007. The results are expressed as percentage of nymph mortality.

## **6. Natural and biological control of** *C. pyri*

In open field and especially in the pear orchard the techniques of biological control are not common because the fruit growers have always aimed to favour and exploit the development of the wild auxiliary insects, thus performing strategies of natural control. *Antochoris nemoralis*, common in all Europe, is known as the main predatory species of pear psylla. This species overwinters as an adult (Fig. 14) and starts to lay eggs in spring, inserting them under the leaf epidermis. The development of the juvenile forms occurs in 5 stages. *A. nemoralis* preys on both eggs and nymphs of psylla and in Emilia-Romagna typically shows three generations. Although generally preferring psylla, this anthocorid may feed on other insects and on the pear trees its activity against aphids and the pear sawfly *Hoplocampa brevis* Klug (Hymenoptera Tenthredinae) is very interesting. Laboratory tests showed an average predation of about 300 psylla nymphs during the entire life of an adult, which lasts about 60 days. The presence of *A. nemoralis* in pear orchards mainly depends on the type of control strategy applied in the farm. As previously mentioned, by limiting the use of pesticides to the minimum required and preferring the selective ones (see next chapter), it is favoured the development of the wild *A. nemoralis* populations which become a relevant factor to control the pest. Indeed, a dynamic equilibrium develops between the predator and *C. pyri* populations, often leading to the solution of the problem without the need for specific chemical treatments, or with treatments only reduced to tree washing. The main problem of this strategy is nevertheless the slow initial development of the predator population, which must have the prey available (in this case *C. pyri*) to rapidly increase in number within the pear orchard. Therefore, in the initial part of the season (around May-June) it is necessary to tolerate some amounts of the pest in the orchard to obtain later a good number of predator anthocorids on the trees. In other words, this means

The results obtained by the spray test on yellow eggs before hatching, using the field doses of spirodiclofen, show low activity on the population collected in the Minotti integrated farm (both on first and second psylla generation). The activity levels in the Minotti farm, expressed as percentage of nymph mortality, are very different from those detected in the other two farms. However, the values detected in the conventional Marchetti farm are unexpectedly similar to those obtained in the organic farm (Table 8): these results were surprising because spirodiclofen was never employed before in none of these farms

(Diamantina) organic 100 94,91 Minotti (S. Martino) integrated 30.62 77.95

(Diamantina) conventional - 94.81

In open field and especially in the pear orchard the techniques of biological control are not common because the fruit growers have always aimed to favour and exploit the development of the wild auxiliary insects, thus performing strategies of natural control. *Antochoris nemoralis*, common in all Europe, is known as the main predatory species of pear psylla. This species overwinters as an adult (Fig. 14) and starts to lay eggs in spring, inserting them under the leaf epidermis. The development of the juvenile forms occurs in 5 stages. *A. nemoralis* preys on both eggs and nymphs of psylla and in Emilia-Romagna typically shows three generations. Although generally preferring psylla, this anthocorid may feed on other insects and on the pear trees its activity against aphids and the pear sawfly *Hoplocampa brevis* Klug (Hymenoptera Tenthredinae) is very interesting. Laboratory tests showed an average predation of about 300 psylla nymphs during the entire life of an adult, which lasts about 60 days. The presence of *A. nemoralis* in pear orchards mainly depends on the type of control strategy applied in the farm. As previously mentioned, by limiting the use of pesticides to the minimum required and preferring the selective ones (see next chapter), it is favoured the development of the wild *A. nemoralis* populations which become a relevant factor to control the pest. Indeed, a dynamic equilibrium develops between the predator and *C. pyri* populations, often leading to the solution of the problem without the need for specific chemical treatments, or with treatments only reduced to tree washing. The main problem of this strategy is nevertheless the slow initial development of the predator population, which must have the prey available (in this case *C. pyri*) to rapidly increase in number within the pear orchard. Therefore, in the initial part of the season (around May-June) it is necessary to tolerate some amounts of the pest in the orchard to obtain later a good number of predator anthocorids on the trees. In other words, this means

Table 8. Results obtained in the spray test with spirodiclofen on *C. pyri* eggs laid by overwintering and summer adults in 2007. The results are expressed as percentage of

Nymph mortality % on eggs laid by overwintering adults

Nymph mortality % on eggs laid by adult summer forms

(unpublished data).

Buondì-Vezzani

Marchetti

nymph mortality.

Population (locality) Field strategy

**6. Natural and biological control of** *C. pyri*

that when the presence of *C. pyri* rapidly increases in the third decade of May, usually we can expect a rapid increase of the predator population about two-three weeks later. This requires for the fruit grower to tolerate a relatively high presence of the pest and "resist" to the temptation to perform specific treatments. Since 1990, other field conditions added to these problems of the natural control, such as the increased chemical treatments against the codling moth, *C. pomonella*. These treatments caused a general weakening of the wild *A. nemoralis* populations, disrupting a very fluctuating natural equilibrium. Another problem recently emerged is that even in equilibrated pear orchards, not undergoing any heavy chemical treatment, for some reason the presence of predators remains low or undergoes high fluctuations over the years or even according to the seasons.

The previously mentioned limits of natural control led to artificially introduce anthocorids in the pear orchard, buying them from biofactories and thus performing a true biological control technique. The aim is to obtain a more numerous presence of the predator in the critical periods, anticipating the reproduction mechanism of the population which could occur naturally but with some delay. The introduction of predators is performed at the end of winter, between the end of March and the beginning of April. About 1000 adult individuals of *A. nemoralis* are placed per hectare of the pear orchard, in three consecutive weekly introductions. This biological control technique was common around 2000 in integrated pear orchards where the active ingredient amitraz was employed for the springsummer control of *C. pyri*, then it was slowly neglected because of the limited results and the relatively high costs.

Fig. 14. Adult of *A. nemoralis.*

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 405

ingredients have significant toxic effects on *A. nemoralis*, thus they must be employed only

Concerning the strategies and methods to control *Cacopsylla pyri* (Hemiptera Psyllidae), and also their effects on the beneficial insects and the development of resistance, during the past decade Italian and European populations of *C. pyri*, in significant decline, have been apparently less capable to induce damage: this could be due to the success of defence programs based on integrated pest management (IPM). For pear, the IPM involves pest population control and auxiliary insect protection, associated to availability of new chemical or microbiological agents specifically targeted to pear key pests (mainly

As far as known about *C. pyri* infestation, the biological control alone is not successful in preventing damage, especially when caused by second generation nymphs that feed on shoots and leaves in late spring and summer; thus chemical pest control strategies are also employed. For example, in Northern Italy the most common defense strategy against *C. pyri* in pear orchards involves chemical treatments on second-generation eggs or nymphs. Traditional treatments with chitin inhibitors, mainly aimed against *C. pomonella*, have also

Specific treatments against *C. pyri* nymphs involve amitraz (now banned), abamectin and spirodiclofen. During spring and summer the treatment with the last two active ingredients, in addition to non-chemical treatments such as tree washing, is usually successful in limiting

During autumn and winter, the *C. pyri* management strategies involve treatments with synthetic pyretroids after leaf fall, to limit adult overwintering population, or at the end of winter, when females are ready to lay eggs. However, these treatments during autumn and winter, common in France and North America, are rarely employed in Italy because of their high toxicity against populations of auxiliary insects (such as Anthocoridae) which could still be present in the pear orchards. In late winter it is also possible to perform a nonchemical treatment against *C. pyri* by distribution of an aqueous suspension of kaolin on

Each one of the above strategies shows favorable and unfavorable aspects in terms of efficacy, side effects on beneficial insects, timing of application and environmental

As for all phytophagous pests, also for *C. pyri* the repeated use of chemical active ingredients causes the development of resistance. Indeed, several insecticides employed in the past to control the pear psylla showed a sharp decline in activity because of resistance development. After the sudden outbreak of *C. pyri* populations in some fruit growing area of Ferrara and Modena (Italy) and Lleida, Girona and Huesca (Spain), abamectin tests were performed on winter form adults and nymphs. The results did not show relevant resistance effects, although LC50 and LC90 values were always higher in populations where abamectin treatment was repeated several times in the year. Overall, the results indicate that no apparent resistance to abamectin has been yet developed in *C. pyri* populations of the most important European areas of pear growth: nevertheless, the pear orchards in which *C. pyri* outbreaks recently occurred are presently under close

for very few treatments along the year.

**8. Conclusions** 

*Cydia pomonella*).

conditions.

some secondary effects on *C. pyri*.

investigation and careful survey.

the honeydew damages caused by *C. pyri*.

trees, in order to obtain a physical barrier to egg laying.

## **7. Selectivity of pesticides against** *A. nemoralis*

In the last 20 years new families of pesticides have been developed with generally lower toxicity towards beneficial species in comparison to the previous ones. These pesticides are more suitable for the new techniques of integrated pest control (IPM) which preserve the contribution of beneficial insects to the natural control against pests, especially against *C. pyri*. Therefore it was necessary to perform tests on the new active ingredients, to verify their different toxicity degree and obtain the data required to improve their use.

Several studies have been recently performed to evaluate the selectivity of the new pesticides towards auxiliaries found in the pear orchards, first of all *A. nemoralis* (Fig. 14), the most relevant in the natural control against *C. pyri*. The active ingredients recently investigated include those directly employed against *C. pyri* (psyllicides) and also those largely employed on other key pests (non-psyllicides).

Concerning the psyllicides, in different toxicity tests performed in Emilia-Romagna since 1997 abamectin showed a medium degree of toxicity against the *A. nemoralis* population, mostly against first and second instar nymphs in comparison to adults. In some cases the mortality of nymphs reached 50% and, according to field data, the total population appears limited in comparison to untreated controls for about two weeks after the treatment. This result must nevertheless take into account the lower presence of prey as food for *A. nemoralis* (Pasqualini & Civolani 2007). Other products specific for *C. pyri*, such as amitraz (now banned), mineral oil, or insecticide soaps did not show relevant effects on the predator population (Civolani & Pasqualini, 1999; Pasqualini et al., 1999). Concerning spirodiclofen, the tests on toxicity towards *A. nemoralis* were performed again in Emilia-Romagna in years 2004-2006. The results show that the anthocorid populations undergoing treatment with spirodiclofen have similar development to the untreated ones, unlike abamectin for which the population first sharply decreases, then recovering according to the amount of prey available (Pasqualini & Civolani, 2007).

Concerning the non-psyllicides, those belonging to the chitin inhibitors, usually characterized by a long period of action, generally show a low toxicity on *A. nemoralis*, even if some of them, such as flufenoxuron, induce a heavy reduction of the anthocorid populations (Girolami et al., 2001, Pasqualini & Civolani 2002).

Among other non-psyllicides employed in pear orchards against the most relevant pests, there is spinosad, a pesticide of natural origin, whose activity derives from a toxin produced by *Saccharopolyspora spinosa* (Bacteria Actinomycetales), the indoxacarb belonging to the family of the oxadiazines, then methoxyfenozide and tebufenozide, synthetic molecules belonging to the family of moulting accelerator compounds (MAC), and the organophosphorates azinphos-methyl (banned in 2007), chlorpyrifos, chlorpyrifos-methyl and phosmet, all employed against relevant lepidopteran pests such as *C. pomonella*, *Cydia molesta* Busck and *Pandemis cerasana* Hübner. All these active ingredients have been more or less tested for toxicity against *A. nemoralis* and the results did not show relevant toxic effects on this predator (Civolani & Pasqualini, 1999).

As previously mentioned, the neonicotinoids are not employed in Italy and Europe as specific psyllicides, on the contrary of what happens in United States where they are employed as an alternative to abamectin against *C. pyricola*. However, a large amount of the above active ingredients are used against other pests, such as aphids and the pear sawfly *H. brevis*: the most relevant are imidacloprid, acetamiprid and tiametoxan, while against the codling moth *C. pomonella* the most frequently used is thiacloprid. All those active ingredients have significant toxic effects on *A. nemoralis*, thus they must be employed only for very few treatments along the year.

## **8. Conclusions**

404 Insecticides – Pest Engineering

In the last 20 years new families of pesticides have been developed with generally lower toxicity towards beneficial species in comparison to the previous ones. These pesticides are more suitable for the new techniques of integrated pest control (IPM) which preserve the contribution of beneficial insects to the natural control against pests, especially against *C. pyri*. Therefore it was necessary to perform tests on the new active ingredients, to verify

Several studies have been recently performed to evaluate the selectivity of the new pesticides towards auxiliaries found in the pear orchards, first of all *A. nemoralis* (Fig. 14), the most relevant in the natural control against *C. pyri*. The active ingredients recently investigated include those directly employed against *C. pyri* (psyllicides) and also those

Concerning the psyllicides, in different toxicity tests performed in Emilia-Romagna since 1997 abamectin showed a medium degree of toxicity against the *A. nemoralis* population, mostly against first and second instar nymphs in comparison to adults. In some cases the mortality of nymphs reached 50% and, according to field data, the total population appears limited in comparison to untreated controls for about two weeks after the treatment. This result must nevertheless take into account the lower presence of prey as food for *A. nemoralis* (Pasqualini & Civolani 2007). Other products specific for *C. pyri*, such as amitraz (now banned), mineral oil, or insecticide soaps did not show relevant effects on the predator population (Civolani & Pasqualini, 1999; Pasqualini et al., 1999). Concerning spirodiclofen, the tests on toxicity towards *A. nemoralis* were performed again in Emilia-Romagna in years 2004-2006. The results show that the anthocorid populations undergoing treatment with spirodiclofen have similar development to the untreated ones, unlike abamectin for which the population first sharply decreases, then recovering according to the amount of prey

Concerning the non-psyllicides, those belonging to the chitin inhibitors, usually characterized by a long period of action, generally show a low toxicity on *A. nemoralis*, even if some of them, such as flufenoxuron, induce a heavy reduction of the anthocorid

Among other non-psyllicides employed in pear orchards against the most relevant pests, there is spinosad, a pesticide of natural origin, whose activity derives from a toxin produced by *Saccharopolyspora spinosa* (Bacteria Actinomycetales), the indoxacarb belonging to the family of the oxadiazines, then methoxyfenozide and tebufenozide, synthetic molecules belonging to the family of moulting accelerator compounds (MAC), and the organophosphorates azinphos-methyl (banned in 2007), chlorpyrifos, chlorpyrifos-methyl and phosmet, all employed against relevant lepidopteran pests such as *C. pomonella*, *Cydia molesta* Busck and *Pandemis cerasana* Hübner. All these active ingredients have been more or less tested for toxicity against *A. nemoralis* and the results did not show relevant toxic effects

As previously mentioned, the neonicotinoids are not employed in Italy and Europe as specific psyllicides, on the contrary of what happens in United States where they are employed as an alternative to abamectin against *C. pyricola*. However, a large amount of the above active ingredients are used against other pests, such as aphids and the pear sawfly *H. brevis*: the most relevant are imidacloprid, acetamiprid and tiametoxan, while against the codling moth *C. pomonella* the most frequently used is thiacloprid. All those active

their different toxicity degree and obtain the data required to improve their use.

**7. Selectivity of pesticides against** *A. nemoralis*

largely employed on other key pests (non-psyllicides).

available (Pasqualini & Civolani, 2007).

on this predator (Civolani & Pasqualini, 1999).

populations (Girolami et al., 2001, Pasqualini & Civolani 2002).

Concerning the strategies and methods to control *Cacopsylla pyri* (Hemiptera Psyllidae), and also their effects on the beneficial insects and the development of resistance, during the past decade Italian and European populations of *C. pyri*, in significant decline, have been apparently less capable to induce damage: this could be due to the success of defence programs based on integrated pest management (IPM). For pear, the IPM involves pest population control and auxiliary insect protection, associated to availability of new chemical or microbiological agents specifically targeted to pear key pests (mainly *Cydia pomonella*).

As far as known about *C. pyri* infestation, the biological control alone is not successful in preventing damage, especially when caused by second generation nymphs that feed on shoots and leaves in late spring and summer; thus chemical pest control strategies are also employed. For example, in Northern Italy the most common defense strategy against *C. pyri* in pear orchards involves chemical treatments on second-generation eggs or nymphs. Traditional treatments with chitin inhibitors, mainly aimed against *C. pomonella*, have also some secondary effects on *C. pyri*.

Specific treatments against *C. pyri* nymphs involve amitraz (now banned), abamectin and spirodiclofen. During spring and summer the treatment with the last two active ingredients, in addition to non-chemical treatments such as tree washing, is usually successful in limiting the honeydew damages caused by *C. pyri*.

During autumn and winter, the *C. pyri* management strategies involve treatments with synthetic pyretroids after leaf fall, to limit adult overwintering population, or at the end of winter, when females are ready to lay eggs. However, these treatments during autumn and winter, common in France and North America, are rarely employed in Italy because of their high toxicity against populations of auxiliary insects (such as Anthocoridae) which could still be present in the pear orchards. In late winter it is also possible to perform a nonchemical treatment against *C. pyri* by distribution of an aqueous suspension of kaolin on trees, in order to obtain a physical barrier to egg laying.

Each one of the above strategies shows favorable and unfavorable aspects in terms of efficacy, side effects on beneficial insects, timing of application and environmental conditions.

As for all phytophagous pests, also for *C. pyri* the repeated use of chemical active ingredients causes the development of resistance. Indeed, several insecticides employed in the past to control the pear psylla showed a sharp decline in activity because of resistance development. After the sudden outbreak of *C. pyri* populations in some fruit growing area of Ferrara and Modena (Italy) and Lleida, Girona and Huesca (Spain), abamectin tests were performed on winter form adults and nymphs. The results did not show relevant resistance effects, although LC50 and LC90 values were always higher in populations where abamectin treatment was repeated several times in the year. Overall, the results indicate that no apparent resistance to abamectin has been yet developed in *C. pyri* populations of the most important European areas of pear growth: nevertheless, the pear orchards in which *C. pyri* outbreaks recently occurred are presently under close investigation and careful survey.

The Past and Present of Pear Protection Against the Pear Psylla, *Cacopsylla pyri* L. 407

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The author wish to thank Dr Milvia Chicca (Department of Biology and Evolution, University of Ferrara) for kindly revising the manuscript and the English style.

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**9. Acknowledgments** 

**10. References** 


**18** 

**in Grape** 

*USA* 

**Effects of Kaolin Particle Film and Imidacloprid** 

**Cicadellidae) Populations and the Prevention** 

K.M. Tubajika1, G.J. Puterka2, N.C. Toscano3, J. Chen4 and E.L. Civerolo4 *1United States Department of Agriculture (USDA), Animal and Plant Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Center for Plant Health Science and* 

*4United States Department of Agriculture (USDA), Agricultural Research Service (ARS),* 

The glassy-winged sharpshooter (GWSS), *Homalodisca vitripennis* (Germar), (Hemiptera: Cicadellidae), is a major pest of important agronomic, horticultural, landscape, ornamental crops and native trees in California (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999). This insect is an invasive species in California that was first detected in the state in 1989 (Sorensen & Gill, 1996). This sharpshooter is a key vector of *Xylella fastidiosa* and has changed the epidemiology of *X. fastidiosa* (*Xf*) diseases affecting important agronomic and horticultural crops as well as landscape, ornamental and native trees in California based on the infection of these crops by the bacteria (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999) (Fig. 1). It is not clear if management strategies for Pierce's Disease (PD) developed in GWSS-free regions of California are suitable to manage the disease in

Data from earlier studies suggest that GWSS has continued to increase in number (population density) and geographical range in California (Purcell & Saunders, 1999; Tubajika et al., 2004). GWSS populations are widely distributed over a large number of hosts including perennial agronomic crops, ornamental plantings, and weedy plant species (Hill & Purcell, 1995; Purcell et al., 1999; Raju et al., 1980). Previous studies by Blua et al. (1999) and Perring et al. (2001) showed that GWSS populations utilize citrus plants as their primary over-wintering host (other plants are available) when grapes, stone fruits, ornamental hosts,

**1. Introduction** 

vineyards where GWSS has become established.

**on Glassy-Winged Sharpshooter** 

**of Spread of** *Xylella fastidiosa*

*Technology (CPHST), Raleigh, North Carolina, 2United States Department of Agriculture (USDA),* 

*Agricultural Research Service (ARS), Stillwater, Oklahoma;* 

*3Department of Entomology, University of California, Riverside, California,* 

*San Joaquin Valley Agricultural Sciences Center (SJVSC), Parlier, California,* 

**(***Homalodisca vitripennis* **) (Hemiptera:** 


## **Effects of Kaolin Particle Film and Imidacloprid on Glassy-Winged Sharpshooter (***Homalodisca vitripennis* **) (Hemiptera: Cicadellidae) Populations and the Prevention of Spread of** *Xylella fastidiosa* **in Grape**

K.M. Tubajika1, G.J. Puterka2, N.C. Toscano3, J. Chen4 and E.L. Civerolo4 *1United States Department of Agriculture (USDA), Animal and Plant Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Center for Plant Health Science and Technology (CPHST), Raleigh, North Carolina, 2United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Stillwater, Oklahoma; 3Department of Entomology, University of California, Riverside, California, 4United States Department of Agriculture (USDA), Agricultural Research Service (ARS), San Joaquin Valley Agricultural Sciences Center (SJVSC), Parlier, California, USA* 

#### **1. Introduction**

408 Insecticides – Pest Engineering

Pasqualini, E.; Civolani, S. & Corelli Grappadelli, L. (2003). Particle film technology:

Pasqualini, E. & Civolani, S. (2006). Difesa della psilla del pero con abamectina. *L'Informatore* 

Pasqualini, E. & Civolani, S. (2007). Spirodiclofen, nuovo insetticida efficace contro la psilla

Pollini, A.; Bariselli, M. & Boselli, M. (1992). La difesa del pero dalla Psilla. *Informatore* 

Puterka, G.; Glenn, D. M.; Sekutowski, D. G.; Unruh, T. R. & Jones S.K. (2000). Progress

Puterka, G.; Farone, W,; Palmer, T. & Barrington, A. (2003). Structure-function relationships

Scaub, L.; Bloesch, B.; Bencheikh, M. & Pigeaud, A. (1996). Spatial distribution of

Schaper, U. & Seemmuller, E. (1982). Condition of the phloem and the persistence of

Souliotis, C. & Moschos, T. (2008). Effectiveness of some pesticides against *Cacopsylla pyri*

Van De Baan H. E., (1988). Factors influencing pesticide resistance in *Psylla pyricola* Foerster

Zwick, R. W. & Westigard, P. H., (1978). Prebloom petroleum oil applications for delaying

toward liquid formulations of particle films for insect and disease control in pear.

affecting the insecticidal and miticidal activity of sugar esters. *Journal of Economic* 

teflubenzuron resistance by pear psylla in Western Switzerland. *OILB/SROP* 

mycoplasma-like organism associated with apple proliferation and pear decline.

and impact on its predator *Anthocoris nemoralis* in pear-orchards. *Bulletin of* 

and susceptibility in its Mirids predator, *Deraeocoris brevis* Knight. *Thesis, Oregon* 

pear psylla (Homptera: Psyllidae) oviposition. *Canandian Entomologist*, 110: 225-236

Northern Italy. *Bulletin of Insectology*, 55: 39-42

del pero. *L'Informatore Agrario*, 11: 89 93

*Environmental Entomology*, 29: 329-339

*Agrario*, 12: 50-54.

*fitopatologico*, 5: 19-24

*Entomology*, 96: 636-644

*Bulletin,* 19 (4): 311-314

*Insectology*, 61: 25-30

*State University*

*Phytopathology*, 72: 736-742

approach for a biorational control of *Cacopsylla pyri* (Rhynchota: Psyllidae) in

The glassy-winged sharpshooter (GWSS), *Homalodisca vitripennis* (Germar), (Hemiptera: Cicadellidae), is a major pest of important agronomic, horticultural, landscape, ornamental crops and native trees in California (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999). This insect is an invasive species in California that was first detected in the state in 1989 (Sorensen & Gill, 1996). This sharpshooter is a key vector of *Xylella fastidiosa* and has changed the epidemiology of *X. fastidiosa* (*Xf*) diseases affecting important agronomic and horticultural crops as well as landscape, ornamental and native trees in California based on the infection of these crops by the bacteria (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999) (Fig. 1). It is not clear if management strategies for Pierce's Disease (PD) developed in GWSS-free regions of California are suitable to manage the disease in vineyards where GWSS has become established.

Data from earlier studies suggest that GWSS has continued to increase in number (population density) and geographical range in California (Purcell & Saunders, 1999; Tubajika et al., 2004). GWSS populations are widely distributed over a large number of hosts including perennial agronomic crops, ornamental plantings, and weedy plant species (Hill & Purcell, 1995; Purcell et al., 1999; Raju et al., 1980). Previous studies by Blua et al. (1999) and Perring et al. (2001) showed that GWSS populations utilize citrus plants as their primary over-wintering host (other plants are available) when grapes, stone fruits, ornamental hosts,

Effects of Kaolin Particle Film and Imidacloprid on

populations.

California.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 411

slowing the development of PD in grape. Contact insecticides offer short-term protection against GWSS infestations because of the continued movement of sharpshooter adults from citrus and non-treated hosts/areas which re-infest the grapevines. In a study on the transmission of *Xf* to grapevines by *H. vitripennis* (syn. *coagulata*), reported that *H. vitripenns*  (syn. *coagulata*) transmitted *Xf* to grapevines in a persistent manner(Almeida & Purcell, 2003). This research also showed that the nymphal lost infectivity during molting, but there was no evidence of a latent period (delay after acquisition) of the pathogen by adult

Likewise, kaolin (Surround®, NovaSource Tessenderlo Kerley, Inc., Phoenix, AZ.) is a potential alternative pest management product with improved safety to pesticide handlers and reduced environmental impact (Glenn et al., 1999)). It protects plants from insect feeding and oviposition by coating the plant surfaces with a protective mineral barrier (aka particle film) (Glenn et al., 1999; Puterka et al., 2000). Kaolin has been shown to suppress pear psylla, *Cacopsylla pyricola* (Forster); the spirea aphid, *Aphis spiraecola* Pagenstecher; the two-spotted spider mite, *Tetranychus urticae* Koch; and the potato leafhopper, *Empasca fabae* (Harris) in previous research (Glenn et al., 1999) (Fig. 2). The mechanism involved in reduction of pest density is reported to be reduced oviposition and feeding on treated plants (Glenn et al., 1999). Puterka et al. (2000) found Surround® reduced *C. pyricola* populations and controlled Fabraea leaf spot, fungal disease caused by *Fabraea maculata* Atk. Moreover, Glenn et al., (1999) reported that kaolin films contributed to control of fungal and bacterial plant pathogens by preventing the formation of a liquid lm on the surface of pear leaves. In this chapter, the effects of kaolin particle film and imidacloprid on *H. vitripennis* (syn. *coagulata*) population levels and the prevention and spread of *Xf* in grape are described.

(a) Field study using kaolin (b) Close-up of kaolin-treated grape leaves Fig. 2. Field study showing the application of kaolin (a) and a close-up of kaolin-treated grape used to control Glassy-winged sharpshooter in Kern County, Bakersfield in

The use of insecticides is common for controlling pest populations (Hemingway & Ranson, 2005). However, the development of resistance and cross-resistance among widely used and newly applied systemic insecticides can affect the development of new strategies in integrated pest management. The GWSS has become a significant problem to California

**2. Impacts of kaolin and imidacloprid on** *Homalodisca vitripennis*

and weedy species are dormant during the winter. This vector may have a great impact on grape-growing areas in the Coachella, Temecula and the lower San Joaquin Valley due to numerous citrus orchards that support overwintering populations. Effective insect and disease management strategies are dependent upon knowledge of inoculum sources, and biology and the ecology of insect vectors and their natural enemies (Blua et al., 1999; Hill & Purcell, 1995; Tubajika et al., 2004). The GWSS transmits *Xf* to grape (Fig. 1), as well as to oleander (Costa et al., 2000) with the transmission efciency greater than that of other vectors such as the green sharpshooter (GSS) [*Draeculacephala mineroa* Ball] and red-headed sharpshooter (RHSS) [*Carneocephala fulgida* Nottingham], RHSS) (Purcell & Saunders, 1999), but much less than that of the blue-green sharpshooter (BGSS) [*Graphocephala atropunctata* (Signoret] (Purcell & Saunders, 1999).

(a) GWSS adult (b) GWSS nymph

(c) GWSS egg (d) PD symptoms in grape

Fig. 1. Glassy-winged sharpshooter (GWSS) adult (a), nymph (b) (photos by B. Stone-Smith), and egg (c) found in grape in Bakersfield, CA. The GWSS transmits the bacterium *Xylella fastidiosa*, which causes Pierce's disease (PD) (d) in grape.

Imidacloprid, (Admire, Bayer Corp., Kansas City, MO, USA), is an neonicotinoid insecticide which interrupts the binding of nicotinergic acetylcholine in post-synaptic receptors of the insect (Hemingway & Ranson, 2005; Romoser & Stoffolano, 1998). In Georgia, treatment of grapevines with Admire slowed the rate of PD spread, but ultimately extended vineyard life by only a year when infestations were severe (Krewer et al., 2002). Imidacloprid is the most widely used neonicotinoid for the prevention of infestation of grapevines by GWSS in California. This product may be useful in reducing *Xf* transmission by the GWSS and in

and weedy species are dormant during the winter. This vector may have a great impact on grape-growing areas in the Coachella, Temecula and the lower San Joaquin Valley due to numerous citrus orchards that support overwintering populations. Effective insect and disease management strategies are dependent upon knowledge of inoculum sources, and biology and the ecology of insect vectors and their natural enemies (Blua et al., 1999; Hill & Purcell, 1995; Tubajika et al., 2004). The GWSS transmits *Xf* to grape (Fig. 1), as well as to oleander (Costa et al., 2000) with the transmission efciency greater than that of other vectors such as the green sharpshooter (GSS) [*Draeculacephala mineroa* Ball] and red-headed sharpshooter (RHSS) [*Carneocephala fulgida* Nottingham], RHSS) (Purcell & Saunders, 1999), but much less than that of the blue-green sharpshooter (BGSS) [*Graphocephala atropunctata*

Fig. 1. Glassy-winged sharpshooter (GWSS) adult (a), nymph (b) (photos by B. Stone-Smith), and egg (c) found in grape in Bakersfield, CA. The GWSS transmits the bacterium *Xylella* 

(c) GWSS egg (d) PD symptoms in grape

(a) GWSS adult (b) GWSS nymph

Imidacloprid, (Admire, Bayer Corp., Kansas City, MO, USA), is an neonicotinoid insecticide which interrupts the binding of nicotinergic acetylcholine in post-synaptic receptors of the insect (Hemingway & Ranson, 2005; Romoser & Stoffolano, 1998). In Georgia, treatment of grapevines with Admire slowed the rate of PD spread, but ultimately extended vineyard life by only a year when infestations were severe (Krewer et al., 2002). Imidacloprid is the most widely used neonicotinoid for the prevention of infestation of grapevines by GWSS in California. This product may be useful in reducing *Xf* transmission by the GWSS and in

*fastidiosa*, which causes Pierce's disease (PD) (d) in grape.

(Signoret] (Purcell & Saunders, 1999).

slowing the development of PD in grape. Contact insecticides offer short-term protection against GWSS infestations because of the continued movement of sharpshooter adults from citrus and non-treated hosts/areas which re-infest the grapevines. In a study on the transmission of *Xf* to grapevines by *H. vitripennis* (syn. *coagulata*), reported that *H. vitripenns*  (syn. *coagulata*) transmitted *Xf* to grapevines in a persistent manner(Almeida & Purcell, 2003). This research also showed that the nymphal lost infectivity during molting, but there was no evidence of a latent period (delay after acquisition) of the pathogen by adult populations.

Likewise, kaolin (Surround®, NovaSource Tessenderlo Kerley, Inc., Phoenix, AZ.) is a potential alternative pest management product with improved safety to pesticide handlers and reduced environmental impact (Glenn et al., 1999)). It protects plants from insect feeding and oviposition by coating the plant surfaces with a protective mineral barrier (aka particle film) (Glenn et al., 1999; Puterka et al., 2000). Kaolin has been shown to suppress pear psylla, *Cacopsylla pyricola* (Forster); the spirea aphid, *Aphis spiraecola* Pagenstecher; the two-spotted spider mite, *Tetranychus urticae* Koch; and the potato leafhopper, *Empasca fabae* (Harris) in previous research (Glenn et al., 1999) (Fig. 2). The mechanism involved in reduction of pest density is reported to be reduced oviposition and feeding on treated plants (Glenn et al., 1999). Puterka et al. (2000) found Surround® reduced *C. pyricola* populations and controlled Fabraea leaf spot, fungal disease caused by *Fabraea maculata* Atk. Moreover, Glenn et al., (1999) reported that kaolin films contributed to control of fungal and bacterial plant pathogens by preventing the formation of a liquid lm on the surface of pear leaves. In this chapter, the effects of kaolin particle film and imidacloprid on *H. vitripennis* (syn. *coagulata*) population levels and the prevention and spread of *Xf* in grape are described.

(a) Field study using kaolin (b) Close-up of kaolin-treated grape leaves

Fig. 2. Field study showing the application of kaolin (a) and a close-up of kaolin-treated grape used to control Glassy-winged sharpshooter in Kern County, Bakersfield in California.

## **2. Impacts of kaolin and imidacloprid on** *Homalodisca vitripennis*

The use of insecticides is common for controlling pest populations (Hemingway & Ranson, 2005). However, the development of resistance and cross-resistance among widely used and newly applied systemic insecticides can affect the development of new strategies in integrated pest management. The GWSS has become a significant problem to California

Effects of Kaolin Particle Film and Imidacloprid on

films in choice versus no-choice experiments.

infested kaolin treated plants.

Puterka 2005, Puterka and Glenn 2008).

**2.2 Mortality effect of kaolin and imidacloprid on GWSS on grape** 

choice environment.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 413

of surviving adults did not decline in untreated trees in the no-choice test indicating they had favorable conditions to colonize and thrive. Some GWSS nymphs that were able to find the few untreated spots on kaolin-treated foliage probably remained at those sites during the study period. In contrast to the above findings, adult survival on kaolin-treated foliage did not change over time in either the choice or no-choice tests. We observed that kaolin treated plants are undesirable hosts to both GWSS adults and nymphs in choice and no-

(a) GWSS nymph counts (b) GWSS adults counts Fig. 3. Mean number of glassy-winged sharpshooter nymph (n=20) (a) and adults (n=50) (b) per tree 4 days after being released on trees that were treated or not treated with kaolin

However, in no-choice experiments, we observed that few nymphs managed to find an untreated site on which to settle when presented with the kaolin-treated plants. This finding implies that good coverage is important when using this material for insect control. When given a choice between kaolin-treated and untreated plants, no GWSS nymphs or adults

The choice study may be more representative of what occurs under field conditions because GWSS can utilize more than 75 species of plants (Turner & Pollard, 1959). Therefore, GWSS could easily move to other plants or plant species if they encountered kaolin treated- plants. Results from other studies indicate that kaolin particle films protect plants from insect feeding, oviposition, and by repelling GWSS nymphs and adults by coating the plant surfaces with a mineral barrier (Puterka et al., 2003). This finding is supported by previous reports using kaolin particle film treatments (Glenn et al., 1999; Puterka et al., 2000; Sisterson et al., 2003). There are numerous examples where kaolin particle films repel insect infestations, thus prevent feeding and oviposition although there are at least six other possible mechanisms of action that depend on specific insect-plant relationships (Glenn and

The mortality effects of kaolin and imidacloprid on GWSS were assessed on grape articially infested by *X. fastidiosa.* The GWSS was caged for 48 h on grape vines that was previously infested by *X. fastidiosa* (Tubajika et al., 2007). After acquisition, GWSS, in groups

agriculture because it feeds readily on grape and, in doing so, transmits *Xf,* the causal agent of Pierce's Disease (PD) in grape (Fig.1d). Prior to the appearance of GWSS, California grape growers were able to manage PD in grape that is vectored by a number of other indigenous sharpshooters (Anon, 1992). Unfortunately, the GWSS more adapted to the citrus/grape agroecosystem than other sharpshooters, thus, making it a serious vector of PD that now threatens the grape industry in California (Purcell & Saunders, 1999). Although contact insecticides offer short-term protection against infestations of GWSS, the continued movement of GWSS adults from citrus and uninfested areas often re-infest grapes. Clearly, there is a need to investigate other approaches or technologies that could repel GWSS infestations or prevent them from feeding on grape vines and transmitting PD (Puterka et al., 2003).

In an effort to assess the efficacy of control using kaolin, experimental potted lemon trees in field cages at the Bakersfield location were used to test the efficacy kaolin on GWSS infestation and survival under no-choice conditions. 'Eureka' lemon trees were either treated with 6% kaolin (60 grams kaolin per liter of water) or left untreated as a control. Trees were sprayed with kaolin by a 4-liter hand-pump sprayer until all of the foliage was wetted and then allowed to dry. Either a treated or untreated tree was placed in screencovered cages measuring 1 m2 by 2 m high, and either 50 GWSS adults (Fig. 1a) or 10 instar nymphs (third to fourth stage) (Fig. 1b) were placed at the bases of the trees to disperse. Numbers of GWSS on trees and within the cages were recorded daily for four days to determine infestation rates and survival. GWSS numbers were converted to percentages and analyzed using analysis of variance (ANOVA) (SAS Institute Inc., Cary, NC).

#### **2.1 Survival effect of kaolin and imidacloprid on GWSS in citrus**

Experimental results showed that treatment of grapes with kaolin significantly affected (P < 0.01) the number of nymph and adult populations. GWSS nymphs and adults were greatly repelled by kaolin application in treatments where choice and no-choice experiments were conducted (Fig.3ab). Very few nymphs or no adults were able to colonize lemon treated with kaolin in experiments where either GWSS nymphs or adults were given no choice for colonization, feeding, and oviposition on plant leaves treated with kaolin and the untreated citrus. There were no significant day or treatment-by-day interactions for nymphs (*P =* 0.4) or adults (*P* = 0.19) in the no-choice tests suggesting that GWSS did not vary over the 4-day period after they had found a suitable site on which to settle (Fig.3ab). Survival of nymphs under no-choice conditions was significantly reduced by kaolin treatments (*P* < 0.001) after being exposed for 4 days, and averaged 8.3 ± 4.2% on kaolin versus 75.0 ± 15.2% on untreated trees. There was no Adult GWSS survival on kaolin-treated lemons and 18.8 ± 4.4% on the untreated lemon 1 day after infestation (Fig.3ab). This relationship did not change over the 4-day period. Adult survival was extremely poor in caged tests because many individuals did not settle on lemon trees under caged conditions and clung to the side of cages until death.

Likewise, the choice tests produced very similar results as the no-choice tests. Neither GWSS nymphs nor adults settled on kaolin-treated lemon, which resulted in no colonization over a 4-day period (Fig.3ab). Treatment preferences of nymphs or adults did not change significantly over time. Survival of nymphs significantly declined over time, from 90.0 ± 4.5% at 1 day to 53.3 ± 6.4% at 4 days after infestation. We hypothesize that this decline in population levels was due to the nymphs falling from PF treated trees or from e movement between treated and untreated foliage and being unable to return to the trees. The number of surviving adults did not decline in untreated trees in the no-choice test indicating they had favorable conditions to colonize and thrive. Some GWSS nymphs that were able to find the few untreated spots on kaolin-treated foliage probably remained at those sites during the study period. In contrast to the above findings, adult survival on kaolin-treated foliage did not change over time in either the choice or no-choice tests. We observed that kaolin treated plants are undesirable hosts to both GWSS adults and nymphs in choice and nochoice environment.

412 Insecticides – Pest Engineering

agriculture because it feeds readily on grape and, in doing so, transmits *Xf,* the causal agent of Pierce's Disease (PD) in grape (Fig.1d). Prior to the appearance of GWSS, California grape growers were able to manage PD in grape that is vectored by a number of other indigenous sharpshooters (Anon, 1992). Unfortunately, the GWSS more adapted to the citrus/grape agroecosystem than other sharpshooters, thus, making it a serious vector of PD that now threatens the grape industry in California (Purcell & Saunders, 1999). Although contact insecticides offer short-term protection against infestations of GWSS, the continued movement of GWSS adults from citrus and uninfested areas often re-infest grapes. Clearly, there is a need to investigate other approaches or technologies that could repel GWSS infestations or prevent them from feeding on grape vines and transmitting PD (Puterka et

In an effort to assess the efficacy of control using kaolin, experimental potted lemon trees in field cages at the Bakersfield location were used to test the efficacy kaolin on GWSS infestation and survival under no-choice conditions. 'Eureka' lemon trees were either treated with 6% kaolin (60 grams kaolin per liter of water) or left untreated as a control. Trees were sprayed with kaolin by a 4-liter hand-pump sprayer until all of the foliage was wetted and then allowed to dry. Either a treated or untreated tree was placed in screencovered cages measuring 1 m2 by 2 m high, and either 50 GWSS adults (Fig. 1a) or 10 instar nymphs (third to fourth stage) (Fig. 1b) were placed at the bases of the trees to disperse. Numbers of GWSS on trees and within the cages were recorded daily for four days to determine infestation rates and survival. GWSS numbers were converted to percentages and

Experimental results showed that treatment of grapes with kaolin significantly affected (P < 0.01) the number of nymph and adult populations. GWSS nymphs and adults were greatly repelled by kaolin application in treatments where choice and no-choice experiments were conducted (Fig.3ab). Very few nymphs or no adults were able to colonize lemon treated with kaolin in experiments where either GWSS nymphs or adults were given no choice for colonization, feeding, and oviposition on plant leaves treated with kaolin and the untreated citrus. There were no significant day or treatment-by-day interactions for nymphs (*P =* 0.4) or adults (*P* = 0.19) in the no-choice tests suggesting that GWSS did not vary over the 4-day period after they had found a suitable site on which to settle (Fig.3ab). Survival of nymphs under no-choice conditions was significantly reduced by kaolin treatments (*P* < 0.001) after being exposed for 4 days, and averaged 8.3 ± 4.2% on kaolin versus 75.0 ± 15.2% on untreated trees. There was no Adult GWSS survival on kaolin-treated lemons and 18.8 ± 4.4% on the untreated lemon 1 day after infestation (Fig.3ab). This relationship did not change over the 4-day period. Adult survival was extremely poor in caged tests because many individuals did not settle on lemon trees under caged conditions and clung to the side

Likewise, the choice tests produced very similar results as the no-choice tests. Neither GWSS nymphs nor adults settled on kaolin-treated lemon, which resulted in no colonization over a 4-day period (Fig.3ab). Treatment preferences of nymphs or adults did not change significantly over time. Survival of nymphs significantly declined over time, from 90.0 ± 4.5% at 1 day to 53.3 ± 6.4% at 4 days after infestation. We hypothesize that this decline in population levels was due to the nymphs falling from PF treated trees or from e movement between treated and untreated foliage and being unable to return to the trees. The number

analyzed using analysis of variance (ANOVA) (SAS Institute Inc., Cary, NC).

**2.1 Survival effect of kaolin and imidacloprid on GWSS in citrus** 

al., 2003).

of cages until death.

Fig. 3. Mean number of glassy-winged sharpshooter nymph (n=20) (a) and adults (n=50) (b) per tree 4 days after being released on trees that were treated or not treated with kaolin films in choice versus no-choice experiments.

However, in no-choice experiments, we observed that few nymphs managed to find an untreated site on which to settle when presented with the kaolin-treated plants. This finding implies that good coverage is important when using this material for insect control. When given a choice between kaolin-treated and untreated plants, no GWSS nymphs or adults infested kaolin treated plants.

The choice study may be more representative of what occurs under field conditions because GWSS can utilize more than 75 species of plants (Turner & Pollard, 1959). Therefore, GWSS could easily move to other plants or plant species if they encountered kaolin treated- plants. Results from other studies indicate that kaolin particle films protect plants from insect feeding, oviposition, and by repelling GWSS nymphs and adults by coating the plant surfaces with a mineral barrier (Puterka et al., 2003). This finding is supported by previous reports using kaolin particle film treatments (Glenn et al., 1999; Puterka et al., 2000; Sisterson et al., 2003). There are numerous examples where kaolin particle films repel insect infestations, thus prevent feeding and oviposition although there are at least six other possible mechanisms of action that depend on specific insect-plant relationships (Glenn and Puterka 2005, Puterka and Glenn 2008).

## **2.2 Mortality effect of kaolin and imidacloprid on GWSS on grape**

The mortality effects of kaolin and imidacloprid on GWSS were assessed on grape articially infested by *X. fastidiosa.* The GWSS was caged for 48 h on grape vines that was previously infested by *X. fastidiosa* (Tubajika et al., 2007). After acquisition, GWSS, in groups

Effects of Kaolin Particle Film and Imidacloprid on

to infective GWSS.

vector abundance.

least significant difference test.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 415

(Minsavage et al., 1997)). In determining the incidence of PD, the number of plants with

The treatment of plants with kaolin and imidacloprid, exposure time of plants to the infectious GWSS, and the interaction of treatment by exposure time had an effect on PD incidence (Tubajika et al., 2007). PD incidence was 4%, 7%, and 32% in kaolin-, imidacloprid-

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Treatment\Feeding time 24-hour 48-hour \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Kaolin-treated plants 8 ± 2bA 0 ± 0bB Imidacloprid-treated plants 9 ± 2bA 4 ± 1bB Positive Control (Infested plants)x 48 ± 12aA 19 ± 3aB Negative Control (Non infested plants)x 0 ± 0bA 0 ± 0bA \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ xUntreated plants control plants consisted of water-treated plants exposed or not exposed, respectively

yCalculated from the means for 5 replications. Within columns (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's

zLeaf samples from each plant were assayed for the presence *X. fastidiosa* using ELISA and IC-PCR.

The incidence of PD did not differ among plants treated with imidacloprid and kaolin. *Xf* detection by ELISA assays was 8%, 11%, and 34% in kaolin-, imidacloprid-treated plants, and the untreated controls, respectively (data not shown). However, incidence of PD was greater when the GWSS were allowed to feed for 24 h versus 48 h. There was no difference in PD incidence on plants treated with imidacloprid and kaolin regardless of the length of the GWSS feeding time. In the untreated control plants, disease incidence was higher in plants exposed to infectious GWSS for 24 h than for 48 h (Table 2). The anti-feedant property of imidacloprid may be important in reducing the acquisition of PD bacterium, decreasing the transmission efciency of infected GWSS, and subsequently, reducing the spread of PD bacterium (Tubajika et al., 2007). This nding is consistent with results obtained by Krewer et al., (2002), who showed that imidacloprid treatment slowed the development of PD in the eld but was not effective in preventing the infection of PD in areas with prevalent sources of inoculum and high

Table 2. Effect of kaolin and imidacloprid treatment and length of glassy winged

**3. Kaolin treatment as a barrier to GWSS movement into vineyard** 

Previous studies by Blua et al., (1999) and Perring et al., (2001) have shown that GWSS population utilize citrus as their primary reproductive host, and citrus is the predominant overwintering host, when grapes are dormant during winter. The application of insecticide

sharpshooter feeding time on Pierce's Disease incidence.

symptoms of PD was expressed as percentage of total plant assessed.

Percent (±SE) Pierce's Disease incidenceyz

treated plants, and in untreated controls, respectively (Table 2).

of 10, were caged for 24 or 48 h in small sleeve cages (45 x 55 cm) containing plants either treated or not treated with kaolin and imidacloprid. After 24hr or 48 hr post-treatment, the number of affected GWSS was recorded. The criterion for mortality was insects without any movement (ataxic)1. The GWSS mortality was then expressed as percentage.

The GWSS mortality (%) was significantly (P < 0.05) impacted by particle film treatment (Tubajika et al., 2007). After 24 hr feeding time, GWSS mortality ranged from 0% (Negative and positive control plants) to 58% (kaolin-treated plants). For the 48 hr feeding time, the mortality ranged from 0% (negative control plants) to 100% (kaolin treated-plants) (Table 1).

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_


xUntreated plants control plants consisted of water-treated plants exposed or not exposed, respectively to infective GWSS.

yCalculated from the means for 5 replications. Within columns (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's least significant difference test.

Table 1. Effect of kaolin and imidacloprid treatments and length of glassy-winged sharpshooter (GWSS) feeding time on GWSS mortality in greenhouse experiments.

Overall, the GWSS mortality was greater on imidacloprid-, kaolin-, and the untreated plants when the GWSS fed for 48 h than when they were allowed to feed for 24 h.

However, mortality of the GWSS on untreated control plants and non-infested plants with *X. fastidiosa* was not impacted by the feeding time (negative controls) in these experiments (Table 1). The occurrence of insecticide resistance in GWSS depends on the insecticide used and duration of exposure (Tubajika et al., 2007). Therefore, effective insecticide management strategies and their implementation are necessary for the prevention of rapid resistance development. The mechanism of action of particle films do not rely on toxicity to insects thus resistance to particle films appears unlikely (Puterka & Glenn 2008).

## **2.3 Pierce's Disease (PD) incidence**

Similarly, the impact of kaolin and Imidacloprid on incidence of PD was also assessed on the same grape vines. Each plant was visually assessed at 30-day intervals for PD symptom development which includes stunting, delayed growth, marginal leaf necrosis, and abscission between the petiole and leaf blade. On each assessment date, the total number of plants exhibiting PD symptoms was recorded. Ten leaf samples from each plant were bulked and assayed for the presence of *X. fastidiosa* using assays enzyme-linked immunosorbent assay (ELISA) and immunocapture polymerase chain reaction (IC-PCR)

<sup>1</sup> An inability to coordinate voluntary muscular movements that is symptomatic of some nervous disorders.

of 10, were caged for 24 or 48 h in small sleeve cages (45 x 55 cm) containing plants either treated or not treated with kaolin and imidacloprid. After 24hr or 48 hr post-treatment, the number of affected GWSS was recorded. The criterion for mortality was insects without any

The GWSS mortality (%) was significantly (P < 0.05) impacted by particle film treatment (Tubajika et al., 2007). After 24 hr feeding time, GWSS mortality ranged from 0% (Negative and positive control plants) to 58% (kaolin-treated plants). For the 48 hr feeding time, the mortality ranged from 0% (negative control plants) to 100% (kaolin treated-plants) (Table 1). \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Percent (±SE) Plant mortalityy \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ xUntreated plants control plants consisted of water-treated plants exposed or not exposed, respectively

yCalculated from the means for 5 replications. Within columns (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's

Overall, the GWSS mortality was greater on imidacloprid-, kaolin-, and the untreated plants

However, mortality of the GWSS on untreated control plants and non-infested plants with *X. fastidiosa* was not impacted by the feeding time (negative controls) in these experiments (Table 1). The occurrence of insecticide resistance in GWSS depends on the insecticide used and duration of exposure (Tubajika et al., 2007). Therefore, effective insecticide management strategies and their implementation are necessary for the prevention of rapid resistance development. The mechanism of action of particle films do not rely on toxicity to insects

Similarly, the impact of kaolin and Imidacloprid on incidence of PD was also assessed on the same grape vines. Each plant was visually assessed at 30-day intervals for PD symptom development which includes stunting, delayed growth, marginal leaf necrosis, and abscission between the petiole and leaf blade. On each assessment date, the total number of plants exhibiting PD symptoms was recorded. Ten leaf samples from each plant were bulked and assayed for the presence of *X. fastidiosa* using assays enzyme-linked immunosorbent assay (ELISA) and immunocapture polymerase chain reaction (IC-PCR)

1 An inability to coordinate voluntary muscular movements that is symptomatic of some nervous

Table 1. Effect of kaolin and imidacloprid treatments and length of glassy-winged sharpshooter (GWSS) feeding time on GWSS mortality in greenhouse experiments.

when the GWSS fed for 48 h than when they were allowed to feed for 24 h.

thus resistance to particle films appears unlikely (Puterka & Glenn 2008).

movement (ataxic)1. The GWSS mortality was then expressed as percentage.

Treatment\Feeding time 24-hour 48-hour

 Kaolin-treated plants 58 ± 14aB 100 ± 25aA Imidacloprid-treated plants 34 ± 1bB 71 ± 21bA Positive Control (Infested plants)x 0 ± 0cA 9 ± 1cA Negative Control (Non infested plants)x 0 ± 0cA 0 ± 0cA

to infective GWSS.

disorders.

least significant difference test.

**2.3 Pierce's Disease (PD) incidence** 

(Minsavage et al., 1997)). In determining the incidence of PD, the number of plants with symptoms of PD was expressed as percentage of total plant assessed.

The treatment of plants with kaolin and imidacloprid, exposure time of plants to the infectious GWSS, and the interaction of treatment by exposure time had an effect on PD incidence (Tubajika et al., 2007). PD incidence was 4%, 7%, and 32% in kaolin-, imidaclopridtreated plants, and in untreated controls, respectively (Table 2).

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

## Percent (±SE) Pierce's Disease incidenceyz \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Treatment\Feeding time 24-hour 48-hour \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Kaolin-treated plants 8 ± 2bA 0 ± 0bB Imidacloprid-treated plants 9 ± 2bA 4 ± 1bB Positive Control (Infested plants)x 48 ± 12aA 19 ± 3aB Negative Control (Non infested plants)x 0 ± 0bA 0 ± 0bA

xUntreated plants control plants consisted of water-treated plants exposed or not exposed, respectively to infective GWSS.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

yCalculated from the means for 5 replications. Within columns (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's least significant difference test.

zLeaf samples from each plant were assayed for the presence *X. fastidiosa* using ELISA and IC-PCR.

Table 2. Effect of kaolin and imidacloprid treatment and length of glassy winged sharpshooter feeding time on Pierce's Disease incidence.

The incidence of PD did not differ among plants treated with imidacloprid and kaolin. *Xf* detection by ELISA assays was 8%, 11%, and 34% in kaolin-, imidacloprid-treated plants, and the untreated controls, respectively (data not shown). However, incidence of PD was greater when the GWSS were allowed to feed for 24 h versus 48 h. There was no difference in PD incidence on plants treated with imidacloprid and kaolin regardless of the length of the GWSS feeding time. In the untreated control plants, disease incidence was higher in plants exposed to infectious GWSS for 24 h than for 48 h (Table 2). The anti-feedant property of imidacloprid may be important in reducing the acquisition of PD bacterium, decreasing the transmission efciency of infected GWSS, and subsequently, reducing the spread of PD bacterium (Tubajika et al., 2007). This nding is consistent with results obtained by Krewer et al., (2002), who showed that imidacloprid treatment slowed the development of PD in the eld but was not effective in preventing the infection of PD in areas with prevalent sources of inoculum and high vector abundance.

## **3. Kaolin treatment as a barrier to GWSS movement into vineyard**

Previous studies by Blua et al., (1999) and Perring et al., (2001) have shown that GWSS population utilize citrus as their primary reproductive host, and citrus is the predominant overwintering host, when grapes are dormant during winter. The application of insecticide

Effects of Kaolin Particle Film and Imidacloprid on

California.

**3.2 GWSS egg counts on grape** 

Distance from edge

0.05) according to Fisher's least significant difference test. zInterface.

Distance in meters from citrus.

25 grape leaves per vine.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 417

Fig. 4. Effects of kaolin and conventional insecticides on the mean number (±SE) of glassywinged sharpshooter adults caught per yellow sticky trap (Trece, Salinas, California placed 1 m high in the grape vines in vineyard bordering citrus (interface) and placed every 30.5 m into 365.7 m transects extending into each treatment block (transect) in Kern County,

GWSS egg counts differed significantly (P = 0.0001) between the insecticide and barrier treatments in leaf samples taken by increasing distances from the grape-orange grove interface into the grape vineyard (P = 0.02). The insecticide treatment had significantly (P < 0.0001) larger numbers of GWSS eggs at the edge (0 meters) than at the distances away from the edge of grape leaves in vineyard bordering the citrus (Table 3). This suggest that oviposition on foliage can be affected by proximity to locales of insecticide application.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ GWSS egg counts (±SE)y \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ xVineyard was partitioned to monitor the movement of glassy-winged sharpshooter into the grapes.

yValues are means of 20 observations. Means followed by the same letter did not differ significantly (*P* <

Table 3. Effects of kaolin barrier and conventional insecticides on the numbers ((±SE) of glassy-winged sharpshooter eggs sampled in Bakersfield vineyard bordering citrus from 0 – 365.7 meters from the edge. Yellow sticky traps were monitored from March 9 to May 9 on

of vineyard (m)x Kaolin Insecticides

 0.0 - 4.3 (block 1)y 0.00 ± 0.02aA 14.1 ± 5.1aB 4.4 - 123.7 (block 2) 0.06 ± 0.02aA 5.3 ± 2bB 123.8 - 247.5 (block 3) 0.06 ± 0.02aA 1.6 ± 0.3cB 248.6 - 365.7 (block 4) 0.90 ± 0.03bA 1.9 ± 0.2cB

to control insects can decrease spread and minimize yield loss in certain pathosystems (Purcell & Finley, 1979). Contact insecticides only offer short-term protection against GWSS infestations because of the continued immigration of sharpshooter adults from citrus which re-infest the grapes (Purcell & Finley, 1979).

The prevention of movement of GWSS from citrus to adjacent vineyards by kaolin particle film treatments as barriers was examined. The vineyard, which was comprised of an assortment of Thompson Seedless, Flames Seedless, and Chenin Blanc grape cultivars, was divided into six blocks 164.6 m wide by 365.7 m long (6.5 ha) and assigned treatments of kaolin or conventional insecticides (Puterka et al., 2003). Kaolin barrier treatments only extended 247.5 m into each block; the remaining 152.4 m was left untreated. The insecticide treatment block s had applications the entire 365.7 m distance of the block. Grapes received three bi-weekly applications of kaolin (11.36 kg kaolin, 378.5 per liter water) and six weekly applications of Dimethoate (Dimethoate 400, Platte Chemical Co., Greeley, CO, USA) applied at 4.67 l per ha, methomyl (Lannate LV, E. I. DuPont de Nemours & Co., Wilmington, DE, USA) applied at 2.33 l per ha, and Naled (Dibrom 8E, AMVAC Chemical Corp., Los Angeles, CA, USA) applied at 0.74 l per ha (Puterka et al., 2003). GWSS adults and nymphs were collected from the yellow sticky traps spaced every 30.5 m along the 365.7 m transects per block. Because of edge effect on movement of GWSS from citrus into grapes, plots were partitioned into four distances (0 to 4.3 m (interface); 4.4 to 123.7 m; 123.8 to 247.5 and 247.6 to 365.7 m) to better estimate the number of GWSS. GWSS adults and nymphs will be assessed by weekly monitoring of GWSS adults and nymphs caught per yellow sticky trap (Trece, Salinas, California placed 1 m high in the grape vines in vineyard bordering citrus (interface) and placed every 30.5 m into 365.7 m transects extending into each treatment block (transect) in Kern County, California. GWSS egg masses were sampled by inspecting 25 leaves per vine every 30.5 m along the sticky trap transects in each block.

#### **3.1 GWSS adult counts from traps**

Traps in kaolin-tread plants at the edge of vineyard (0-4.3 m) caught fewer GWSS than traps in the insecticide-treated plants based on one experiment from 9 March to 6 April, except on 16 March (Fig. 4a). After 6 April, there were no differences in Interface trap catches between treatments. Transect traps in kaolin barrier treatment resulted in fewer GWSS on 22 March and 6 April. Data from all other trap dates showed that treatment effects were not significantly different. Counts of GWSS adults were significantly different between sample dates, treatments, and blocks (Fig. 4ab).

Visual counts of in the grape-orange grove interface (0-4.3 meters) revealed higher number of GWSS adult in the insecticide treatment than in the kaolin treatment on week 1 (22 March) (kaolin = 0.06 ± 0.06, insecticides = 0.6 ± 0.6), week 2 (29 March) (kaolin = 0.14 ± 0.02, insecticide = 0.56 ± 0.10), and week 3 (6 April) (kaolin = 0.0 ± 0.0, insecticides = 0.9 ± 0.5). Overall, the GWSS adult counts were much lower in traps beyond the particle film barrier in grape (Fig. 4b). The treatment comparison of three applications of kaolin to six insecticide applications over a 2-month period showed that the kaolin barrier was equally effective or better than numerous insecticide applications in reducing GWSS movement into grapes (Puterka et al., 2003). This may appear to be contrary given the fact that the insecticide used was systemic; however, it appears that the kaolin was a sufficient barrier to prevent GWSS movement into the grape vineyards.

Fig. 4. Effects of kaolin and conventional insecticides on the mean number (±SE) of glassywinged sharpshooter adults caught per yellow sticky trap (Trece, Salinas, California placed 1 m high in the grape vines in vineyard bordering citrus (interface) and placed every 30.5 m into 365.7 m transects extending into each treatment block (transect) in Kern County, California.

## **3.2 GWSS egg counts on grape**

416 Insecticides – Pest Engineering

to control insects can decrease spread and minimize yield loss in certain pathosystems (Purcell & Finley, 1979). Contact insecticides only offer short-term protection against GWSS infestations because of the continued immigration of sharpshooter adults from citrus which

The prevention of movement of GWSS from citrus to adjacent vineyards by kaolin particle film treatments as barriers was examined. The vineyard, which was comprised of an assortment of Thompson Seedless, Flames Seedless, and Chenin Blanc grape cultivars, was divided into six blocks 164.6 m wide by 365.7 m long (6.5 ha) and assigned treatments of kaolin or conventional insecticides (Puterka et al., 2003). Kaolin barrier treatments only extended 247.5 m into each block; the remaining 152.4 m was left untreated. The insecticide treatment block s had applications the entire 365.7 m distance of the block. Grapes received three bi-weekly applications of kaolin (11.36 kg kaolin, 378.5 per liter water) and six weekly applications of Dimethoate (Dimethoate 400, Platte Chemical Co., Greeley, CO, USA) applied at 4.67 l per ha, methomyl (Lannate LV, E. I. DuPont de Nemours & Co., Wilmington, DE, USA) applied at 2.33 l per ha, and Naled (Dibrom 8E, AMVAC Chemical Corp., Los Angeles, CA, USA) applied at 0.74 l per ha (Puterka et al., 2003). GWSS adults and nymphs were collected from the yellow sticky traps spaced every 30.5 m along the 365.7 m transects per block. Because of edge effect on movement of GWSS from citrus into grapes, plots were partitioned into four distances (0 to 4.3 m (interface); 4.4 to 123.7 m; 123.8 to 247.5 and 247.6 to 365.7 m) to better estimate the number of GWSS. GWSS adults and nymphs will be assessed by weekly monitoring of GWSS adults and nymphs caught per yellow sticky trap (Trece, Salinas, California placed 1 m high in the grape vines in vineyard bordering citrus (interface) and placed every 30.5 m into 365.7 m transects extending into each treatment block (transect) in Kern County, California. GWSS egg masses were sampled by inspecting 25 leaves per vine every 30.5 m

Traps in kaolin-tread plants at the edge of vineyard (0-4.3 m) caught fewer GWSS than traps in the insecticide-treated plants based on one experiment from 9 March to 6 April, except on 16 March (Fig. 4a). After 6 April, there were no differences in Interface trap catches between treatments. Transect traps in kaolin barrier treatment resulted in fewer GWSS on 22 March and 6 April. Data from all other trap dates showed that treatment effects were not significantly different. Counts of GWSS adults were significantly different between sample

Visual counts of in the grape-orange grove interface (0-4.3 meters) revealed higher number of GWSS adult in the insecticide treatment than in the kaolin treatment on week 1 (22 March) (kaolin = 0.06 ± 0.06, insecticides = 0.6 ± 0.6), week 2 (29 March) (kaolin = 0.14 ± 0.02, insecticide = 0.56 ± 0.10), and week 3 (6 April) (kaolin = 0.0 ± 0.0, insecticides = 0.9 ± 0.5). Overall, the GWSS adult counts were much lower in traps beyond the particle film barrier in grape (Fig. 4b). The treatment comparison of three applications of kaolin to six insecticide applications over a 2-month period showed that the kaolin barrier was equally effective or better than numerous insecticide applications in reducing GWSS movement into grapes (Puterka et al., 2003). This may appear to be contrary given the fact that the insecticide used was systemic; however, it appears that the kaolin was a sufficient barrier to prevent GWSS

re-infest the grapes (Purcell & Finley, 1979).

along the sticky trap transects in each block.

**3.1 GWSS adult counts from traps** 

dates, treatments, and blocks (Fig. 4ab).

movement into the grape vineyards.

GWSS egg counts differed significantly (P = 0.0001) between the insecticide and barrier treatments in leaf samples taken by increasing distances from the grape-orange grove interface into the grape vineyard (P = 0.02). The insecticide treatment had significantly (P < 0.0001) larger numbers of GWSS eggs at the edge (0 meters) than at the distances away from the edge of grape leaves in vineyard bordering the citrus (Table 3). This suggest that oviposition on foliage can be affected by proximity to locales of insecticide application.


xVineyard was partitioned to monitor the movement of glassy-winged sharpshooter into the grapes. Distance in meters from citrus.

yValues are means of 20 observations. Means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's least significant difference test. zInterface.

Table 3. Effects of kaolin barrier and conventional insecticides on the numbers ((±SE) of glassy-winged sharpshooter eggs sampled in Bakersfield vineyard bordering citrus from 0 – 365.7 meters from the edge. Yellow sticky traps were monitored from March 9 to May 9 on 25 grape leaves per vine.

Effects of Kaolin Particle Film and Imidacloprid on

**4.2 GWSS adult counts in citrus** 

untreated orange trees.

untreated lemon trees (Fig 5b).

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 419

nymphal counts were so low (Catle et al., 2005). However the variability in the application through the irrigation system and/or the rate of uptake could also accounted for the

significant variation in number of nymphs or adults among trees (Catle et al., 2005).

 (a) GWSS nymph counts in 2001 (b) GWSS nymph counts in 2002 Fig. 5. Mean (±SE) number of (a) GWSS nymphs in 2001 and (b) in 2002 on oranges trees treated with imidacloprid compared with untreated trees. Sample size each week was n=12

GWSS Adults were not observed through the rst eight weeks post treatment (Fig 4b). With maturation of the rst nymphs and emergence to adults, numbers of adults rapidly increased in both treated and untreated oranges trees between weeks 9 and 12 (15 June–6 July). Contributing to the inux of young adults into the imidacloprid-treated oranges was the absence of any buffer zones between the treated and untreated trees (two rows of treated trees only). By week 14, however, a divergence in the mean number of adults caught in each treatment had begun, reaching its greatest difference in week 18 (17 August). Difference in adult densities was greater throughout week 25 (5 October) after which treated and untreated GWSS adult counts began to converge (Fig 4b). The mean number of GWSS adults between 15 June and 30 November 2001 was signicant (*P* < 0.0001) between treated and

In contrast to the mean number of nymphs observed in 2001 in orange trees, the difference in number of GWSS nymphs in lemon trees in 2002 was inconsistent treated and untreated lemon trees (Fig 5a). Numbers of GWSS nymphs in untreated lemons were especially erratic, thus making it difficult to observe any clear treatment effect (*P* = 0.40). However, a very similar pattern to the orange trees was observed for GWSS adult counts in treated and

It is clear that protection by imidacloprid was not sporadic or spatially uneven, but rather was confronted by a phenomenon where mass emergence of adults coupled with heightened ight activity simply overwhelmed both treated and untreated trees in the orchard (Catle et al., 2005). After a few weeks, GWSS adult numbers began to decline and population remained consistently and signicantly lower than the untreated orange and lemon trees. Similarly, a rapid increase in nymphal densities occurred in both treated and untreated orange trees in 2001, much as they were observed in Temecula in 2000 (Catle et

trees using a bucker sampler thrusted a t five different locations per tree.

In contrast, the oviposition averaged 0.0 to 0.9 GWSS eggs per block in the kaolin barrier treatment and did not differ (P = 0.50) from one another. Overall, the kaolin barrier resulted in undetectable levels of oviposition, whereas insecticides did not prevent oviposition (Puterka et al., 2003). The egg mass sampling has been shown as the best measure of how kaolin barrier treatments and insecticide treatments affected GWSS activity and host suitability (Puterka et al., 2003). This study suggests that unlike other vectors of Pierce's Disease, the GWSS disperse well into vineyard and is able to vector *Xf*, agent causal of PD in grape. This is consistent with the pattern of PD spread that we observed in Kern County, Bakersfield, CA (Tubajika et al., 2004).

## **4. Impact of imidacloprid on GWSS in citrus**

The epidemic of PD in the vineyards of Temecula in Riverside County, California brought into focus the urgent need to control GWSS populations in CA (Castle et al., 2005) around vineyards. Prior to the first applications of imidacloprid which was made in the spring of 2000 for control of GWSS infestations in Temecula, CA; there had been very limited experience with imidacloprid in citrus or against GWSS in any crop (Catle et al., 2005)). Experiments with imidacloprid treatment were carried out at the University of California's Agricultural Operations at Riverside, CA during 2001 and 2002. The experiment was conducted in a block of 10 rows (6.4 m centers) which were split equally between 30 yearold orange trees (var Frost Valencia grafted on Troyer citrange) and lemons (var Lupe grafted on Cook) situated in the center of a 12-ha orchard. The GWSS nymphs and adults were collected in each bag and counted to determine the effect of imidacloprid on GWSS infestations between treated and untreated citrus trees. A sample consisted of four to six rapid thrusts at ve locations around each tree. The contents of the collecting jar were then emptied into pre-labeled ziplock bags before moving on to the next tree

#### **4.1 GWSS nymph counts in citrus**

Based on the counts in the above experiments, sampling date proved to be a source of variation a two year study. In 2001, ifferences among the three sampling dates from April to June September. GWSS adults and nymphs were signicantly (*P* < 0.0001) greater as observed among four dates in 2002 (P < 0.0001). Differences in GWSS counts between imidacloprid-treated and untreated trees were observed in 2001 (Fig. 5a) when populations were much larger than in 2002 (Fig. 5b). For the rst four weeks following treatment, nymphal counts were high in both treated and untreated trees (Fig 4a). At week 6 posttreatment, a sharp decline in nymphal counts occurred in the imidacloprid-treated oranges coinciding with mean titers of imidacloprid surpassing 5 µg per liter (data not shown). Mean nymphal counts fell to 4.4 (±1.4) by week 8 compared with 30.49 (±4.4) in the untreated control.

The decline on number of nymphs at the beginning of week 9 may be due natural mortality, emigration and emergence to the adult stage, as observed in untreated control. However, mean number of nymphs remained between 30 and 40 through week 10 in the untreated trees while mean counts dropped below 2 at week 10 in the imidacloprid-treated orange trees (Fig 4a). Season-long differences between treated and untreated nymphal counts were signicantly (P < 0.0001) high based on a repeated measures MANOVA. The decline in nymphal counts in 2001 corresponded nicely with the rise of imidacloprid titers in oranges, but this was not apparent in lemons or during the evaluation the following year when nymphal counts were so low (Catle et al., 2005). However the variability in the application through the irrigation system and/or the rate of uptake could also accounted for the significant variation in number of nymphs or adults among trees (Catle et al., 2005).

Fig. 5. Mean (±SE) number of (a) GWSS nymphs in 2001 and (b) in 2002 on oranges trees treated with imidacloprid compared with untreated trees. Sample size each week was n=12 trees using a bucker sampler thrusted a t five different locations per tree.

## **4.2 GWSS adult counts in citrus**

418 Insecticides – Pest Engineering

In contrast, the oviposition averaged 0.0 to 0.9 GWSS eggs per block in the kaolin barrier treatment and did not differ (P = 0.50) from one another. Overall, the kaolin barrier resulted in undetectable levels of oviposition, whereas insecticides did not prevent oviposition (Puterka et al., 2003). The egg mass sampling has been shown as the best measure of how kaolin barrier treatments and insecticide treatments affected GWSS activity and host suitability (Puterka et al., 2003). This study suggests that unlike other vectors of Pierce's Disease, the GWSS disperse well into vineyard and is able to vector *Xf*, agent causal of PD in grape. This is consistent with the pattern of PD spread that we observed in Kern County,

The epidemic of PD in the vineyards of Temecula in Riverside County, California brought into focus the urgent need to control GWSS populations in CA (Castle et al., 2005) around vineyards. Prior to the first applications of imidacloprid which was made in the spring of 2000 for control of GWSS infestations in Temecula, CA; there had been very limited experience with imidacloprid in citrus or against GWSS in any crop (Catle et al., 2005)). Experiments with imidacloprid treatment were carried out at the University of California's Agricultural Operations at Riverside, CA during 2001 and 2002. The experiment was conducted in a block of 10 rows (6.4 m centers) which were split equally between 30 yearold orange trees (var Frost Valencia grafted on Troyer citrange) and lemons (var Lupe grafted on Cook) situated in the center of a 12-ha orchard. The GWSS nymphs and adults were collected in each bag and counted to determine the effect of imidacloprid on GWSS infestations between treated and untreated citrus trees. A sample consisted of four to six rapid thrusts at ve locations around each tree. The contents of the collecting jar were then

Based on the counts in the above experiments, sampling date proved to be a source of variation a two year study. In 2001, ifferences among the three sampling dates from April to June September. GWSS adults and nymphs were signicantly (*P* < 0.0001) greater as observed among four dates in 2002 (P < 0.0001). Differences in GWSS counts between imidacloprid-treated and untreated trees were observed in 2001 (Fig. 5a) when populations were much larger than in 2002 (Fig. 5b). For the rst four weeks following treatment, nymphal counts were high in both treated and untreated trees (Fig 4a). At week 6 posttreatment, a sharp decline in nymphal counts occurred in the imidacloprid-treated oranges coinciding with mean titers of imidacloprid surpassing 5 µg per liter (data not shown). Mean nymphal counts fell to 4.4 (±1.4) by week 8 compared with 30.49 (±4.4) in the

The decline on number of nymphs at the beginning of week 9 may be due natural mortality, emigration and emergence to the adult stage, as observed in untreated control. However, mean number of nymphs remained between 30 and 40 through week 10 in the untreated trees while mean counts dropped below 2 at week 10 in the imidacloprid-treated orange trees (Fig 4a). Season-long differences between treated and untreated nymphal counts were signicantly (P < 0.0001) high based on a repeated measures MANOVA. The decline in nymphal counts in 2001 corresponded nicely with the rise of imidacloprid titers in oranges, but this was not apparent in lemons or during the evaluation the following year when

Bakersfield, CA (Tubajika et al., 2004).

**4.1 GWSS nymph counts in citrus** 

untreated control.

**4. Impact of imidacloprid on GWSS in citrus** 

emptied into pre-labeled ziplock bags before moving on to the next tree

GWSS Adults were not observed through the rst eight weeks post treatment (Fig 4b). With maturation of the rst nymphs and emergence to adults, numbers of adults rapidly increased in both treated and untreated oranges trees between weeks 9 and 12 (15 June–6 July). Contributing to the inux of young adults into the imidacloprid-treated oranges was the absence of any buffer zones between the treated and untreated trees (two rows of treated trees only). By week 14, however, a divergence in the mean number of adults caught in each treatment had begun, reaching its greatest difference in week 18 (17 August). Difference in adult densities was greater throughout week 25 (5 October) after which treated and untreated GWSS adult counts began to converge (Fig 4b). The mean number of GWSS adults between 15 June and 30 November 2001 was signicant (*P* < 0.0001) between treated and untreated orange trees.

In contrast to the mean number of nymphs observed in 2001 in orange trees, the difference in number of GWSS nymphs in lemon trees in 2002 was inconsistent treated and untreated lemon trees (Fig 5a). Numbers of GWSS nymphs in untreated lemons were especially erratic, thus making it difficult to observe any clear treatment effect (*P* = 0.40). However, a very similar pattern to the orange trees was observed for GWSS adult counts in treated and untreated lemon trees (Fig 5b).

It is clear that protection by imidacloprid was not sporadic or spatially uneven, but rather was confronted by a phenomenon where mass emergence of adults coupled with heightened ight activity simply overwhelmed both treated and untreated trees in the orchard (Catle et al., 2005). After a few weeks, GWSS adult numbers began to decline and population remained consistently and signicantly lower than the untreated orange and lemon trees. Similarly, a rapid increase in nymphal densities occurred in both treated and untreated orange trees in 2001, much as they were observed in Temecula in 2000 (Catle et

Effects of Kaolin Particle Film and Imidacloprid on

incidence in either year (Tables 3, 4).

according to Fisher's least significant difference test.

year at Bakersfield, CA.

per ha and naled (Dibrom 8E) applied at 0.74 liter per ha.

previously described.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 421

presence of *X. fastidiosa* using ELISA and IC-PCR and PD incidence was assessed as

When results were averaged across years, the incidence of PD was signicantly (P < 0.05) lower (6%) in plots treated with kaolin than in plots treated with conventional insecticides (14%) (Tables 3, 4). There was no signicant difference in the incidence of PD among grape cultivars in both years (P = 0.67). Also, chemical by cultivar interaction did not affect PD

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Mean (± SE) percent PD incidencexy \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ xValues are means of eight observations (Four replications x two years). Within rows (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (P< 0.05)

yPathogen identity was conrmed by ELISA and IC-PCR assays. *Xylella fastidiosa* strain Temecula (ATCC 700964) collected from grape in Temecula, California was used as a reference control. zdimethoate (Dimethoate 400) applied at 4.67 liter per ha, methomyl (Lannate LV) applied at 2.33 liter

Table 3. Effects of kaolin and conventional insecticides on Pierce's Disease incidence on Thompson Seedless, Flame Seedless, and Chenin Blanc cultivars during the 2001 production

plants treated with insecticides and 4% on kaolin-treated plants (Table 4).

Overall incidence of PD was 43% higher in 2001 than in 2002. All of the infected grape plants were removed from the plots. The inoculum sources for the 2002 season were subsequently reduced (Tubajika et al., 2007). In 2001, PD incidence was 18% on plants treated with insecticides and 4% in kaolin-treated plants (Table3). In 2002; the incidence of PD was 8% on

Our previous study on the spatial patterns of incidence of PD in the lower San Joaquin Valley, CA indicates that GWSS may not be infective but their movements within the vineyard after arriving into the vineyard are important in the spread of the infection, and resulted in the symptoms we recorded (Tubajika et al., 2004). Field studies showed that plants treated with kaolin were less likely to become infected with *X. fastidiosa* and had a lower incidence of PD symptoms than untreated control plants (Tubajika et al., 2007). There are limited reports on application of particle film to control plant diseases caused by the vector population in the elds (Glenn et al., 1999; Blua et al., 1999; Puterka et al., 2000; Puterka et al., 2003). Also, data showed that the GWSS had a lower rate of survival following exposure to kaolin-treated plants for 48 h. This nding is similar to previous reports where kaolin completely protected plants from insect feeding (Blua et al., 1999; Puterka et al., 2000; Puterka et al., 2003). They suggested that the kaolin protects hosts against GWSS by camouaging the plant with a white coating making them visually

unperceivable, or by reecting sunlight, which repels leafhoppers as well as aphids.

Cultivars Kaolin Conventional insecticidesz

 Thompson Seedless 8.4 ±1.1 aB 18.7 ± 2.4 aA Flames Seedless 8.1 ± 1.1 aB 19.2 ± 2.1 aA Chenin Blanc 7.8 ± 0.5 aB 17.5 ± 2.9 aA Mean 8.1 ± 0.4 18. 5 ± 2.2

al., 2005). The antifeedant effects of imidacloprid on other herbivores belonging to Hemiptera (Sternorrhyncha) are well established (Nauen et al., 1999).

Fig. 6. Mean (±SE) number of (a) GWSS adults in 2001 and (b) in 2002 on oranges trees treated with imidacloprid compared with untreated trees. Sample size each week was n=12 trees using a bucker sampler thrusted a t five different locations per tree.

Results from these experiments showed that a recruitment of adults from surrounding orchards during week increased densities in imidacloprid and untreated controls but at levels that were significantly lower for imidacloprid treated–trees. Moreover, Variability in the application through the irrigation system and/or the rate of uptake could have accounted for the signicant variation observed among trees. Also, substantial reductions in GWSS nymphs and adults in imidacloprid treated-trees observed during 2001 production year were sustained for 4-5 months post treatment. Overall, GWSS infestations were reduced in imidacloprid-treated trees than in untreated-trees. Data on the number of GWSS adult and nymph counts from grapevines and citrus studies confirmed that citrus is the primary and preferred host for GWSS as observed in Temecula valley and when given other choices such as grape, almond, cherry, stone fruit, the insect feeds on these hosts as Kern County provides a perfect variety of hosts (Tubajika et al., 2007; Blua et al., 1999; Purcell & Saunder, 1999; Raju et al., 1980).

## **5. Pierce's Disease incidence**

Our previous study on the analysis of the spatial patterns of PD incidence in the lower San Joaquin Valley, CA indicated that GWSS may not be infective but their movements within the vineyard after arrival are important in the spread of the infection, which resulted in the symptoms we recorded (Tubajika et al., 2004).

Experimental plots consisting of Thompson Seedless, Flame Seedless and Chenin Blanc cultivars which were approximately 15-years-old were assessed for Pierce's disease (PD) incidence using a visual evaluation of disease symptoms such as stunted shoot growth, leaf scorch, and persistent petioles, a condition that occurs when the leaf blades scald and abscise, leaving only petioles attached to the shoot (Anon, 1992; Purcell, 1974). Plants were assessed at 30-d intervals. Ten leaf samples from each plant were bulked and assayed for the

al., 2005). The antifeedant effects of imidacloprid on other herbivores belonging to

 (a) GWSS adult counts in 2001 (b) GWSS adult counts in 2002 Fig. 6. Mean (±SE) number of (a) GWSS adults in 2001 and (b) in 2002 on oranges trees treated with imidacloprid compared with untreated trees. Sample size each week was n=12

Results from these experiments showed that a recruitment of adults from surrounding orchards during week increased densities in imidacloprid and untreated controls but at levels that were significantly lower for imidacloprid treated–trees. Moreover, Variability in the application through the irrigation system and/or the rate of uptake could have accounted for the signicant variation observed among trees. Also, substantial reductions in GWSS nymphs and adults in imidacloprid treated-trees observed during 2001 production year were sustained for 4-5 months post treatment. Overall, GWSS infestations were reduced in imidacloprid-treated trees than in untreated-trees. Data on the number of GWSS adult and nymph counts from grapevines and citrus studies confirmed that citrus is the primary and preferred host for GWSS as observed in Temecula valley and when given other choices such as grape, almond, cherry, stone fruit, the insect feeds on these hosts as Kern County provides a perfect variety of hosts (Tubajika et al., 2007; Blua et al., 1999; Purcell &

Our previous study on the analysis of the spatial patterns of PD incidence in the lower San Joaquin Valley, CA indicated that GWSS may not be infective but their movements within the vineyard after arrival are important in the spread of the infection, which resulted in the

Experimental plots consisting of Thompson Seedless, Flame Seedless and Chenin Blanc cultivars which were approximately 15-years-old were assessed for Pierce's disease (PD) incidence using a visual evaluation of disease symptoms such as stunted shoot growth, leaf scorch, and persistent petioles, a condition that occurs when the leaf blades scald and abscise, leaving only petioles attached to the shoot (Anon, 1992; Purcell, 1974). Plants were assessed at 30-d intervals. Ten leaf samples from each plant were bulked and assayed for the

trees using a bucker sampler thrusted a t five different locations per tree.

Saunder, 1999; Raju et al., 1980).

**5. Pierce's Disease incidence** 

symptoms we recorded (Tubajika et al., 2004).

Hemiptera (Sternorrhyncha) are well established (Nauen et al., 1999).

presence of *X. fastidiosa* using ELISA and IC-PCR and PD incidence was assessed as previously described.

When results were averaged across years, the incidence of PD was signicantly (P < 0.05) lower (6%) in plots treated with kaolin than in plots treated with conventional insecticides (14%) (Tables 3, 4). There was no signicant difference in the incidence of PD among grape cultivars in both years (P = 0.67). Also, chemical by cultivar interaction did not affect PD incidence in either year (Tables 3, 4).


xValues are means of eight observations (Four replications x two years). Within rows (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (P< 0.05) according to Fisher's least significant difference test.

yPathogen identity was conrmed by ELISA and IC-PCR assays. *Xylella fastidiosa* strain Temecula (ATCC 700964) collected from grape in Temecula, California was used as a reference control. zdimethoate (Dimethoate 400) applied at 4.67 liter per ha, methomyl (Lannate LV) applied at 2.33 liter per ha and naled (Dibrom 8E) applied at 0.74 liter per ha.

Table 3. Effects of kaolin and conventional insecticides on Pierce's Disease incidence on Thompson Seedless, Flame Seedless, and Chenin Blanc cultivars during the 2001 production year at Bakersfield, CA.

Overall incidence of PD was 43% higher in 2001 than in 2002. All of the infected grape plants were removed from the plots. The inoculum sources for the 2002 season were subsequently reduced (Tubajika et al., 2007). In 2001, PD incidence was 18% on plants treated with insecticides and 4% in kaolin-treated plants (Table3). In 2002; the incidence of PD was 8% on plants treated with insecticides and 4% on kaolin-treated plants (Table 4).

Our previous study on the spatial patterns of incidence of PD in the lower San Joaquin Valley, CA indicates that GWSS may not be infective but their movements within the vineyard after arriving into the vineyard are important in the spread of the infection, and resulted in the symptoms we recorded (Tubajika et al., 2004). Field studies showed that plants treated with kaolin were less likely to become infected with *X. fastidiosa* and had a lower incidence of PD symptoms than untreated control plants (Tubajika et al., 2007). There are limited reports on application of particle film to control plant diseases caused by the vector population in the elds (Glenn et al., 1999; Blua et al., 1999; Puterka et al., 2000; Puterka et al., 2003). Also, data showed that the GWSS had a lower rate of survival following exposure to kaolin-treated plants for 48 h. This nding is similar to previous reports where kaolin completely protected plants from insect feeding (Blua et al., 1999; Puterka et al., 2000; Puterka et al., 2003). They suggested that the kaolin protects hosts against GWSS by camouaging the plant with a white coating making them visually unperceivable, or by reecting sunlight, which repels leafhoppers as well as aphids.

Effects of Kaolin Particle Film and Imidacloprid on

management.

**7. References** 

*Publ. No. 3343*.

35, 1265–1267.

and ornamentals. *Calif. Agric., 53*, 22-25.

populations. . *Pest Manag. Sci, 61*, 75-84.

and plant diseases. J. Econ. Entomol. 92, 759–771.

637). San Francisco: Elsevier Academic Press.

Glassy-Winged Sharpshooter (*Homalodisca vitripennis* ) (Hemiptera: Cicadellidae)… 423

The application of kaolin and imidaclorprid impacted GWSS mortality and PD incidence. Higher GWSS mortality rates were observed on kaolin-treated plants than on untreatedplants. The reduction in PD incidence observed in eld studies suggest that either kaolin treatments as barriers to GWSS infestation or imidacloprid can be effective in reducing GWSS population and PD symptoms, and can be valuable tools for PD management. Both treatments strategies could also be used where insect resistance to imidacloprid would be a concern since insect resistance to particle films would not be a concern. Additionally, these treatments could be combined with other PD management approaches in integrated pest

The costs of chemicals and applications by either commercial applicators or growers were not determined in these studies. However, the choice of chemical to apply (alone or in combination) is important and the level of net returns will depend on this as well as other factors such as frequency of applications, disease intensity, and growth stage at which the chemical sprays are initiated. Additional work assessing these and other potential benets are needed to fully determine the economic value of these treatments for grape production.

Almeida, R. P. P., & Purcell, A. H. (2003). Transmission of *Xylella fastidiosa* to grapevines by Homalodisca coagulata (Hemiptera cicadellidae). J. Econ. Entomol. 96, 264–271. Anon. (1992). *Grape pest management, 2nd ed. Univ. of California, Div. Nat. Res., Okland, CA.* 

Blua, M. J., Phillips, P. A., & Redak, R. A. (1999). A new sharpshooter threatens both crops

Castle, S. J., F.J, B., Jian, B. L., & Toscan, N. C. (2005). Spatial and temporal distribution of

Costa, H. S., Blua, M. S., Bethke, A., & Redak, R. A. (2000). Transmission of *Xylella fastidiosa*

Glenn, D. M., Puterka, F. G., van der Zwet, T., Byers, R. E., & Feldhake, C. (1999).

Glenn, D., and G. J. Puterka. (2005) Particle Films: A new technology for Agriculture. Pp. 1- 44. In J. Janick [ed.] HortReviews. Vol.31. John Wiley & Sons, Inc., Hoboken, NJ. Hemingway, J., & Ranson, H. (2005). Chemical Control of Vectors and Mechanisms of

Hill, B. L., & Purcell, A. H. (1995). Multiplication and movement of *Xylella fastidiosa* within

Krewer, G., Dutcher, J. D., & Chang, C. J. (2002). Imidacloprid 2F insecticide slows development of Pierce's disease bunch grapes. J. Entomol. Sci. 37, 101–112. . Minsavage, G. V., Thompson, C. M., Hopkins, D. L., Leite, B. C., & Stall, R. E. (1994).

Nauen, R., Koob, B., & Elbert, A. (1999). Antifeedant effects of sublethal dosages of

grape and four other plants. Phytopathology 85, 1368–1372. .

imidacloprid on *Bemissia tabaci*. *Entomol. Exp. Appl., 88*, 287-293.

*fastidiosa* in plant tissue. Phytopathology 84, 456–461. .

imidacloprid and thiamethoxam in citrus and impact on *Homalodisca coagulata*

to oleander by the glassy-winged sharpshooter, *Homalodisca coagulata*. HortScience

Hydrophobic particle lms: a new paradigm for suppression of arthropod pests

Resistance. In W. C. Marquardt (Ed.), *Biology of Disease Vectors* (second ed., pp. 627-

Development of a polymerase chain reaction protocol for detection of *Xylella* 


\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Mean (± SEM) percent PD incidencexy

xValues are means of eight observations (Four replications x two years). Within rows (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05) according to Fisher's least significant difference test.

yPathogen identity was conrmed by ELISA and IC-PCR assays. *Xylella fastidiosa* strain Temecula (ATCC 700964) collected from grape in Temecula, CA was used as a reference control culture. zdimethoate (Dimethoate 400) applied at 4.67 liter per ha, methomyl (Lannate LV) applied at 2.33 liter per ha and naled (Dibrom 8E) applied at 0.74 liter per ha.

Table 4. Effects of kaolin and conventional insecticides on Pierce's Disease incidence on Thompson Seedless, Flame Seedless, and Chenin Blanc cultivars during the 2002 production year at Bakersfield, CA.

## **6. Conclusion**

The GWSS was recently introduced into California and poses a serious threat to the grape industry because it is a very effective vector of the bacterium that causes Pierce's disease. This introduction of the GWSS has changed the epidemiology of *Xf* diseases affecting important agronomic and horticultural crops as well as landscape ornamental and native trees in CA by infesting these crops with the bacteria. The epidemic of PD in the vineyards brought into focus the urgent need to control GWSS populations, especially around vineyards. A new technology, called particle film and a systemic insecticide, imidacloprid were assessed both in greenhouse and field.

Results showed that kaolin protects plants from insect feeding, oviposition, and infestation by coating the plant surfaces with a protective mineral barrier. In caged field studies, we found that GWSS nymphs and adults were highly repelled by lemon trees treated with kaolin. In field studies that compared three biweekly kaolin treatments to six weekly contact insecticide treatments, kaolin performed as well as insecticides in reducing GWSS adult numbers and oviposition. A good coverage of plant leaves with kaolin is important when using this material for insect control. In greenhouse studies, GWSS adult counts were reduced greatly in kaolin-treated plants versus untreated control trees.

Based on data on GWSS nymph and adults counts in citrus, the persistence of imidacloprid in citrus varied as near-peak levels of imidacloprid were sustained for 6–10weeks before gradually declining as substantial reductions in GWSS nymphs and adults were observed in imidacloprid-treated trees during the 2001 trial and were sustained for 4–5months posttreatment. Imidacloprid effect on GWSS nymphs was not as well pronounced in the 2002 trial, when overall GWSS infestations were much reduced from the previous year. However, consistently lower adult infestations were observed in 2002 for imidacloprid compared with untreated trees.

The application of kaolin and imidaclorprid impacted GWSS mortality and PD incidence. Higher GWSS mortality rates were observed on kaolin-treated plants than on untreatedplants. The reduction in PD incidence observed in eld studies suggest that either kaolin treatments as barriers to GWSS infestation or imidacloprid can be effective in reducing GWSS population and PD symptoms, and can be valuable tools for PD management. Both treatments strategies could also be used where insect resistance to imidacloprid would be a concern since insect resistance to particle films would not be a concern. Additionally, these treatments could be combined with other PD management approaches in integrated pest management.

The costs of chemicals and applications by either commercial applicators or growers were not determined in these studies. However, the choice of chemical to apply (alone or in combination) is important and the level of net returns will depend on this as well as other factors such as frequency of applications, disease intensity, and growth stage at which the chemical sprays are initiated. Additional work assessing these and other potential benets are needed to fully determine the economic value of these treatments for grape production.

## **7. References**

422 Insecticides – Pest Engineering

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Mean (± SEM) percent PD incidencexy \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ xValues are means of eight observations (Four replications x two years). Within rows (small letters) and across rows (capital letters), means followed by the same letter did not differ significantly (*P* < 0.05)

yPathogen identity was conrmed by ELISA and IC-PCR assays. *Xylella fastidiosa* strain Temecula (ATCC 700964) collected from grape in Temecula, CA was used as a reference control culture. zdimethoate (Dimethoate 400) applied at 4.67 liter per ha, methomyl (Lannate LV) applied at 2.33 liter

Table 4. Effects of kaolin and conventional insecticides on Pierce's Disease incidence on Thompson Seedless, Flame Seedless, and Chenin Blanc cultivars during the 2002 production

The GWSS was recently introduced into California and poses a serious threat to the grape industry because it is a very effective vector of the bacterium that causes Pierce's disease. This introduction of the GWSS has changed the epidemiology of *Xf* diseases affecting important agronomic and horticultural crops as well as landscape ornamental and native trees in CA by infesting these crops with the bacteria. The epidemic of PD in the vineyards brought into focus the urgent need to control GWSS populations, especially around vineyards. A new technology, called particle film and a systemic insecticide, imidacloprid

Results showed that kaolin protects plants from insect feeding, oviposition, and infestation by coating the plant surfaces with a protective mineral barrier. In caged field studies, we found that GWSS nymphs and adults were highly repelled by lemon trees treated with kaolin. In field studies that compared three biweekly kaolin treatments to six weekly contact insecticide treatments, kaolin performed as well as insecticides in reducing GWSS adult numbers and oviposition. A good coverage of plant leaves with kaolin is important when using this material for insect control. In greenhouse studies, GWSS adult counts were

Based on data on GWSS nymph and adults counts in citrus, the persistence of imidacloprid in citrus varied as near-peak levels of imidacloprid were sustained for 6–10weeks before gradually declining as substantial reductions in GWSS nymphs and adults were observed in imidacloprid-treated trees during the 2001 trial and were sustained for 4–5months posttreatment. Imidacloprid effect on GWSS nymphs was not as well pronounced in the 2002 trial, when overall GWSS infestations were much reduced from the previous year. However, consistently lower adult infestations were observed in 2002 for imidacloprid compared with

reduced greatly in kaolin-treated plants versus untreated control trees.

Cultivars Kaolin Conventional insecticides

 Thompson Seedless 4.3 ±0.5 aB 8.8 ± 1.4 aA Flames Seedless 4.2 ± 0.5 aB 8.1 ± 1.1 aA Chenin Blanc 3.9 ± 0.2 aB 7.9 ± 0.9 aA Mean 4.1 ± 0.3 8.3 ± 1.3

according to Fisher's least significant difference test.

were assessed both in greenhouse and field.

year at Bakersfield, CA.

**6. Conclusion** 

untreated trees.

per ha and naled (Dibrom 8E) applied at 0.74 liter per ha.


**19** 

*USA* 

**Use and Management of Pesticides in** 

Carlos García Salazar1, Anamaría Gómez Rodas2 and John C. Wise2

Historically, pesticide management has been an inefficient process for pest control in tree fruit production. The amount of active ingredient released into the pest habitat is thousands of times greater than the amount of pesticide required to kill it if the treated population were confined in a jar or a small space. To illustrate this, lets take the oral LD50 for methyl parathion in rats (18 to 50 mg/kg) (1, 2) and the field dose for controlling the Brown Stink Bug (BSB) in pecans (Penncap-M, 227 grams of active ingredient (AI) per acre) (3). If the weight of the population of BSB in one acre of pecans were the equivalent of 1 kg, taking the LD50 of 50 mg/kg for methyl parathion we may need only 100 mg to kill the entire population if treated in a jar or a small confined space. But since they are distributed all over the field it takes thousand times more product than needed to kill every insect present in the treated field. In today's world, due to the increased cost of the pesticides a more efficient approach is needed, as well as more potent poisons requiring smaller application rates and new environmental legislations. In 1996, the enactment of the Food Quality Protection Act (FQPA) in the United States of America changed for good the way we controlled pests in agriculture in general (4). The enactment of the FQPA legislation demanded the reregistration of all pesticides used in agriculture until 1996. One of the major changes was the introduction of a 10 X safety margin for children for pesticides shown to have a deleterious

The most important changes occurring under FQPA were that pesticides had to be evaluated in relation to the aggregated exposure taking into consideration all potential sources of exposures to residues in food, water and residential use. Pesticides with a common mode of action potential exposure had to be assessed as a cumulative exposure.

Organophosphate (OP) or conventional insecticides used extensively in fruit production were some of the most affected by FQPA. This created the need for new chemicals to substitute those products that were removed from the list of approved materials. Products that were not cancelled required mitigation measures that restricted their use. These substitute pesticides are less effective and require multiple applications to obtain similar results than the ones obtained with conventional OP's. This difference in the performance of the new pesticides increased the cost of pest control. New products are more pest-specific, a

Finally, potential risk must be assessed without considering the benefits.

**1. Introduction** 

effect on children and woman during pregnancy.

*2Department of Entomology, Trevor Nichols Research Center, Fennville, MI* 

**Small Fruit Production** 

*1Agriculture & Agribusiness Institute,* 

*Michigan State University Extension, West Olive, MI* 


## **Use and Management of Pesticides in Small Fruit Production**

Carlos García Salazar1, Anamaría Gómez Rodas2 and John C. Wise2 *1Agriculture & Agribusiness Institute, Michigan State University Extension, West Olive, MI 2Department of Entomology, Trevor Nichols Research Center, Fennville, MI USA* 

## **1. Introduction**

424 Insecticides – Pest Engineering

Perring, T. M., Farrar, C. A., & Blua, M. J. (2001). Proximity to citrus inuences Pierce's

Pooler, M. R., Myung, I. S., Bentz, J., Sherald, J., & Hartung, J. S. (1997). Detection of *Xylella* 

Purcell, A. H. (1974). Spatial patterns of Pierce's disease in the Napa Valley. Am. J. Enol.

Purcell, A. H., & Finley, A. H. (1979). Evidence for noncirculative transmis¬sion of Pierce's

Purcell, A. H., Saunders, R. S., Hendson, M., Grebus, M. E., & Henry, M. J. (1999). Causal role of *Xylella fastidiosa* in Oleander leaf scorch disease. Phytopathology 89, 53–58. Purcell, A. H., & Saunders, S. R. (1999). Glassy-winged sharpshooters expected to increase

Puterka, G. J., Glenn, D. M., Sekutowski, D. G., Unruh, T. R., & Jones, S. K. (2000). Progress

Puterka, G. J., Reinke, M., Luvisi, D., Ciomperlik, M. A., Bartels, D., Wendel, L., et al., (2003).

Puterka, G. J., and D. M. Glenn. (2008) Kaolin-Based Particle Films for Arthropod Control.

Raju, B. C., Nome, S. F., Docampo, D. M., Gohen, A. C., Nyland, G., & Lowe, S. K. (1980).

Romoser, W. S., & Stoffolano, G. J. (1998). *The Science of Entomology.* Boston, MA:

Sisterson, M. S., Liu, Y. B., Kerns, D. L., & Tabashinik, B. A. (2003). Effects of kaolin particle

Sorensen, S. J., & Gill, R. J. (1996). A range extension of *Homalodosca coagulata* (Say)

Tubajika, K. M., Civerolo, E. L., Ciomperlik, M. A., Hashim, J. M., & Luvisi, D. A. (2004).

Tubajika, K. M., Civerolo, E. L., Puterka, G. J., Hashim, J. M., & Luvisi, D. A. (2007). The

Turner, W. F., & Pollard, H. N. (1959). Life histories and behavior of five insect vectors of

Unruh, T. R., Knight, A. L., Upton, J., Glenn, D. M., & Puturka, G. J. (2000). Particle-film for

growing areas in California. Am. J. Enol. Viticult. 31, 144–148.

(Lepidoptyera: Gelechiidae). *J. Econ. Entomol., 96*(3), 805-810.

Joaquin Valley in California. Phytopathology 94, 1136–1144

toward liquid formulations of particle lms for insect and disease control in pear.

Particle lm, Surround WP, effects on glassy-winged sharpshooter behavior and its utility as a barrier to sharpshooter infestations in grapes. Plant Health Prog.Online,

pp. 2075-2080, In (J. L. Capinera, Ed.) Encyclopedia of Entomology, Kluwer

Alternative hosts of Pierce's disease of grapevines that occur adjacent to grape

film on oviposition, larval mining, and infestation of cotton by pink bollworm

(Hemiptera: Clypeorrhyncha: Cicadellidae) to southern California *Pan-Pac* 

Analysis of the spatial patterns of Pierce's disease incidence in the lower San

effects of kaolin, harpin, and imidacloprid on development of Pierce's disease in

suppression of the codling moth in apple and pear orchards. *J. Econ.Entomol., 93*,

*fastidiosa* in potential insects by immunomagnetic separation and nested

disease in Temecula Valley vineyards. Calif. Agric. 55, 13–18.

polymerase chain reaction. Lett. Appl. Micro¬biol. 25, 123–126.

disease bacterium by sharpshooter vectors. Science 206, 839–841.

Viticutt. 25, 162–167. .

plant disease. Calif. Agric. 53, 26–27.

Environ. Entomol. 29, 329–339.

doi: 10.1094/PHP-2003-0321-RS. .

Academic Publishers.

WCB/McGraw-Hill.

*Entomol., 72*, 160-161.

737-743.

grape *Crop Protection, 26*, 92-99.

phony peach disease. USDA Techn. Bull.1188.

Historically, pesticide management has been an inefficient process for pest control in tree fruit production. The amount of active ingredient released into the pest habitat is thousands of times greater than the amount of pesticide required to kill it if the treated population were confined in a jar or a small space. To illustrate this, lets take the oral LD50 for methyl parathion in rats (18 to 50 mg/kg) (1, 2) and the field dose for controlling the Brown Stink Bug (BSB) in pecans (Penncap-M, 227 grams of active ingredient (AI) per acre) (3). If the weight of the population of BSB in one acre of pecans were the equivalent of 1 kg, taking the LD50 of 50 mg/kg for methyl parathion we may need only 100 mg to kill the entire population if treated in a jar or a small confined space. But since they are distributed all over the field it takes thousand times more product than needed to kill every insect present in the treated field.

In today's world, due to the increased cost of the pesticides a more efficient approach is needed, as well as more potent poisons requiring smaller application rates and new environmental legislations. In 1996, the enactment of the Food Quality Protection Act (FQPA) in the United States of America changed for good the way we controlled pests in agriculture in general (4). The enactment of the FQPA legislation demanded the reregistration of all pesticides used in agriculture until 1996. One of the major changes was the introduction of a 10 X safety margin for children for pesticides shown to have a deleterious effect on children and woman during pregnancy.

The most important changes occurring under FQPA were that pesticides had to be evaluated in relation to the aggregated exposure taking into consideration all potential sources of exposures to residues in food, water and residential use. Pesticides with a common mode of action potential exposure had to be assessed as a cumulative exposure. Finally, potential risk must be assessed without considering the benefits.

Organophosphate (OP) or conventional insecticides used extensively in fruit production were some of the most affected by FQPA. This created the need for new chemicals to substitute those products that were removed from the list of approved materials. Products that were not cancelled required mitigation measures that restricted their use. These substitute pesticides are less effective and require multiple applications to obtain similar results than the ones obtained with conventional OP's. This difference in the performance of the new pesticides increased the cost of pest control. New products are more pest-specific, a

Use and Management of Pesticides in Small Fruit Production 427

C per 100 meters). Cool air tends to sink, displacing lower warm air and causing vertical mixing. As a warm air layer rises, suspended droplets rise with it and dissipate into the upper layers by normal air turbulence and vertical mixing. Vertical mixing is the result of the ADIABATIC effect. Adiabatic effect refers to the behavior of a parcel of air when it moves up and down through a gradient of pressure in the atmosphere—this being greatest at the surface and diminishes with height. This process occurs as follows: **Rising Air Parcel.** This movement causes *adiabatic cooling*. As the air parcel rises it enters a low pressure region and this causes the parcel to expand. This sudden expansion causes a decrease in the parcel's temperature and it starts cooling down. This effect is similar to opening a canister of propane gas, while it escapes the gas forms a frosty cap at the tip of the exit. **Falling Air Parcel.** This creates the *adiabatic warming*. As the parcel of air starts falling down, it enters a region of high pressure. This change in atmospheric pressure compresses the parcel of air and causes an increase in its temperature. The parcel of air continues descending until its

When the rising parcel of air gets cooler than the surrounding air, it stops rising. This produces a situation in which the temperature at ground level and the temperature several hundred meters above the ground are the same. There is no vertical movement of the air and the atmosphere is in a condition of stability. Conversely, if the rising parcel gets warmer than the surrounding air, it continues rising creating the condition of atmospheric instability

*Movement of pesticide droplets.* When water-encapsulated pesticide droplets exit the nozzle their final deposition depends on the droplet size, the air and water temperature, and the environmental relative humidity. Big droplets (400-500 microns) will reach the foliage before losing substantial amount of volume. But all other droplets that do not reach the foliage are affected by the adiabatic effect. When those droplets are falling to the ground they are compressed by the atmosphere, their temperature rises beyond the surrounding air and they become airborne until their temperature equals the surrounding air. As those droplets cool

where parcels of air move up and down as they warm up and cool down.

down they fall back and the process is repeated again (Figure 2).

pressure and temperature equals the surrounding air (Figure 1).

Fig. 1. Adiabatic effect.

small amount of a.i. is required per application (< 1 oz /acre) and intensive site-specific IPM pest scouting is required to effectively use them.

Under this new paradigm, an effective use and management of pesticides require growers and IPM practitioners to know the chemistry of the pesticide being used, the biology of the pest and its behavior, the influence of weather, plant structure, and equipment used to release the pesticide into the pest habitat.

## **2. Problems associated with the use of pesticides in agriculture**

Every year there are a number of claims to farm insurance companies that are the result of errors during the application of pesticides. The main causes for losses of crops due to misapplications are equipment failure (24%), improper tank mixes (33%) and drift (33%). Other causes are application in the wrong field and problems reading the label of the product (5). The most important main factors affecting the efficacy of pest control are**,** the prevailing weather conditions during the application, the type of nuzzles and conditions of the spray equipment, the characteristics of the crop and the behavior of the pest. Although the applicator can not control some of these factors there are always measures that may help reduce the pesticide environmental impact and increase their effectiveness.

#### **3. Weather conditions during the application**

*Wind velocity.* It is usually the most critical factor of all meteorological conditions affecting the efficacy of the pesticide application. The greater the wind speed, the farther off-target a droplet of a given size will be carried. The larger the droplet, the less it is affected by the wind and the faster it will fall. High winds however, can cause even larger droplets to move off-target. Therefore, spraying operations should be stopped if wind speeds are excessively high (> 10 miles/hour or 16 Km/hour). In 2001, the US Environmental Protection Agency (EPA) issued a recommendation that indicated that for pesticide applications in orchards and vineyards and other fruit crops the maximum wind speed during the application should be between 3 and 10 miles/hour (4.5-16 Km/hour) (EPA 730-N-01-006). To illustrate the effect of wind velocity on pesticide droplets we may take the data from Rose and Lambi (1985) (6). They showed that droplets 100 microns in diameter travel 4.6 m in a wind speed of 1.6 Km/hour. However, in a wind speed of 8 Km/hour they travel 23.1 m. A droplet of 400 microns under the same conditions travels 1 and 5 m, respectively.

*Relative Humidity*. As droplets fall through the air, they evaporate into the atmosphere. This evaporation reduces the size and mass of the particle enabling it to remain airborne longer and, under the right conditions, to drift farther from the application site. The rate at which water evaporates from the spray particles depends primarily on air temperature and relative humidity. At 70 % relative humidity and 78°F, a 100-micron droplet will fall 5 feet and hit the ground before evaporating to half its original diameter. However, at 30% relative humidity and 78°F, a 100-micron droplet quickly evaporates and becomes one-eighth of its original volume while falling only 2.5 feet. While evaporative loss of spray materials occurs under almost all atmospheric conditions, these losses are less pronounced under the environmental conditions that occur during the cooler parts of the day - early morning and late afternoon. The relative humidity is usually highest during these cooler periods (7).

*Atmospheric stability*. It is an important factor influencing drift. Under normal (stable) meteorological conditions, the air temperature decreases by 5.4°F per 1,000 feet of height (1°

small amount of a.i. is required per application (< 1 oz /acre) and intensive site-specific IPM

Under this new paradigm, an effective use and management of pesticides require growers and IPM practitioners to know the chemistry of the pesticide being used, the biology of the pest and its behavior, the influence of weather, plant structure, and equipment used to

Every year there are a number of claims to farm insurance companies that are the result of errors during the application of pesticides. The main causes for losses of crops due to misapplications are equipment failure (24%), improper tank mixes (33%) and drift (33%). Other causes are application in the wrong field and problems reading the label of the product (5). The most important main factors affecting the efficacy of pest control are**,** the prevailing weather conditions during the application, the type of nuzzles and conditions of the spray equipment, the characteristics of the crop and the behavior of the pest. Although the applicator can not control some of these factors there are always measures that may help

*Wind velocity.* It is usually the most critical factor of all meteorological conditions affecting the efficacy of the pesticide application. The greater the wind speed, the farther off-target a droplet of a given size will be carried. The larger the droplet, the less it is affected by the wind and the faster it will fall. High winds however, can cause even larger droplets to move off-target. Therefore, spraying operations should be stopped if wind speeds are excessively high (> 10 miles/hour or 16 Km/hour). In 2001, the US Environmental Protection Agency (EPA) issued a recommendation that indicated that for pesticide applications in orchards and vineyards and other fruit crops the maximum wind speed during the application should be between 3 and 10 miles/hour (4.5-16 Km/hour) (EPA 730-N-01-006). To illustrate the effect of wind velocity on pesticide droplets we may take the data from Rose and Lambi (1985) (6). They showed that droplets 100 microns in diameter travel 4.6 m in a wind speed of 1.6 Km/hour. However, in a wind speed of 8 Km/hour they travel 23.1 m. A droplet of

*Relative Humidity*. As droplets fall through the air, they evaporate into the atmosphere. This evaporation reduces the size and mass of the particle enabling it to remain airborne longer and, under the right conditions, to drift farther from the application site. The rate at which water evaporates from the spray particles depends primarily on air temperature and relative humidity. At 70 % relative humidity and 78°F, a 100-micron droplet will fall 5 feet and hit the ground before evaporating to half its original diameter. However, at 30% relative humidity and 78°F, a 100-micron droplet quickly evaporates and becomes one-eighth of its original volume while falling only 2.5 feet. While evaporative loss of spray materials occurs under almost all atmospheric conditions, these losses are less pronounced under the environmental conditions that occur during the cooler parts of the day - early morning and late afternoon. The relative humidity is usually highest during these cooler periods (7). *Atmospheric stability*. It is an important factor influencing drift. Under normal (stable) meteorological conditions, the air temperature decreases by 5.4°F per 1,000 feet of height (1°

**2. Problems associated with the use of pesticides in agriculture** 

reduce the pesticide environmental impact and increase their effectiveness.

400 microns under the same conditions travels 1 and 5 m, respectively.

**3. Weather conditions during the application** 

pest scouting is required to effectively use them.

release the pesticide into the pest habitat.

C per 100 meters). Cool air tends to sink, displacing lower warm air and causing vertical mixing. As a warm air layer rises, suspended droplets rise with it and dissipate into the upper layers by normal air turbulence and vertical mixing. Vertical mixing is the result of the ADIABATIC effect. Adiabatic effect refers to the behavior of a parcel of air when it moves up and down through a gradient of pressure in the atmosphere—this being greatest at the surface and diminishes with height. This process occurs as follows: **Rising Air Parcel.** This movement causes *adiabatic cooling*. As the air parcel rises it enters a low pressure region and this causes the parcel to expand. This sudden expansion causes a decrease in the parcel's temperature and it starts cooling down. This effect is similar to opening a canister of propane gas, while it escapes the gas forms a frosty cap at the tip of the exit. **Falling Air Parcel.** This creates the *adiabatic warming*. As the parcel of air starts falling down, it enters a region of high pressure. This change in atmospheric pressure compresses the parcel of air and causes an increase in its temperature. The parcel of air continues descending until its pressure and temperature equals the surrounding air (Figure 1).

#### Fig. 1. Adiabatic effect.

When the rising parcel of air gets cooler than the surrounding air, it stops rising. This produces a situation in which the temperature at ground level and the temperature several hundred meters above the ground are the same. There is no vertical movement of the air and the atmosphere is in a condition of stability. Conversely, if the rising parcel gets warmer than the surrounding air, it continues rising creating the condition of atmospheric instability where parcels of air move up and down as they warm up and cool down.

*Movement of pesticide droplets.* When water-encapsulated pesticide droplets exit the nozzle their final deposition depends on the droplet size, the air and water temperature, and the environmental relative humidity. Big droplets (400-500 microns) will reach the foliage before losing substantial amount of volume. But all other droplets that do not reach the foliage are affected by the adiabatic effect. When those droplets are falling to the ground they are compressed by the atmosphere, their temperature rises beyond the surrounding air and they become airborne until their temperature equals the surrounding air. As those droplets cool down they fall back and the process is repeated again (Figure 2).

Use and Management of Pesticides in Small Fruit Production 429

Under stable air conditions since the temperature at ground level is the same over the next few hundred meters above the ground, vertical air movement is hindered and there is little or no atmospheric mixing. Consequently, fine droplets will hang in the air and slowly

**5. Spray application under stable conditions** 

Fig. 4. Spray application early in the morning with no vertical mixing.

hours when the ground cools the air layer immediately above it.

In Figure 4 the effect of atmospheric stability is presented. Fine spray droplets remain aloft traveling horizontally away from the target area. In this picture, pesticide drift goes over the road falling on passing by vehicles. Applications under these circumstances are the most problematic because they cause dangerous drifts that end up creating environmental

A very common type of atmospheric stability is the one created by an inversion of the gradient of temperature occurring in the atmosphere. Under conditions of instability the air temperature decreases with an increase in altitude, 5.4°F per 1,000 feet of height (1° C per 100 meters, approximately). Very often the air temperature near the ground is cooler than the air a few meters above ground; the surrounding air gets warmer with increasing altitude. This creates a layer of hot air that acts like a cap that does not allow the vertical movement of the air holding down cooler air underneath. This phenomenon is what we call thermal inversion and is very frequent very early in the morning and during the evening

**Spray application under thermal inversion conditions**. Pesticide applications conducted under conditions of thermal inversion are prone to produce large quantities of dangerous drifts. Pesticide particles suspended in the cool layer cannot move anywhere except laterally, possibly for several miles. **Do not spray** during thermal inversions, when air close to the ground is colder than the air above it. You should not spray early in the morning in still air if sensitive areas are nearby (especially down gradient if the terrain is sloped). Applicators concerned about these adverse spraying conditions should wait until late

diffuse superficially on relatively level terrain.

pollution.

Fig. 2. Adiabatic effect on droplets containing the pesticides as they exit the nuzzle.

## **4. Spray application under unstable conditions**

As warm air rises, suspended droplets rise with it and dissipate into the upper layers by normal air turbulence and vertical mixing. Under these conditions, the opportunity for crop injury at any specific off target site is very small because the pesticide is dispersed and diluted into the atmosphere. **Figure 3**, shows the application with a cannon sprayer in blueberries under unstable conditions. The picture shows that most of the drift remains confined to the first 5-6 rows facing the sprayer and the cloud of droplets that constituted drift moves up with little horizontal movement out of the target area

Fig. 3. Spray application under unstable conditions in a blueberry field.

Fig. 2. Adiabatic effect on droplets containing the pesticides as they exit the nuzzle.

As warm air rises, suspended droplets rise with it and dissipate into the upper layers by normal air turbulence and vertical mixing. Under these conditions, the opportunity for crop injury at any specific off target site is very small because the pesticide is dispersed and diluted into the atmosphere. **Figure 3**, shows the application with a cannon sprayer in blueberries under unstable conditions. The picture shows that most of the drift remains confined to the first 5-6 rows facing the sprayer and the cloud of droplets that constituted

**4. Spray application under unstable conditions** 

drift moves up with little horizontal movement out of the target area

Fig. 3. Spray application under unstable conditions in a blueberry field.

## **5. Spray application under stable conditions**

Under stable air conditions since the temperature at ground level is the same over the next few hundred meters above the ground, vertical air movement is hindered and there is little or no atmospheric mixing. Consequently, fine droplets will hang in the air and slowly diffuse superficially on relatively level terrain.

Fig. 4. Spray application early in the morning with no vertical mixing.

In Figure 4 the effect of atmospheric stability is presented. Fine spray droplets remain aloft traveling horizontally away from the target area. In this picture, pesticide drift goes over the road falling on passing by vehicles. Applications under these circumstances are the most problematic because they cause dangerous drifts that end up creating environmental pollution.

A very common type of atmospheric stability is the one created by an inversion of the gradient of temperature occurring in the atmosphere. Under conditions of instability the air temperature decreases with an increase in altitude, 5.4°F per 1,000 feet of height (1° C per 100 meters, approximately). Very often the air temperature near the ground is cooler than the air a few meters above ground; the surrounding air gets warmer with increasing altitude. This creates a layer of hot air that acts like a cap that does not allow the vertical movement of the air holding down cooler air underneath. This phenomenon is what we call thermal inversion and is very frequent very early in the morning and during the evening hours when the ground cools the air layer immediately above it.

**Spray application under thermal inversion conditions**. Pesticide applications conducted under conditions of thermal inversion are prone to produce large quantities of dangerous drifts. Pesticide particles suspended in the cool layer cannot move anywhere except laterally, possibly for several miles. **Do not spray** during thermal inversions, when air close to the ground is colder than the air above it. You should not spray early in the morning in still air if sensitive areas are nearby (especially down gradient if the terrain is sloped). Applicators concerned about these adverse spraying conditions should wait until late

Use and Management of Pesticides in Small Fruit Production 431

Different air-assisted equipment produces different types of spray which in turn have different distribution on the target crop. Table 1 shows the properties of different air-

**Technology Droplet size Canopy Penetration Field Distribution**

Aerial Application Fine – Medium Medium Vertically Variable Proptec Sprayer Fine – Medium High Uniform

Show (1996) gave a very good definition of every type of spray depending on the droplet size (5). Accordingly, a very fine spray (VF) is made of droplets with a diameter < 182 microns (µm); A fine spray (F) is made of droplets with a diameter ranging from 183 to 280 µm; Medium spray (M) is made of droplets ranging from 281 to 429 µm; A coarse spray (C)

The type of spray produced by the equipment determines the volume of water needed to spray the recommended dose of pesticide. The following table indicates how much water is

Coarse High 500 108 Medium Medium 200 - 500 32 -108 Fine Low 100 - 200 6 - 32 Table 2. Type of spray and its effect on the amount of water needed to spray one acre of

After selecting the right environmental conditions and the equipment for the spray application the next factors determining the success of your applications will be, type of

Plant structure is important in determining the final destination of your spray. It also influences the success of pest control. Canopy is the place where the pest is found scouting for food, mating, looking for oviposition sites or escaping from natural enemies and adverse environmental factors. This also includes escaping from the applications of pesticides. Therefore, in determining the amount of water and the equipment required to control a pest

In blueberries, for example, the structure of the bush is important in the efficacy of the application of insecticides and fungicides using air blast sprayers. A well pruned bush will allow for better penetration of the pesticide. Thus, you could achieve better pest control and

Table 1. Pesticide application equipment and characteristics of spray deposition.

is made of droplets ranging from 430 to 531 µm in diameter, etc.

needed to cover one acre of blueberries using different types of sprays.

Spray type Volume type Droplet diameter

**7. Plant structure and canopy: where do your chemicals end up?** 

problem, type of target crop and plant structure are crucial.

Coarse High Uniform

Medium Medium Horizontally-

(µm)

Variable

Application rate gal/acre

assisted equipment on the deposition of pesticides on the target canopy.

**Application** 

blueberries.

canopy and plant structure.

Air-blast Sprayer Fine – Very

Cannon Sprayer Very Fine –

afternoon or early evening to spray, when there is less chance of the atmosphere being inverted and conditions are more favorable.

## **6. Spray equipment used for pesticide applications in small fruit crops**

The most important factor in providing an efficient pesticide cover is type of equipment. The efficacy of your pest control depends on the right selection of your spraying equipment. Different types of spraying equipment are used in small fruit crops for pest control. Airblast spraying equipment is the most common equipment employed for pesticide application (Figure 5).

Fig. 5. Air assisted sprayers: right, a cannon sprayer; left, conventional air-blast sprayer.

However, when the crop canopy makes it difficult to apply pesticides using ground equipment fixed-wing aircrafts are mainly used (Figure 6).

Fig. 6. Aerial application of insecticide in blueberries near harvest with fixed-wing aircrafts (Fennville, Michigan 2006).

afternoon or early evening to spray, when there is less chance of the atmosphere being

The most important factor in providing an efficient pesticide cover is type of equipment. The efficacy of your pest control depends on the right selection of your spraying equipment. Different types of spraying equipment are used in small fruit crops for pest control. Airblast spraying equipment is the most common equipment employed for pesticide

Fig. 5. Air assisted sprayers: right, a cannon sprayer; left, conventional air-blast sprayer.

equipment fixed-wing aircrafts are mainly used (Figure 6).

However, when the crop canopy makes it difficult to apply pesticides using ground

Fig. 6. Aerial application of insecticide in blueberries near harvest with fixed-wing aircrafts

**6. Spray equipment used for pesticide applications in small fruit crops** 

inverted and conditions are more favorable.

application (Figure 5).

(Fennville, Michigan 2006).


Different air-assisted equipment produces different types of spray which in turn have different distribution on the target crop. Table 1 shows the properties of different airassisted equipment on the deposition of pesticides on the target canopy.

Table 1. Pesticide application equipment and characteristics of spray deposition.

Show (1996) gave a very good definition of every type of spray depending on the droplet size (5). Accordingly, a very fine spray (VF) is made of droplets with a diameter < 182 microns (µm); A fine spray (F) is made of droplets with a diameter ranging from 183 to 280 µm; Medium spray (M) is made of droplets ranging from 281 to 429 µm; A coarse spray (C) is made of droplets ranging from 430 to 531 µm in diameter, etc.

The type of spray produced by the equipment determines the volume of water needed to spray the recommended dose of pesticide. The following table indicates how much water is needed to cover one acre of blueberries using different types of sprays.


Table 2. Type of spray and its effect on the amount of water needed to spray one acre of blueberries.

## **7. Plant structure and canopy: where do your chemicals end up?**

After selecting the right environmental conditions and the equipment for the spray application the next factors determining the success of your applications will be, type of canopy and plant structure.

Plant structure is important in determining the final destination of your spray. It also influences the success of pest control. Canopy is the place where the pest is found scouting for food, mating, looking for oviposition sites or escaping from natural enemies and adverse environmental factors. This also includes escaping from the applications of pesticides. Therefore, in determining the amount of water and the equipment required to control a pest problem, type of target crop and plant structure are crucial.

In blueberries, for example, the structure of the bush is important in the efficacy of the application of insecticides and fungicides using air blast sprayers. A well pruned bush will allow for better penetration of the pesticide. Thus, you could achieve better pest control and

Use and Management of Pesticides in Small Fruit Production 433

Van Ee, found that spray penetration and deposition in all types of canopies was greater in the bush's side facing the sprayer. However, spray penetration and coverage was greatly reduced at the far side of the bush when the field was lightly pruned. Conversely, medium or heavy pruning allowed more penetration and deposition than in light pruned bushes. Moderate or heavy pruning allowed similar deposition, except at the top of the bush. In the heavily pruned bushes pesticide deposition was greater than at any other canopy structure

In 2006, we conducted a study to determine the spray deposition patterns in mature blueberries using different spray equipment. A mature Jersey field was sprayed with Surround WP (kaolin clay) utilizing a cannon sprayer, an airblast sprayer and a 20-gallon

We found that a spray application of 50 gal/acre in light pruned bushes resulted in high deposition at the bottom and middle portion of the canopy and less deposition on the top of the bush. The same volume applied with a cannon sprayer resulted in higher spray deposition on top of the bush but less deposition on the middle section and much less at the

The application of a spray volume of 20 gallons to the same field with fixed-wing aircraft

These results were very important to establish what type of equipment we need to use to

Finally, the purpose of the spray application is to place the pesticide in contact with the pest. This objective can be achieved in two ways; 1) depositing the pesticide in the habitat of the pest were the pest may enter in contact with the pesticide or 2) placing the pesticide in contact with the pest by taking advantage of its behavior. In this case the pest "finds" the

For plant diseases and insects that do not move a lot and remain confined to a small area (a leaf, branch, bush or section of the canopy) we need to bring the pesticide to the pest by targeting their habitat. Spray cover has to be very good with enough number of droplets per square centimeter of foliage to maximize the probability of the pest getting in contact with the spray. In the case of blueberry aphids and fruitworms, pest control measures require placing the insecticide in the habitat of the pest. Blueberry aphids live at the bottom, inside

apply pesticides in blueberry fields in order to optimize the efficacy of the pesticide.

resulted in an even deposition of the spray from top to bottom of the bush.

Fig. 7.3. Heavily pruned bushes.

aerial application (Wise et al. unpublished results).

product while moving around the canopy of the crop.

(7.3).

bottom of the bush.

**8. Pest habitat and behavior** 

savings in product. On the other hand, thick canopies prevent the placement of the product in the habitat of the pest resulting in poor and more expensive pest control. Van Ee et al.(2000) demonstrated how different types of pruning affected pesticide deposition in "highbush" blueberries (*Vaccinium coymbosnm* L) at different times during the plant growth season. Their goal was to determine how sprayer type, pruning severity, and canopy development interacted to affect spray deposition patterns. Deposition was measured as the percentage of the surface area of card targets that was covered following applications of black dye. Light measurements indicated that the canopy of blueberry bushes, regardless of pruning treatment, closed by the middle of June, and light levels within the canopy changed little from then until fruit harvest in August. They used a standard airblast sprayer that pushed spray up and white kromcoat cards clipped to each target. Targets were clipped to branches in a random orientation in the same two bushes used for the light measurements. Figures 7.1, 7.2, and 7.3 showed the conditions of pruning of treated bushed before treatments were applied.

Fig. 7.1. Lightly pruned bushes.

Fig. 7.2. Moderately pruned bushes.

Fig. 7.3. Heavily pruned bushes.

432 Insecticides – Pest Engineering

savings in product. On the other hand, thick canopies prevent the placement of the product in the habitat of the pest resulting in poor and more expensive pest control. Van Ee et al.(2000) demonstrated how different types of pruning affected pesticide deposition in "highbush" blueberries (*Vaccinium coymbosnm* L) at different times during the plant growth season. Their goal was to determine how sprayer type, pruning severity, and canopy development interacted to affect spray deposition patterns. Deposition was measured as the percentage of the surface area of card targets that was covered following applications of black dye. Light measurements indicated that the canopy of blueberry bushes, regardless of pruning treatment, closed by the middle of June, and light levels within the canopy changed little from then until fruit harvest in August. They used a standard airblast sprayer that pushed spray up and white kromcoat cards clipped to each target. Targets were clipped to branches in a random orientation in the same two bushes used for the light measurements. Figures 7.1, 7.2, and 7.3 showed the conditions of pruning of treated bushed before

treatments were applied.

Fig. 7.1. Lightly pruned bushes.

Fig. 7.2. Moderately pruned bushes.

Van Ee, found that spray penetration and deposition in all types of canopies was greater in the bush's side facing the sprayer. However, spray penetration and coverage was greatly reduced at the far side of the bush when the field was lightly pruned. Conversely, medium or heavy pruning allowed more penetration and deposition than in light pruned bushes. Moderate or heavy pruning allowed similar deposition, except at the top of the bush. In the heavily pruned bushes pesticide deposition was greater than at any other canopy structure (7.3).

In 2006, we conducted a study to determine the spray deposition patterns in mature blueberries using different spray equipment. A mature Jersey field was sprayed with Surround WP (kaolin clay) utilizing a cannon sprayer, an airblast sprayer and a 20-gallon aerial application (Wise et al. unpublished results).

We found that a spray application of 50 gal/acre in light pruned bushes resulted in high deposition at the bottom and middle portion of the canopy and less deposition on the top of the bush. The same volume applied with a cannon sprayer resulted in higher spray deposition on top of the bush but less deposition on the middle section and much less at the bottom of the bush.

The application of a spray volume of 20 gallons to the same field with fixed-wing aircraft resulted in an even deposition of the spray from top to bottom of the bush.

These results were very important to establish what type of equipment we need to use to apply pesticides in blueberry fields in order to optimize the efficacy of the pesticide.

## **8. Pest habitat and behavior**

Finally, the purpose of the spray application is to place the pesticide in contact with the pest. This objective can be achieved in two ways; 1) depositing the pesticide in the habitat of the pest were the pest may enter in contact with the pesticide or 2) placing the pesticide in contact with the pest by taking advantage of its behavior. In this case the pest "finds" the product while moving around the canopy of the crop.

For plant diseases and insects that do not move a lot and remain confined to a small area (a leaf, branch, bush or section of the canopy) we need to bring the pesticide to the pest by targeting their habitat. Spray cover has to be very good with enough number of droplets per square centimeter of foliage to maximize the probability of the pest getting in contact with the spray. In the case of blueberry aphids and fruitworms, pest control measures require placing the insecticide in the habitat of the pest. Blueberry aphids live at the bottom, inside

Use and Management of Pesticides in Small Fruit Production 435

**Equipment**. This affects pesticide deposition in the target site (amount of product and

The conditions of the sprayer will limit the biological effect of the spray. Important factors to

**Crop conditions.** This affects the penetration of chemicals and pesticide deposition. Proper

In the case of crop conditions, the canopy density limits the spray's penetration and deposition. It will be more difficult to implement a successful pesticide management program in a slightly pruned field than in a field that is properly pruned. A thick canopy provides many places where the pest can be protected from natural enemies and from our pesticides! Under these circumstances, only a limited amount of our spray will reach the

**Pest behavior.** Each pest species has its particular behavior. Therefore, the efficacy of your

Knowing the pest is important because it is the objective of our pest control program. If we do not understand the pest biology and behavior, chances are we will miss the critical times

Finally, the success of your **Pesticide management program will depend on** your ability in

[1] Hayes, W.J. and E.R. Laws (ed.). 1990. Handbook of Pesticide Toxicology, Vol. 3, Classes

[2] Meister, R.T. (ed.). 1992. Farm Chemicals Handbook '92. Meister Publishing Company,

[4] U.S. Food and Drug Administration. 2009. The Food Quality Protection Act of 1996.

http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugandC osmeticActFDCAct/SignificantAmendmentstotheFDCAct/ucm148008.htm. [5] Show, B.W. 1996. Minimizing Spray Drift. Texas Agricultural Extension Service, The


consider are droplet size, volume and distribution of the application

pest habitat limiting the effectiveness of our pest control program.



when the pest is more susceptible to our pesticides.


Willoughby, OH.

putting the pesticide in contact with the pest either by: - Placing the pesticide in the pest habitat and/or,

of Pesticides. Academic Press, Inc., NY.

[3] Crop Data Management Systems, Inc. 2007. PennCap-M. http://www.cdms.net/LDat/ld403009.pdf

> Texas A&M University System. College Station, Texas. agsafety.tamu.edu/Programs/Ag-Chemical/drift.ppt


percentage of surface area covered).




pest control will depend on:

**10. References** 

pruning facilitates:


of the bush. Thus, spray applications should reach the bottom of the bush. If the product applied is systemic, droplet size does not really matter, the product will be translocated into de plant and the insect will acquire the lethal dose while sucking up sap. With the Cranberry fruitworm (*Acrobasis vaccinii* Riley) and the Cherry fruitworm (*Grapholita packardi* Zeller), if pest control is directed against eggs and larvae it should be directed into the calyx of the fruit. The product needs to be placed also into the calyx where larvae will hatch. In this case a large volume of spray needs to be deposited on the top of the blueberry bush where the fruit is produced and the fruitworms mate and deposit their eggs. In both cases, a large number of small droplets of pesticide need to be applied into the habitat of the insect. With an insect or pest that moves a lot or explores intensively the canopy of the crop, like the Blueberry Maggot (*Rhagoletis mendex*) or the Cranberry fruitworm first instar larvae. A few large droplets will be enough to have a good control of these pests. Adult blueberry maggot explores intensively the blueberry canopy in search of mates, food and sites to oviposit. Also, first instar Cranberry fruitworm larvae after hatching crawl out of the berry calix to search for a place to enter the berry at the site where the peduncle and the fruit meet. In both insects the wandering period is critical to put the pesticide in contact with the pest. Table 3 presents a summary of the habitat and behavior of several insects that attack blueberries in Michigan.


Table 3. Habitat and behavior of several insect pest of "Highbush" blueberries.

## **9. Pesticide management summary**

We can summarize the recommendations for optimizing the efficacy of the pesticides as follow:

The pesticide efficacy and efficiency will depend on:



**Equipment**. This affects pesticide deposition in the target site (amount of product and percentage of surface area covered).


434 Insecticides – Pest Engineering

of the bush. Thus, spray applications should reach the bottom of the bush. If the product applied is systemic, droplet size does not really matter, the product will be translocated into de plant and the insect will acquire the lethal dose while sucking up sap. With the Cranberry fruitworm (*Acrobasis vaccinii* Riley) and the Cherry fruitworm (*Grapholita packardi* Zeller), if pest control is directed against eggs and larvae it should be directed into the calyx of the fruit. The product needs to be placed also into the calyx where larvae will hatch. In this case a large volume of spray needs to be deposited on the top of the blueberry bush where the fruit is produced and the fruitworms mate and deposit their eggs. In both cases, a large number of small droplets of pesticide need to be applied into the habitat of the insect. With an insect or pest that moves a lot or explores intensively the canopy of the crop, like the Blueberry Maggot (*Rhagoletis mendex*) or the Cranberry fruitworm first instar larvae. A few large droplets will be enough to have a good control of these pests. Adult blueberry maggot explores intensively the blueberry canopy in search of mates, food and sites to oviposit. Also, first instar Cranberry fruitworm larvae after hatching crawl out of the berry calix to search for a place to enter the berry at the site where the peduncle and the fruit meet. In both insects the wandering period is critical to put the pesticide in contact with the pest. Table 3 presents a summary of the habitat and behavior of several insects that attack

> **Pest Behavioral Activity Level**

Fruitworms Egg/larva Low Fruit Cluster Short

Leafrollers Larva Medium Upper Canopy Long

Maggot Adult High Upper Canopy Short

Japanese Beetle Adult High Upper Canopy Long

Blueberry Aphid Adult/Nymphs Low Lower Canopy Long

Table 3. Habitat and behavior of several insect pest of "Highbush" blueberries.

**Weather conditions** that affect the movement of pesticides in the environment.

Bud mites Adult/Nymphs Low Buds Short

We can summarize the recommendations for optimizing the efficacy of the pesticides as

**Location on the** 

**Plant Exposure Period** 

blueberries in Michigan.

Blueberry

follow:

**Pest Targeted Life** 

**9. Pesticide management summary** 

The pesticide efficacy and efficiency will depend on:



**Stage** 


The conditions of the sprayer will limit the biological effect of the spray. Important factors to consider are droplet size, volume and distribution of the application

**Crop conditions.** This affects the penetration of chemicals and pesticide deposition. Proper pruning facilitates:


In the case of crop conditions, the canopy density limits the spray's penetration and deposition. It will be more difficult to implement a successful pesticide management program in a slightly pruned field than in a field that is properly pruned. A thick canopy provides many places where the pest can be protected from natural enemies and from our pesticides! Under these circumstances, only a limited amount of our spray will reach the pest habitat limiting the effectiveness of our pest control program.

**Pest behavior.** Each pest species has its particular behavior. Therefore, the efficacy of your pest control will depend on:


Knowing the pest is important because it is the objective of our pest control program. If we do not understand the pest biology and behavior, chances are we will miss the critical times when the pest is more susceptible to our pesticides.

Finally, the success of your **Pesticide management program will depend on** your ability in putting the pesticide in contact with the pest either by:


## **10. References**


**20** 

*U.S.A.* 

Allan T. Showler

**The Conundrum of Chemical Boll Weevil** 

Originally a Mesoamerican insect, the boll weevil, *Anthonomus grandis grandis* Boheman (Coleoptera: Curculionidae), has spread from the tropics, where it evolved on cotton, *Gossypium hirsutum* L., and other malvaceous plant species (Burke et al., 1986; Brubaker & Wendel, 1994; Showler, 2009b), north to temperate cotton producing areas of the United States and south to northern provinces of Argentina (Cuadrado, 2002; Showler, 2009b). The pest was first detected in United States cotton in 1892 (Parencia, 1978) and infested the Cotton Belt such that by 1917, every cotton-producing county in Georgia, for example, was infested (Hunter, 1917). Adults oviposit inside cotton buds or "squares" (usually one egg per square), and the hatched larva causes the square to abscise before it can flower (Showler and Cantú, 2005). If an egg is deposited within a young boll (older bolls become too hard to penetrate), or if mouthparts penetrate the rind of squares to the inner reproductive portion, fiber-producing locks can be injured or completely destroyed, but not necessarily all four locks (Showler, 2006a; Showler & Cantú, 2008). Boll weevil losses have been valued at \$83.34 billion and insecticide-based control costs at \$18.67 billion between 1893 and 1999, and infestations became so injurious that cotton-free winter periods were instituted by law in some areas (Haney, 2001). Later, insecticide-based eradication programs were launched in the United States and in Argentina (Dickerson & Haney, 2001; Haney et al., 2001; Johnson &

Martin, 2001; Texas Department of Agriculture, 2002; Carmona et al., 2003).

Natural enemies indigenous to the United States are not considered to be important as mortality factors against boll weevils (Jones & Sterling, 1979; Showler & Greenberg, 2003), although the imported fire ant, *Solenopsis invicta* Buren, native to South America (Buren et al., 1974; Lofgren, 1986), can account for up to 58% of boll weevil mortality in relatively wet regions where the predator thrives (Sturm & Sterling, 1990). In one study imported fire ant predation on immature boll weevils averaged 84% compared with 0.14% and 6.9% mortality caused by parasitism and desiccation, respectively (Fillman & Sterling, 1983). But in drier cotton growing areas, lack of sufficient predation to help govern populations in some new habitats outside Mesoamerica (Showler, 2007) permitted rapid dispersal (Burke et al., 1986; Showler, 2009a). While certain cultural practices, such as early planting (Showler et al. 2005) can help avoid large populations that typically accumulate in the summer (Showler, 2003, 2005), chemical intervention against building infestations has been the chief control tactic.

**1. Introduction** 

**Control in Subtropical Regions** 

*USDA-ARS, Kika de la Garza Subtropical Agricultural Research Center, Weslaco, Texas* 


## **The Conundrum of Chemical Boll Weevil Control in Subtropical Regions**

## Allan T. Showler

*USDA-ARS, Kika de la Garza Subtropical Agricultural Research Center, Weslaco, Texas U.S.A.* 

## **1. Introduction**

436 Insecticides – Pest Engineering

[6] Ross, Merrill A. and Carole A. Lembi. 1985. Applied Weed Science. Burgess Publishing

[7] H. Erdal Ozkan. 1998. Effect of Major Variables on Drift Distances of Spray Droplets. The

[8] Van Ee, G., Richard Ledebuhr, Eric Hanson, Jim Hancock, and Donald Ramsdell (2000)

Ohio State University Extension (AEX-525-98). http://ohioline.osu.edu/aex-

Canopy Development and Spray Deposition in Highbush Blueberry.

Company, Minneapolis, MN.

HortTechnology 10(2) 354-357.

fact/0525.html.

Originally a Mesoamerican insect, the boll weevil, *Anthonomus grandis grandis* Boheman (Coleoptera: Curculionidae), has spread from the tropics, where it evolved on cotton, *Gossypium hirsutum* L., and other malvaceous plant species (Burke et al., 1986; Brubaker & Wendel, 1994; Showler, 2009b), north to temperate cotton producing areas of the United States and south to northern provinces of Argentina (Cuadrado, 2002; Showler, 2009b). The pest was first detected in United States cotton in 1892 (Parencia, 1978) and infested the Cotton Belt such that by 1917, every cotton-producing county in Georgia, for example, was infested (Hunter, 1917). Adults oviposit inside cotton buds or "squares" (usually one egg per square), and the hatched larva causes the square to abscise before it can flower (Showler and Cantú, 2005). If an egg is deposited within a young boll (older bolls become too hard to penetrate), or if mouthparts penetrate the rind of squares to the inner reproductive portion, fiber-producing locks can be injured or completely destroyed, but not necessarily all four locks (Showler, 2006a; Showler & Cantú, 2008). Boll weevil losses have been valued at \$83.34 billion and insecticide-based control costs at \$18.67 billion between 1893 and 1999, and infestations became so injurious that cotton-free winter periods were instituted by law in some areas (Haney, 2001). Later, insecticide-based eradication programs were launched in the United States and in Argentina (Dickerson & Haney, 2001; Haney et al., 2001; Johnson & Martin, 2001; Texas Department of Agriculture, 2002; Carmona et al., 2003).

Natural enemies indigenous to the United States are not considered to be important as mortality factors against boll weevils (Jones & Sterling, 1979; Showler & Greenberg, 2003), although the imported fire ant, *Solenopsis invicta* Buren, native to South America (Buren et al., 1974; Lofgren, 1986), can account for up to 58% of boll weevil mortality in relatively wet regions where the predator thrives (Sturm & Sterling, 1990). In one study imported fire ant predation on immature boll weevils averaged 84% compared with 0.14% and 6.9% mortality caused by parasitism and desiccation, respectively (Fillman & Sterling, 1983). But in drier cotton growing areas, lack of sufficient predation to help govern populations in some new habitats outside Mesoamerica (Showler, 2007) permitted rapid dispersal (Burke et al., 1986; Showler, 2009a). While certain cultural practices, such as early planting (Showler et al. 2005) can help avoid large populations that typically accumulate in the summer (Showler, 2003, 2005), chemical intervention against building infestations has been the chief control tactic.

The Conundrum of Chemical Boll Weevil Control in Subtropical Regions 439

Weevil Eradication Foundation, 2011)]. Eradication involves three techniques over a 4–5 year period: pheromone trap detection, reduction of boll weevil food supply, and malathion treatments (USDA-APHIS, 2007). The process starts with a series (often seven) of fall malathion applications that were once [and probably erroneously (Showler, 2010)] termed "diapause sprays" (McKibben et al., 2001). The aim is to reduce late season populations that will be further, and dramatically, reduced during cold temperate winters when food is unavailable and temperatures can be lethal (Showler, 2009b,c, 2010). Pheromone traps are then deployed around all cotton fields during the following spring planting and spraying of each field begins 5–6 weeks later based on trap captures; the process is repeated annually until the boll weevil is no longer found (USDA-APHIS, 2007). Still, there are 262,000 ha of cotton in east and subtropical south Texas where eradication has not been achieved (Texas Boll Weevil Eradication Foundation, 2011). While the chance of boll weevil dispersal from infested areas on wind and in hurricane systems exists (Texas Boll Weevil Eradication Foundation, 2011), particularly from nearby Mexico where eradication efforts are not underway, there are other overriding factors contributing to the pest's persistence in the subtropics (Showler, 2007, 2009b). Misconceptions relating to boll weevil ecology and biology (Showler, 2009c), while immaterial in cold temperate areas, appear to underlie the

challenges to eradication efforts under subtropical conditions (Showler, 2009b, 2010).

There have been misconceptions pertaining to fundamental aspects of boll weevil survival outside its native Mesoamerican region that involve dietary habits, overwintering, and diapause, all of which interrelate (Showler, 2007, 2009b,c, 2010), presenting obstacles to temperate eradication approaches when used in the subtropics. Ultimately, the problem resides in numbers of boll weevils (including offspring from overwintering weevils) that can survive cotton-free winters to feed and reproduce in large cotton plantings of the following season. In south Texas, large end-of-season populations can be observed by trapping at the edges of cotton fields that are disrupted by defoliant application, consequent host plant desiccation, harvest, and stalk shredding (Showler, 2003). Those populations move into surrounding habitats where, under temperate winter conditions, the boll weevils that survived the first-year series of late-season eradication program sprays must survive frequently severe and extended cold conditions for which the tropical insect had not evolved, as well as starvation due to lack of viable winter plant hosts (Showler, 2009b,c). Boll weevils have long been assumed to feed solely on pollen of certain malvaceous plants (Burke & Earle, 1965; Cate & Skinner, 1978), and later, pollens of other plants were recognized (Jones et al., 1992, 1993; Hardee et al., 1999), but recent research has revealed that adult boll weevils can consume cotton leaves and bracts, citrus and cactus fruit, and likely nectar (Showler & Abrigo, 2007; Showler, 2009b). In the subtropics, adult boll weevils can survive and reproduce during the winter on small patches of volunteer cotton that, despite surveillance, are overlooked, and adults can be trapped in substantial numbers around grapefruit, *Citrus paradisi* Macfad., and orange, *C*. *sinensis* (L.) Osbeck., orchards (Showler, 2006b). The edible endocarps of grapefruits and oranges of those citrus species can sustain up to 25% of adult boll weevils in nonreproductive condition for longer than five months (completing the cotton-free period); the maximum longevity (246 days) was only seven days less than boll weevils fed large cotton squares (Showler & Abrigo, 2007). The fruit of prickly pear cacti, *Opuntia* spp. [114 species in Mexico alone (Vigueras & Portillo, 2001)], which is

**2. Misunderstandings** 

In subtropical south Texas, before the boll weevil eradication program was re-instated in the fall of 2005 (after a halted attempt in 1995), crop protection against boll weevils was approached using three tactics: mandatory cotton stalk destruction before 1 September, prohibition on planting until 1 February and elimination of volunteer cotton during the cotton-free winter period (Texas Department of Agriculture, 2002). Insecticides were the only in-season control approach (Showler, 2007). Some growers sprayed 2–3 "pre-emptive" treatments starting at the "pinhead" square size (1–2-mm diameter) (Heilman et al. 1979) followed by insecticide applications (often azinphosmethyl, cyfluthrin, bifenthrin, or oxamyl) whenever 10% of randomly selected medium (3–5.4-mm diameter) or large (5.5–8 mm diameter) squares (Showler, 2005; Showler et al., 2005) had oviposition punctures (Showler et al., 2005). While Heilman et al. (1979) suggested that pre-emptive spraying delays insecticide applications later into the season, other research found no beneficial effect (Showler, 2004a) and the economic value of the practice is debatable. Pre-emptive sprays might kill some adult boll weevils that have entered the field after overwintering elsewhere, but the sparse numbers of weevils at that time and the presence of less-preferred and nutritionally inferior small squares contribute relatively little to field-level population buildups, and injury to such small squares has negligible impact on lint yield (Showler, 2004b; Showler et al., 2005). Late-season spraying for immediate crop protection (not eradication) purposes is also of questionable utility because, although feeding and oviposition punctures can be abundant on bolls, older bolls (14 days old) are less vulnerable to attack (because they harden) than younger (10 days old) bolls and bolls do not abscise in response to boll weevil oviposition (Showler, 2006a). When injury to a boll does occur, usually because of prior adult feeding during the square stage or larval infestation of the boll, damage is often limited to individual lint-bearing locks, of which there are four (Showler 2006a). Insecticides applied in the context of crop protection after cut-out (Guinn, 1986; Cothren, 1999), when bolls predominate, generally fail to measurably suppress boll weevil infestations (Showler & Robinson, 2005; Showler, 2008a).

When cotton is forming medium and large squares, which are most vulnerable to and useful for boll weevil reproduction (Showler, 2004b), the 10% spray intervention threshold is compromised by variability in total numbers of squares over time (Showler, 2007). Declining abundance of squares coupled with surges in boll weevil populations contribute toward the likelihood of triggering interventions based on randomly sampled squares; hence, spraying later protects fewer and fewer squares (Showler, 2007). A better estimate of infestation would involve comparing numbers of oviposition-punctured squares to total squares within, for example, three-meter (or some other length) sections of rows (Showler, 2007). In a study in south Texas, the standard approach, including three pre-emptive sprays, involved nine applications that failed to increase yield and economic return (Showler & Robinson, 2005). Once 10% of the squares harbor a boll weevil egg, protecting it from contact insecticides (Showler & Scott, 2004), it is too late to expect good control.

In temperate areas of the United States, the boll weevil eradication program has had remarkable successes since its beginning in North Carolina and Virginia in 1978 to Georgia to California (Dickerson et al., 2001) and the pest has been eradicated from northern and central regions of Texas as well (USDA-APHIS, 2007; Texas Boll Weevil Eradication Foundation, 2011). The boll weevil is "functionally" eradicated in other areas of Texas (USDA-APHIS, 2007), whereby <0.001 weevils/trap/week were found during the most recently completed growing season, indicating that boll weevils are not reproducing or causing economic damage in an area [*e.g.*, >1.5 million ha of cotton in 2010 (Texas Boll Weevil Eradication Foundation, 2011)]. Eradication involves three techniques over a 4–5 year period: pheromone trap detection, reduction of boll weevil food supply, and malathion treatments (USDA-APHIS, 2007). The process starts with a series (often seven) of fall malathion applications that were once [and probably erroneously (Showler, 2010)] termed "diapause sprays" (McKibben et al., 2001). The aim is to reduce late season populations that will be further, and dramatically, reduced during cold temperate winters when food is unavailable and temperatures can be lethal (Showler, 2009b,c, 2010). Pheromone traps are then deployed around all cotton fields during the following spring planting and spraying of each field begins 5–6 weeks later based on trap captures; the process is repeated annually until the boll weevil is no longer found (USDA-APHIS, 2007). Still, there are 262,000 ha of cotton in east and subtropical south Texas where eradication has not been achieved (Texas Boll Weevil Eradication Foundation, 2011). While the chance of boll weevil dispersal from infested areas on wind and in hurricane systems exists (Texas Boll Weevil Eradication Foundation, 2011), particularly from nearby Mexico where eradication efforts are not underway, there are other overriding factors contributing to the pest's persistence in the subtropics (Showler, 2007, 2009b). Misconceptions relating to boll weevil ecology and biology (Showler, 2009c), while immaterial in cold temperate areas, appear to underlie the challenges to eradication efforts under subtropical conditions (Showler, 2009b, 2010).

## **2. Misunderstandings**

438 Insecticides – Pest Engineering

In subtropical south Texas, before the boll weevil eradication program was re-instated in the fall of 2005 (after a halted attempt in 1995), crop protection against boll weevils was approached using three tactics: mandatory cotton stalk destruction before 1 September, prohibition on planting until 1 February and elimination of volunteer cotton during the cotton-free winter period (Texas Department of Agriculture, 2002). Insecticides were the only in-season control approach (Showler, 2007). Some growers sprayed 2–3 "pre-emptive" treatments starting at the "pinhead" square size (1–2-mm diameter) (Heilman et al. 1979) followed by insecticide applications (often azinphosmethyl, cyfluthrin, bifenthrin, or oxamyl) whenever 10% of randomly selected medium (3–5.4-mm diameter) or large (5.5–8 mm diameter) squares (Showler, 2005; Showler et al., 2005) had oviposition punctures (Showler et al., 2005). While Heilman et al. (1979) suggested that pre-emptive spraying delays insecticide applications later into the season, other research found no beneficial effect (Showler, 2004a) and the economic value of the practice is debatable. Pre-emptive sprays might kill some adult boll weevils that have entered the field after overwintering elsewhere, but the sparse numbers of weevils at that time and the presence of less-preferred and nutritionally inferior small squares contribute relatively little to field-level population buildups, and injury to such small squares has negligible impact on lint yield (Showler, 2004b; Showler et al., 2005). Late-season spraying for immediate crop protection (not eradication) purposes is also of questionable utility because, although feeding and oviposition punctures can be abundant on bolls, older bolls (14 days old) are less vulnerable to attack (because they harden) than younger (10 days old) bolls and bolls do not abscise in response to boll weevil oviposition (Showler, 2006a). When injury to a boll does occur, usually because of prior adult feeding during the square stage or larval infestation of the boll, damage is often limited to individual lint-bearing locks, of which there are four (Showler 2006a). Insecticides applied in the context of crop protection after cut-out (Guinn, 1986; Cothren, 1999), when bolls predominate, generally fail to measurably

suppress boll weevil infestations (Showler & Robinson, 2005; Showler, 2008a).

insecticides (Showler & Scott, 2004), it is too late to expect good control.

When cotton is forming medium and large squares, which are most vulnerable to and useful for boll weevil reproduction (Showler, 2004b), the 10% spray intervention threshold is compromised by variability in total numbers of squares over time (Showler, 2007). Declining abundance of squares coupled with surges in boll weevil populations contribute toward the likelihood of triggering interventions based on randomly sampled squares; hence, spraying later protects fewer and fewer squares (Showler, 2007). A better estimate of infestation would involve comparing numbers of oviposition-punctured squares to total squares within, for example, three-meter (or some other length) sections of rows (Showler, 2007). In a study in south Texas, the standard approach, including three pre-emptive sprays, involved nine applications that failed to increase yield and economic return (Showler & Robinson, 2005). Once 10% of the squares harbor a boll weevil egg, protecting it from contact

In temperate areas of the United States, the boll weevil eradication program has had remarkable successes since its beginning in North Carolina and Virginia in 1978 to Georgia to California (Dickerson et al., 2001) and the pest has been eradicated from northern and central regions of Texas as well (USDA-APHIS, 2007; Texas Boll Weevil Eradication Foundation, 2011). The boll weevil is "functionally" eradicated in other areas of Texas (USDA-APHIS, 2007), whereby <0.001 weevils/trap/week were found during the most recently completed growing season, indicating that boll weevils are not reproducing or causing economic damage in an area [*e.g.*, >1.5 million ha of cotton in 2010 (Texas Boll There have been misconceptions pertaining to fundamental aspects of boll weevil survival outside its native Mesoamerican region that involve dietary habits, overwintering, and diapause, all of which interrelate (Showler, 2007, 2009b,c, 2010), presenting obstacles to temperate eradication approaches when used in the subtropics. Ultimately, the problem resides in numbers of boll weevils (including offspring from overwintering weevils) that can survive cotton-free winters to feed and reproduce in large cotton plantings of the following season. In south Texas, large end-of-season populations can be observed by trapping at the edges of cotton fields that are disrupted by defoliant application, consequent host plant desiccation, harvest, and stalk shredding (Showler, 2003). Those populations move into surrounding habitats where, under temperate winter conditions, the boll weevils that survived the first-year series of late-season eradication program sprays must survive frequently severe and extended cold conditions for which the tropical insect had not evolved, as well as starvation due to lack of viable winter plant hosts (Showler, 2009b,c). Boll weevils have long been assumed to feed solely on pollen of certain malvaceous plants (Burke & Earle, 1965; Cate & Skinner, 1978), and later, pollens of other plants were recognized (Jones et al., 1992, 1993; Hardee et al., 1999), but recent research has revealed that adult boll weevils can consume cotton leaves and bracts, citrus and cactus fruit, and likely nectar (Showler & Abrigo, 2007; Showler, 2009b). In the subtropics, adult boll weevils can survive and reproduce during the winter on small patches of volunteer cotton that, despite surveillance, are overlooked, and adults can be trapped in substantial numbers around grapefruit, *Citrus paradisi* Macfad., and orange, *C*. *sinensis* (L.) Osbeck., orchards (Showler, 2006b). The edible endocarps of grapefruits and oranges of those citrus species can sustain up to 25% of adult boll weevils in nonreproductive condition for longer than five months (completing the cotton-free period); the maximum longevity (246 days) was only seven days less than boll weevils fed large cotton squares (Showler & Abrigo, 2007). The fruit of prickly pear cacti, *Opuntia* spp. [114 species in Mexico alone (Vigueras & Portillo, 2001)], which is

The Conundrum of Chemical Boll Weevil Control in Subtropical Regions 441

warm and can support populations with food until the spring (Showler, 2009b). In February, when cotton can be planted, boll weevil numbers near south Texas citrus orchards were found to be substantial (Showler, 2006b). Loss of major winter attrition as an eradication tool will likely require adjustments to the customary approach. Chance movement of boll weevils on wind or farm vehicles into active eradication program areas might cause setbacks to eradication, but the ecological reasons for the boll weevil's persistence in subtropical

The spray regimen for cotton crop protection against boll weevils and the reasons it was sometimes not sufficient across all growing areas have been discussed, but aspects of eradication involving insecticide application are also weakened in the subtropical context. Monitoring in-season boll weevil populations, for example, is important for determining whether to intervene and to assess efficacy. It is surprising that boll weevil surveillance fails to account for in-season changes in adult boll weevil response to grandlure largely predicated by cotton plant phenology and associated volatiles. One change occurs as cotton begins to square; then, even while boll weevils are accumulating in cotton fields, few are collected in the traps (Parajulee et al., 2001), presumably a result of competing plant volatiles from large fields of cotton versus a point pheromone source. Further, the trap's physical design presents a series of obstacles that boll weevils must negotiate before finding their way into a plastic cap on top where the weevils are counted (Showler, 2007). At low ambient populations in south Texas, differences in numbers of boll weevils captured in the conventional trap versus a sticky board trap were not detected, both traps using the same pheromone lure, but at higher populations sticky board traps collected 9-fold more weevils than the conventional trap, and 30% of the conventional traps collected no boll weevils when corresponding sticky boards accumulated from 82 to 511 weevils at the same locations and time; on one occasion, the conventional trap had two boll weevils compared with 2,228 on a sticky board (Showler, 2003). This is not to suggest that sticky board traps should replace the conventional trap unless their deployment can be made less labor intensive, but a more sensitive trap design would refine spray timing for greater effect as a result of more

Because the boll weevil's life cycle includes 18 days in immature life stages protected within squares (Showler & Cantú, 2005), commonly-used insecticides with relatively short residual effects (4 days) can miss that cohort (Showler & Scott, 2004). To ensure lethal exposure to a larger proportion of the population, such insecticides would have to be sprayed *at least* once every four days. Yield increases in experimental plots were reported where some were sprayed "proactively" every 7–8 days starting when 2% of randomly selected squares were large (Showler & Robinson, 2005). It is unlikely, however, that the proactive spray regime would be as effective in larger commercial fields on an area-wide scale; in the study, applications were meticulous and tractor-mounted drop nozzles provided complete coverage even when the plants were high. For large boll weevil populations likes those encountered in the Lower Rio Grande Valley (Showler 2003), insecticides would have to be applied every three or four days from the time medium-sized

areas presents broader and more difficult challenges.

**3. Chemical tactics: no easy answers** 

**3.1 Insensitive trigger** 

accurate population detection.

**3.2 Spray timing** 

widespread and abundant in south Texas, can support 10% of adult boll weevils over the winter period, and there are likely other as yet unreported food sources (Showler, 2009b). Hence, in subtropical areas of North and South America where cotton is grown in proximity with citrus, persistent boll weevil populations have been reported even after cotton growing was eliminated or where eradication programs have begun (Cuadrado, 2002; Carmona et al., 2003; Mas et al., 2007; Texas Boll Weevil Eradication Foundation, 2011). Despite the availability of *Opuntia* spp. and other host plants in Mexico and south Texas (Gaines, 1935; Lukefahr, 1956; Lukefahr & Martin, 1962; Stoner, 1968; Cross et al., 1975; Vigueras & Portillo, 2001), cotton in the Lower Rio Grande Valley remained free of boll weevils for 30 years of commercial production beginning 1860 (Garza & Long, 2001) even though cotton around Monclova, Coahuila, Mexico, 45 minutes latitude north and 220 km west of the Lower Rio Grande Valley, was so heavily infested that the crop was abandoned in 1862 (Howard, 1897). Boll weevil food sources under orchard conditions are concentrated and support substantial active populations through winter (Showler, 2006b) because endocarps are accessible through cracks, holes, or lesions while the fruit is attached to the plant or fallen (Showler, 2007; Showler & Abrigo, 2007). Establishment of boll weevils in Lower Rio Grande Valley cotton during the early 1890s (Parencia, 1978; Haney, 2001) may have been connected to a simultaneous citrus industry boom (Waibel, 1953). The author has witnessed, in mid January, large flying populations of boll weevils in and around nonsanitized (fallen fruit on the orchard floor not removed) orange and grapefruit orchards in south Texas that were so abundant that they were a nuisance. Boll weevils are also known to reproduce in volunteer cotton during Lower Rio Grande Valley winters (Summy et al., 1988), which also contradicts widely accepted, but apparently erroneous, dogma regarding the existence of winter diapause (Showler, 2009c, 2010).

For more than 50 years, boll weevils have been assumed to enter a state of winter diapause (Brazzel & Newsom, 1959), but diapause-induction studies involved weak experimental methods and dubious interpretations of results, and recent research in the subtropics indicates that boll weevils, being of tropical origin, did not evolve a diapause mechanism for surviving temperate winters (Showler, 2007, 2009c, 2010). Sterling and Adkisson's (1966) finding that boll weevils in the Texas High Plains "diapause" earlier and in greater percentages than in Central Texas (at a lower latitude) implies that boll weevil dormancy is not seasonal (a criterion for diapause), but it is instead responsive to dormancy-triggering conditions whenever they occur (Showler, 2010). Brazzel and Newsom (1959), however, claimed that, in the instance of boll weevils, diapause could be a "facultative" response to harsh, unfamiliar, conditions such as cold temperate winters. It is more likely, however, that the response is merely a metabolic and locomotory slowing caused by declining temperature (Fye et al., 1969; Jones & Sterling, 1979; Watson et al., 1986), giving the appearance of being facultative. As winter temperatures cool, a threshold for quiescence (Koštál, 2006; Guerra et al., 1984) or some other nondiapause expression of dormancy is reached first, followed later, if temperatures become sufficiently cold, by mortality (Showler, 2010). Whatever words are employed to describe the insect's response to temperate winters, eradication strategy involving "diapause spraying" has been effective where temperate winter attrition is substantial even if "diapause" might not be the technically correct term (Showler, 2010).

The inescapable point is that under subtropical conditions, particularly in the presence of relatively large plantings of citrus throughout the agricultural landscape, boll weevil mortality is not as great as in cold-winter temperate areas because winters are generally warm and can support populations with food until the spring (Showler, 2009b). In February, when cotton can be planted, boll weevil numbers near south Texas citrus orchards were found to be substantial (Showler, 2006b). Loss of major winter attrition as an eradication tool will likely require adjustments to the customary approach. Chance movement of boll weevils on wind or farm vehicles into active eradication program areas might cause setbacks to eradication, but the ecological reasons for the boll weevil's persistence in subtropical areas presents broader and more difficult challenges.

## **3. Chemical tactics: no easy answers**

## **3.1 Insensitive trigger**

440 Insecticides – Pest Engineering

widespread and abundant in south Texas, can support 10% of adult boll weevils over the winter period, and there are likely other as yet unreported food sources (Showler, 2009b). Hence, in subtropical areas of North and South America where cotton is grown in proximity with citrus, persistent boll weevil populations have been reported even after cotton growing was eliminated or where eradication programs have begun (Cuadrado, 2002; Carmona et al., 2003; Mas et al., 2007; Texas Boll Weevil Eradication Foundation, 2011). Despite the availability of *Opuntia* spp. and other host plants in Mexico and south Texas (Gaines, 1935; Lukefahr, 1956; Lukefahr & Martin, 1962; Stoner, 1968; Cross et al., 1975; Vigueras & Portillo, 2001), cotton in the Lower Rio Grande Valley remained free of boll weevils for 30 years of commercial production beginning 1860 (Garza & Long, 2001) even though cotton around Monclova, Coahuila, Mexico, 45 minutes latitude north and 220 km west of the Lower Rio Grande Valley, was so heavily infested that the crop was abandoned in 1862 (Howard, 1897). Boll weevil food sources under orchard conditions are concentrated and support substantial active populations through winter (Showler, 2006b) because endocarps are accessible through cracks, holes, or lesions while the fruit is attached to the plant or fallen (Showler, 2007; Showler & Abrigo, 2007). Establishment of boll weevils in Lower Rio Grande Valley cotton during the early 1890s (Parencia, 1978; Haney, 2001) may have been connected to a simultaneous citrus industry boom (Waibel, 1953). The author has witnessed, in mid January, large flying populations of boll weevils in and around nonsanitized (fallen fruit on the orchard floor not removed) orange and grapefruit orchards in south Texas that were so abundant that they were a nuisance. Boll weevils are also known to reproduce in volunteer cotton during Lower Rio Grande Valley winters (Summy et al., 1988), which also contradicts widely accepted, but apparently erroneous, dogma regarding the existence of winter

For more than 50 years, boll weevils have been assumed to enter a state of winter diapause (Brazzel & Newsom, 1959), but diapause-induction studies involved weak experimental methods and dubious interpretations of results, and recent research in the subtropics indicates that boll weevils, being of tropical origin, did not evolve a diapause mechanism for surviving temperate winters (Showler, 2007, 2009c, 2010). Sterling and Adkisson's (1966) finding that boll weevils in the Texas High Plains "diapause" earlier and in greater percentages than in Central Texas (at a lower latitude) implies that boll weevil dormancy is not seasonal (a criterion for diapause), but it is instead responsive to dormancy-triggering conditions whenever they occur (Showler, 2010). Brazzel and Newsom (1959), however, claimed that, in the instance of boll weevils, diapause could be a "facultative" response to harsh, unfamiliar, conditions such as cold temperate winters. It is more likely, however, that the response is merely a metabolic and locomotory slowing caused by declining temperature (Fye et al., 1969; Jones & Sterling, 1979; Watson et al., 1986), giving the appearance of being facultative. As winter temperatures cool, a threshold for quiescence (Koštál, 2006; Guerra et al., 1984) or some other nondiapause expression of dormancy is reached first, followed later, if temperatures become sufficiently cold, by mortality (Showler, 2010). Whatever words are employed to describe the insect's response to temperate winters, eradication strategy involving "diapause spraying" has been effective where temperate winter attrition is substantial even if "diapause" might not be the technically correct term

The inescapable point is that under subtropical conditions, particularly in the presence of relatively large plantings of citrus throughout the agricultural landscape, boll weevil mortality is not as great as in cold-winter temperate areas because winters are generally

diapause (Showler, 2009c, 2010).

(Showler, 2010).

The spray regimen for cotton crop protection against boll weevils and the reasons it was sometimes not sufficient across all growing areas have been discussed, but aspects of eradication involving insecticide application are also weakened in the subtropical context. Monitoring in-season boll weevil populations, for example, is important for determining whether to intervene and to assess efficacy. It is surprising that boll weevil surveillance fails to account for in-season changes in adult boll weevil response to grandlure largely predicated by cotton plant phenology and associated volatiles. One change occurs as cotton begins to square; then, even while boll weevils are accumulating in cotton fields, few are collected in the traps (Parajulee et al., 2001), presumably a result of competing plant volatiles from large fields of cotton versus a point pheromone source. Further, the trap's physical design presents a series of obstacles that boll weevils must negotiate before finding their way into a plastic cap on top where the weevils are counted (Showler, 2007). At low ambient populations in south Texas, differences in numbers of boll weevils captured in the conventional trap versus a sticky board trap were not detected, both traps using the same pheromone lure, but at higher populations sticky board traps collected 9-fold more weevils than the conventional trap, and 30% of the conventional traps collected no boll weevils when corresponding sticky boards accumulated from 82 to 511 weevils at the same locations and time; on one occasion, the conventional trap had two boll weevils compared with 2,228 on a sticky board (Showler, 2003). This is not to suggest that sticky board traps should replace the conventional trap unless their deployment can be made less labor intensive, but a more sensitive trap design would refine spray timing for greater effect as a result of more accurate population detection.

## **3.2 Spray timing**

Because the boll weevil's life cycle includes 18 days in immature life stages protected within squares (Showler & Cantú, 2005), commonly-used insecticides with relatively short residual effects (4 days) can miss that cohort (Showler & Scott, 2004). To ensure lethal exposure to a larger proportion of the population, such insecticides would have to be sprayed *at least* once every four days. Yield increases in experimental plots were reported where some were sprayed "proactively" every 7–8 days starting when 2% of randomly selected squares were large (Showler & Robinson, 2005). It is unlikely, however, that the proactive spray regime would be as effective in larger commercial fields on an area-wide scale; in the study, applications were meticulous and tractor-mounted drop nozzles provided complete coverage even when the plants were high. For large boll weevil populations likes those encountered in the Lower Rio Grande Valley (Showler 2003), insecticides would have to be applied every three or four days from the time medium-sized

The Conundrum of Chemical Boll Weevil Control in Subtropical Regions 443

There are a number of ways in which chemical boll weevil control might be improved. First, a more sensitive trap would permit increasingly timely responses to the early in-season presence of adult boll weevils (but not while squares are still match-head size). At that point, spraying should provide continuous protection of vulnerable and nutritious mediumand large-sized squares. Even if sprays occur weekly, achieving acceptable control on an area-wide scale is improbable, which suggests that using a more sensitive trap design could result in more appropriately-timed, and likely increased, spray applications for the subtropics (unless spraying is conducted at 4-day intervals between late match-head to cutout stages) where overwintering populations are relatively large (Showler, 2007). Both crop protection strategies and the eradication approach should evolve to incorporate emerging information on boll weevil ecology to find tactics that can help mitigate population buildups, such as avoiding late planting, use of earlier-maturing varieties, and development of longer-residual insecticides to reduce numbers of applications and to enhance protection of squares. Because subtropical boll weevil populations are active during winter and can sustain themselves on citrus, removal of such plentiful food through post-harvest orchard sanitation would augment the ban on cotton. Another overlooked tactic is plant resistance. While cotton has been bred for a variety of traits, no cultivars have been developed to resist boll weevil attack. Efforts in this direction might include altering square rind thickness or consistency to make the inner portion, where the immature weevil stages develop, less accessible, or changing the availability of certain nutrients that can affect egg production (Showler, 2009a). Eradicating a tropical pest like the boll weevil in temperate areas was achievable, but the subtropics are more akin to the insect's native habitat in terms of temperature and host plants. For this reason, adjustments to the temperate eradication strategy might have to involve tailoring insecticides, application timing, and the circumstances under which they are applied (*e.g.*, as influenced by planting dates and phenological stages of the crop) for extending prophylactic crop protection and decimating boll weevil populations as selectively as possible to avoid the possibility of for secondary

However, even were all of the issues surrounding subtropical boll weevil eradication to be resolved, the feasibility of remaining boll weevil-free in areas along international borders is compromised by boll weevil populations breeding on the other side of the border where attention to eradication, for a complex of reasons, may not be in synchrony. Hence, the success of eradication in subtropical border areas depends to a great extent on the coordinated efforts of both countries. In the instance of a somewhat analogous pest, the desert locust, *Schistocerca gregaria* (Forskål), which can move long distances as massive swarms in Africa and the Middle East, breeding in one country can put crops in neighboring countries at risk, resulting in perpetually reactive and increasingly insecticide-based, rather than preventive maintenance strategies (Steedman, 1988; Showler & Potter 1991; Showler,

Bottrell, D.G.; Wade, L.J. & Bruce, D.L. (1973). Boll weevils fail to develop resistance to

*Economic Entomology* 66: 791-792, ISSN 0022-0493.

malathion after several years of heavy exposure in Texas High Plains. *Journal of* 

**4. Possibilities** 

pest outbreaks.

1995).

**5. References** 

squares (3–5.4-mm- diameter) first develop (before 2%) until cut-out when square production declines rapidly (Guinn, 1986; Cothren, 1999).

Under subtropical field conditions, feeding on pinhead- and match-head-sized squares is negligible, and large squares are preferred to medium-sized squares regardless of planting date (Showler, 2005). Boll weevil feeding punctures on large squares were 7.8- and 25-fold more abundant compared with match-head squares and bolls, respectively (Showler, 2004b). In terms of nutritional value, medium and large squares promote greater egg production and longevity of adult boll weevils than any other stage of cotton fruiting body (Showler, 2008b), and in terms of providing enough food and space for the immature stages of the boll weevil to develop, pinhead and match-head squares are generally too small (Showler, 2004b). Hence, spraying insecticides well before medium and large square sizes are available is of little value to crop protection and for impeding boll weevil reproduction, which agrees with the recommendation by Norman and Sparks (1998) for beginning boll weevil control in the Lower Rio Grande Valley when one-third-grown squares appear. Once large squares blossom and form post-bloom, young, and hardened older bolls, the nutritional value for longevity and egg production declines to nil when the rind can no longer be penetrated (Showler, 2004b). This explains why adult boll weevil populations plateau following cut-out through harvest (Showler et al., 2005). While spraying during the late season, particularly the series of late season eradication program sprays that occur in the first year (USDA-APHIS, 2007), can likely reduce boll weevil numbers, warm winters with plentiful food can ensure the survival of many until after spring cotton planting.

Scott et al. (1998) reported that, in the Lower Rio Grande Valley, early- and mediummaturing cotton varieties produce the best yields. In a similar vein, square production in early-planted cotton is lower than in later plantings and avoids the high numbers of weevils occurring in later-planted cotton (Showler et al., 2005). Although late-planted cotton produces more squares than early-planted cotton, this advantage is off-set by losses from heavy boll weevil infestations (Showler et al., 2005). The best time for planting was found to be intermediate between early and late for an optimal balance between increasing square production while avoiding the greatest accumulations of boll weevils, thereby reducing insecticide applications as well (Showler et al., 2005).

Harvest timing can also influence insecticide use. From a crop protection perspective, although harvesting late (at 75% boll splitting) rather than earlier (at 40% boll split) can require an extra insecticide application where using the proactive approach, particularly when boll weevil populations were relatively large, but harvesting late captures greater quantities of lint when more bolls have matured, resulting in better economic return, even if the late season insecticide treatment is superfluous (Showler & Robinson, 2008). Mixing the defoliant with an insecticide was found to be relatively ineffective and unreliable (Showler, 2008a).

#### **3.3 Resistance**

Boll weevil tolerance to organophosphorus, carbamate, and pyrethroid insecticides was reported by Kanga et al. (1995), but analyses of field populations have not detected resistance to malathion. It is conceivable, however, that under continual insecticide pressure from malathion only, resistance might develop (Bottrell et al., 1973), and because the boll weevil eradication program relies exclusively upon malathion, exposed boll weevil populations should be assessed intermittently for signs of resistance. For the time being, malathion remains toxic to boll weevils, even at reduced rates (Showler et al., 2002).

## **4. Possibilities**

442 Insecticides – Pest Engineering

squares (3–5.4-mm- diameter) first develop (before 2%) until cut-out when square

Under subtropical field conditions, feeding on pinhead- and match-head-sized squares is negligible, and large squares are preferred to medium-sized squares regardless of planting date (Showler, 2005). Boll weevil feeding punctures on large squares were 7.8- and 25-fold more abundant compared with match-head squares and bolls, respectively (Showler, 2004b). In terms of nutritional value, medium and large squares promote greater egg production and longevity of adult boll weevils than any other stage of cotton fruiting body (Showler, 2008b), and in terms of providing enough food and space for the immature stages of the boll weevil to develop, pinhead and match-head squares are generally too small (Showler, 2004b). Hence, spraying insecticides well before medium and large square sizes are available is of little value to crop protection and for impeding boll weevil reproduction, which agrees with the recommendation by Norman and Sparks (1998) for beginning boll weevil control in the Lower Rio Grande Valley when one-third-grown squares appear. Once large squares blossom and form post-bloom, young, and hardened older bolls, the nutritional value for longevity and egg production declines to nil when the rind can no longer be penetrated (Showler, 2004b). This explains why adult boll weevil populations plateau following cut-out through harvest (Showler et al., 2005). While spraying during the late season, particularly the series of late season eradication program sprays that occur in the first year (USDA-APHIS, 2007), can likely reduce boll weevil numbers, warm winters with plentiful food can ensure the survival of many until after spring cotton planting. Scott et al. (1998) reported that, in the Lower Rio Grande Valley, early- and mediummaturing cotton varieties produce the best yields. In a similar vein, square production in early-planted cotton is lower than in later plantings and avoids the high numbers of weevils occurring in later-planted cotton (Showler et al., 2005). Although late-planted cotton produces more squares than early-planted cotton, this advantage is off-set by losses from heavy boll weevil infestations (Showler et al., 2005). The best time for planting was found to be intermediate between early and late for an optimal balance between increasing square production while avoiding the greatest accumulations of boll weevils, thereby reducing

Harvest timing can also influence insecticide use. From a crop protection perspective, although harvesting late (at 75% boll splitting) rather than earlier (at 40% boll split) can require an extra insecticide application where using the proactive approach, particularly when boll weevil populations were relatively large, but harvesting late captures greater quantities of lint when more bolls have matured, resulting in better economic return, even if the late season insecticide treatment is superfluous (Showler & Robinson, 2008). Mixing the defoliant with an insecticide was found to be relatively ineffective and unreliable (Showler,

Boll weevil tolerance to organophosphorus, carbamate, and pyrethroid insecticides was reported by Kanga et al. (1995), but analyses of field populations have not detected resistance to malathion. It is conceivable, however, that under continual insecticide pressure from malathion only, resistance might develop (Bottrell et al., 1973), and because the boll weevil eradication program relies exclusively upon malathion, exposed boll weevil populations should be assessed intermittently for signs of resistance. For the time being, malathion remains toxic to boll weevils, even at reduced rates (Showler et

production declines rapidly (Guinn, 1986; Cothren, 1999).

insecticide applications as well (Showler et al., 2005).

2008a).

al., 2002).

**3.3 Resistance** 

There are a number of ways in which chemical boll weevil control might be improved. First, a more sensitive trap would permit increasingly timely responses to the early in-season presence of adult boll weevils (but not while squares are still match-head size). At that point, spraying should provide continuous protection of vulnerable and nutritious mediumand large-sized squares. Even if sprays occur weekly, achieving acceptable control on an area-wide scale is improbable, which suggests that using a more sensitive trap design could result in more appropriately-timed, and likely increased, spray applications for the subtropics (unless spraying is conducted at 4-day intervals between late match-head to cutout stages) where overwintering populations are relatively large (Showler, 2007). Both crop protection strategies and the eradication approach should evolve to incorporate emerging information on boll weevil ecology to find tactics that can help mitigate population buildups, such as avoiding late planting, use of earlier-maturing varieties, and development of longer-residual insecticides to reduce numbers of applications and to enhance protection of squares. Because subtropical boll weevil populations are active during winter and can sustain themselves on citrus, removal of such plentiful food through post-harvest orchard sanitation would augment the ban on cotton. Another overlooked tactic is plant resistance. While cotton has been bred for a variety of traits, no cultivars have been developed to resist boll weevil attack. Efforts in this direction might include altering square rind thickness or consistency to make the inner portion, where the immature weevil stages develop, less accessible, or changing the availability of certain nutrients that can affect egg production (Showler, 2009a). Eradicating a tropical pest like the boll weevil in temperate areas was achievable, but the subtropics are more akin to the insect's native habitat in terms of temperature and host plants. For this reason, adjustments to the temperate eradication strategy might have to involve tailoring insecticides, application timing, and the circumstances under which they are applied (*e.g.*, as influenced by planting dates and phenological stages of the crop) for extending prophylactic crop protection and decimating boll weevil populations as selectively as possible to avoid the possibility of for secondary pest outbreaks.

However, even were all of the issues surrounding subtropical boll weevil eradication to be resolved, the feasibility of remaining boll weevil-free in areas along international borders is compromised by boll weevil populations breeding on the other side of the border where attention to eradication, for a complex of reasons, may not be in synchrony. Hence, the success of eradication in subtropical border areas depends to a great extent on the coordinated efforts of both countries. In the instance of a somewhat analogous pest, the desert locust, *Schistocerca gregaria* (Forskål), which can move long distances as massive swarms in Africa and the Middle East, breeding in one country can put crops in neighboring countries at risk, resulting in perpetually reactive and increasingly insecticide-based, rather than preventive maintenance strategies (Steedman, 1988; Showler & Potter 1991; Showler, 1995).

## **5. References**

Bottrell, D.G.; Wade, L.J. & Bruce, D.L. (1973). Boll weevils fail to develop resistance to malathion after several years of heavy exposure in Texas High Plains. *Journal of Economic Entomology* 66: 791-792, ISSN 0022-0493.

The Conundrum of Chemical Boll Weevil Control in Subtropical Regions 445

Guerra, A.A.; Garcia, R.F.; Bodegas, P.R. & De Coss, M.E. (1984). The quiescent physiological

Guinn, G. (1986). Hormonal relations during reproduction. In: *Cotton Physiology*, J.R.

Haney, P.B. (2001). The cotton boll weevil in the United States: impact on cotton production

Haney, P.B.; Herzog, G. & Roberts, P.M. (2001). Boll weevil eradication in Georgia. In: *Boll* 

Hardee, D.D.; Jones, G.D. & Adams, L.C. (1999). Emergence, movement, and host plants of

Heilman, M.D.; Namken, L.N.; Norman, J.W. & Lukefahr, M.J. (1979). Evaluation of an

Howard, L.O. (1897). Insects affecting the cotton plant. *USDA Farmers' Bulletin* No. 47: 1-31. Hunter, W.D. (1917). The boll weevil problem with special reference to means of reducing

Johnson, D.R. & Martin, G. (2001). Boll weevil eradication in Arkansas. In: *Boll Weevil* 

Jones, D. & Sterling, W.L. (1979). Manipulation of red imported fire ants in a trap crop for boll weevil suppression. *Environmental Entomology* 8: 1073-1077, ISSN 0046-225X. Jones, R.W.; Cate, J.R.; Hernandez, S.M. & Navarro, R.T. (1992). Hosts and seasonal activity

northeastern Mexico. *Environmental Entomology* 22: 99-108, ISSN 0046-225X. Kanga, L.H.B.; Plapp, F.W., Jr.; Wall, M.L.; Karner, M.A.; Huffman, R.L.; Fuchs, T.W.; Elzen,

Koštál, V. (2006). Eco-physiological phases of insect diapause. *Journal of Insect Physiology* 52:

Cotton Foundation, ISBN 0939809060, Memphis, Tennessee, USA.

Cotton Foundation, ISBN 0939809060, Memphis, Tennessee, USA.

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**21** 

*Botswana* 

**in Northern Botswana** 

*1Water Affairs, Private Bag 002, Maun* 

*2Animal Health, Box 14, Maun,* 

C. N. Kurugundla1, P. M. Kgori2 and N. Moleele3

*3Biokavango Project, Okavango Research Institute, Maun,* 

**Management of Tsetse Fly Using Insecticides** 

The tsetse fly (*Glossina* spp.) is only found in Africa and carries trypanosomes (the disease agents causing human sleeping sickness and animal trypanosomosis- *Nagana*), reaching their southern limits (particularly the *morsitans* group) in Botswana and Kwazulu Natal in South Africa. The tsetse fly transmitted disease is a serious problem in Sub-Saharan Africa and it is estimated that the removal of this disease could double livestock production and markedly increase cultivation levels. It is estimated that potential distribution of tsetse fly in

Generally, the economic and social impacts of *nagana* and sleeping sickness on animal production and human health are severe, estimates put annual cattle production losses at US\$2.7 billion and >55, 000 people dying from sleeping sickness annually (Budd, 1999). The disease is usually of chronic and debilitating form. It is essentially a problem in rural areas, where the threat and burden of such is a significant contributor to rural poverty and

In Botswana, the tsetse infested area covered ≤ 5% but has had significant impact on livestock and human populations, particularly in Ngamiland and Chobe regions, largely because of the wet areas in an otherwise dry country. Prior to the rinderpest pandemic of 1896 which significantly reduced tsetse populations in much of east and southern Africa as a result of the critical loss of food source, the distribution of tsetse fly in Botswana had reached its historical limits of approximately >20,000 km2 (Ford, 1971; Jordon, 1986). As the tsetse populations recovered from the rinderpest epizootic the incidence of trypanosomosis increased. For instance, between 1949 and 1960, the cattle populations in Chobe District declined by ≤ 95% (Lambrecht, 1972). Ploughing along the flood plains is a common local practice in these areas, and whilst beneficial in the sense of optimal exploitation of soil moisture, this practice carries the risk of potential exposure of farm workers to tsetse fly bites. An average of 50 (geometric mean range = 13-272) trypanosomosis cases was recorded between 1957 and 1977 in Maun Hospital each year. Today the Okavango Delta, Kwando-Linyanti-Chobe areas are the most important destinations for international tourists and one of the major sources of revenue for the country. The risk of sleeping sickness within these areas is perceived as potential threat to the tourism industry, both locally and nationally

Africa is 300,000 km2 (Mathiessen & douthwaite, 1985).

**1. Introduction** 

malnutrition.

(RTTCP, 1995).


## **Management of Tsetse Fly Using Insecticides in Northern Botswana**

C. N. Kurugundla1, P. M. Kgori2 and N. Moleele3 *1Water Affairs, Private Bag 002, Maun 2Animal Health, Box 14, Maun, 3Biokavango Project, Okavango Research Institute, Maun, Botswana* 

## **1. Introduction**

448 Insecticides – Pest Engineering

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The tsetse fly (*Glossina* spp.) is only found in Africa and carries trypanosomes (the disease agents causing human sleeping sickness and animal trypanosomosis- *Nagana*), reaching their southern limits (particularly the *morsitans* group) in Botswana and Kwazulu Natal in South Africa. The tsetse fly transmitted disease is a serious problem in Sub-Saharan Africa and it is estimated that the removal of this disease could double livestock production and markedly increase cultivation levels. It is estimated that potential distribution of tsetse fly in Africa is 300,000 km2 (Mathiessen & douthwaite, 1985).

Generally, the economic and social impacts of *nagana* and sleeping sickness on animal production and human health are severe, estimates put annual cattle production losses at US\$2.7 billion and >55, 000 people dying from sleeping sickness annually (Budd, 1999). The disease is usually of chronic and debilitating form. It is essentially a problem in rural areas, where the threat and burden of such is a significant contributor to rural poverty and malnutrition.

In Botswana, the tsetse infested area covered ≤ 5% but has had significant impact on livestock and human populations, particularly in Ngamiland and Chobe regions, largely because of the wet areas in an otherwise dry country. Prior to the rinderpest pandemic of 1896 which significantly reduced tsetse populations in much of east and southern Africa as a result of the critical loss of food source, the distribution of tsetse fly in Botswana had reached its historical limits of approximately >20,000 km2 (Ford, 1971; Jordon, 1986). As the tsetse populations recovered from the rinderpest epizootic the incidence of trypanosomosis increased. For instance, between 1949 and 1960, the cattle populations in Chobe District declined by ≤ 95% (Lambrecht, 1972). Ploughing along the flood plains is a common local practice in these areas, and whilst beneficial in the sense of optimal exploitation of soil moisture, this practice carries the risk of potential exposure of farm workers to tsetse fly bites. An average of 50 (geometric mean range = 13-272) trypanosomosis cases was recorded between 1957 and 1977 in Maun Hospital each year. Today the Okavango Delta, Kwando-Linyanti-Chobe areas are the most important destinations for international tourists and one of the major sources of revenue for the country. The risk of sleeping sickness within these areas is perceived as potential threat to the tourism industry, both locally and nationally (RTTCP, 1995).

Management of Tsetse Fly Using Insecticides in Northern Botswana 451

The earliest methods used for reducing tsetse numbers in Botswana (and throughout Africa), were large-scale clearing of bush and vegetation where tsetse fly rested, thus denying them shelter, and elimination of wild game animals which provided the natural food source of the fly. However, both these methods had huge environmental implications and were gradually phased out when alternative methods became available. Nonetheless, bush clearing continued as a complementary method until the mid 1960's (Davis, 1980). Game fencing also emerged as alternative, especially to limit the incursion of game animals

The method of controlling tsetse using chemicals became common in the 1940s following the discovery of chlorinated hydrocarbon insecticides. In Botswana, residual insecticides such as DDT were introduced in 1967 by applying it to selected tsetse resting sites using knapsack spraying machines. This technique, known as *selective ground spraying*, targeted only about 20% of all potential tsetse resting sites in the woodland, including tree trunks, lower large branches, rot holes and holes on the ground such as ant-bear holes, springhare and hyena dens (Davies, 1980). The residual insecticides would remain available (even after spraying) to tsetse which emerged from underground pupal sites for 2 to 3 months after spray treatment. Given the accessibility challenges within the Okavango Delta, the ground spraying method of control got restricted only to the peripheral areas around the western Delta, the 'Maun Front' or along the Savuti Channel, and it was found to be unsuited to the

Following preliminary trials carried out in 1971, Botswana's tsetse control strategy switched almost entirely to aerial spraying of insecticide using the Sequential Aerosol Technique (SAT). Very low dosages of endosulfan, and later a cocktail of endosulfan and synthetic pyrethroid insecticide were applied several times over the tsetse habitat to cover all the emerging tsetse pupal period. At best, the early SAT campaign by aerial spraying (Davies, 1979) reduced the tsetse distribution limits from 20,000km2 to 5,000km2 but still tsetse could

not be eliminated completely despite that being the primary objective from the onset.

In 1992 Botswana (following other African countries) adopted the use of chemicallyimpregnated tsetse screens or targets and traps known as the *odour-bait technique* (Vale and Torr, 2004). The system was pioneered in Zimbabwe in the 1980s and became wildly used due to its perceived environmental sensitivity. Targets were treated with synthetic pyrethroid insecticides, particularly deltamethrin suspension concentrate formulation of 0.6% (w/v) following the treatment procedure (Kgori et al., 2006). In the technique the insecticide is applied only to the target screens and not the surrounding vegetation. All traces of the insecticide would therefore disappear once the targets are removed. The concept was well received and particularly suited for the Okavango's pristine wilderness. Overall, the effectiveness of targets depended on the management and institutional capacity of the government's Tsetse Control Department. Effective distribution of targets and their regular maintenance in the Okavango Delta became a problem as the access was severely impeded by vegetation and terrain. However, a long period of drought ending in 1999 allowed some 25,000

into the settled areas of the Okavango Delta fringes.

**2.2 1960-1972 residual ground spraying** 

swamps and island mosaics of the delta interior.

**2.3 1970-1990 non-residual aerial spraying** 

**2.4 1990-2000 traps and targets** 

However, at the end of the rinderpest epidemic, a rebound of the tsetse population in northern Botswana and the subsequent disease challenges led to the introduction of intensified control efforts in the mid-twentieth century using conventional and improved insecticide based methods. This chapter therefore reviews the history of management of tsetse fly control in Botswana, with specific focus on the factors that influence tsetse fly distribution in Botswana, methods of insecticide applications to control tsetse fly, effectiveness of the control methods, and monitored environmental impacts.

## **2. History, distribution, control and management of tsetse fly in Botswana**

Comprehensive historical review of tsetse distribution in Botswana up to the 1980s is provided by Davies (1980). Early records largely from European explorers suggest that tsetse occurred in a continuous belt from Angola, through Caprivi Strip (Namibia) and then the Kwando-Linyanti and Chobe River systems, then via the Selinda Spillway to cover most of northern Botswana's Okavango Delta and its immediate surroundings including Nxai Pan N. P (Figure 1). The history of control and management of tsetse in Botswana is detailed below.

Fig. 1. Distribution of tsetse flies in Botswana (NG = Ngamiland District – Okavango Delta, Maun, Kwando Linyanti; CH = Chobe District – Chobe and Kasane; and CD = Central District – Nxai Pan N. P)

## **2.1 1940-1960 early methods of tsetse control**

The disease Trypanosomosis (sleeping sickness and *Nagana*) is the primary reason for controlling tsetse fly in Botswana. In the early 1940s, cattle *Nagana* in parts of Ngamiland was so bad that a government tsetse control department was formed to combat the disease. The earliest methods used for reducing tsetse numbers in Botswana (and throughout Africa), were large-scale clearing of bush and vegetation where tsetse fly rested, thus denying them shelter, and elimination of wild game animals which provided the natural food source of the fly. However, both these methods had huge environmental implications and were gradually phased out when alternative methods became available. Nonetheless, bush clearing continued as a complementary method until the mid 1960's (Davis, 1980). Game fencing also emerged as alternative, especially to limit the incursion of game animals into the settled areas of the Okavango Delta fringes.

### **2.2 1960-1972 residual ground spraying**

450 Insecticides – Pest Engineering

However, at the end of the rinderpest epidemic, a rebound of the tsetse population in northern Botswana and the subsequent disease challenges led to the introduction of intensified control efforts in the mid-twentieth century using conventional and improved insecticide based methods. This chapter therefore reviews the history of management of tsetse fly control in Botswana, with specific focus on the factors that influence tsetse fly distribution in Botswana, methods of insecticide applications to control tsetse fly,

**2. History, distribution, control and management of tsetse fly in Botswana** 

Comprehensive historical review of tsetse distribution in Botswana up to the 1980s is provided by Davies (1980). Early records largely from European explorers suggest that tsetse occurred in a continuous belt from Angola, through Caprivi Strip (Namibia) and then the Kwando-Linyanti and Chobe River systems, then via the Selinda Spillway to cover most of northern Botswana's Okavango Delta and its immediate surroundings including Nxai Pan N. P (Figure

NG

CH

CD

Fig. 1. Distribution of tsetse flies in Botswana (NG = Ngamiland District – Okavango Delta, Maun, Kwando Linyanti; CH = Chobe District – Chobe and Kasane; and CD = Central

The disease Trypanosomosis (sleeping sickness and *Nagana*) is the primary reason for controlling tsetse fly in Botswana. In the early 1940s, cattle *Nagana* in parts of Ngamiland was so bad that a government tsetse control department was formed to combat the disease.

District – Nxai Pan N. P)

**2.1 1940-1960 early methods of tsetse control** 

effectiveness of the control methods, and monitored environmental impacts.

1). The history of control and management of tsetse in Botswana is detailed below.

The method of controlling tsetse using chemicals became common in the 1940s following the discovery of chlorinated hydrocarbon insecticides. In Botswana, residual insecticides such as DDT were introduced in 1967 by applying it to selected tsetse resting sites using knapsack spraying machines. This technique, known as *selective ground spraying*, targeted only about 20% of all potential tsetse resting sites in the woodland, including tree trunks, lower large branches, rot holes and holes on the ground such as ant-bear holes, springhare and hyena dens (Davies, 1980). The residual insecticides would remain available (even after spraying) to tsetse which emerged from underground pupal sites for 2 to 3 months after spray treatment. Given the accessibility challenges within the Okavango Delta, the ground spraying method of control got restricted only to the peripheral areas around the western Delta, the 'Maun Front' or along the Savuti Channel, and it was found to be unsuited to the swamps and island mosaics of the delta interior.

## **2.3 1970-1990 non-residual aerial spraying**

Following preliminary trials carried out in 1971, Botswana's tsetse control strategy switched almost entirely to aerial spraying of insecticide using the Sequential Aerosol Technique (SAT). Very low dosages of endosulfan, and later a cocktail of endosulfan and synthetic pyrethroid insecticide were applied several times over the tsetse habitat to cover all the emerging tsetse pupal period. At best, the early SAT campaign by aerial spraying (Davies, 1979) reduced the tsetse distribution limits from 20,000km2 to 5,000km2 but still tsetse could not be eliminated completely despite that being the primary objective from the onset.

### **2.4 1990-2000 traps and targets**

In 1992 Botswana (following other African countries) adopted the use of chemicallyimpregnated tsetse screens or targets and traps known as the *odour-bait technique* (Vale and Torr, 2004). The system was pioneered in Zimbabwe in the 1980s and became wildly used due to its perceived environmental sensitivity. Targets were treated with synthetic pyrethroid insecticides, particularly deltamethrin suspension concentrate formulation of 0.6% (w/v) following the treatment procedure (Kgori et al., 2006). In the technique the insecticide is applied only to the target screens and not the surrounding vegetation. All traces of the insecticide would therefore disappear once the targets are removed. The concept was well received and particularly suited for the Okavango's pristine wilderness.

Overall, the effectiveness of targets depended on the management and institutional capacity of the government's Tsetse Control Department. Effective distribution of targets and their regular maintenance in the Okavango Delta became a problem as the access was severely impeded by vegetation and terrain. However, a long period of drought ending in 1999 allowed some 25,000

Management of Tsetse Fly Using Insecticides in Northern Botswana 453

Fig. 2. Operations to control tsetse in the Okavango Delta region of Botswana conducted between 2001 and 2002, and the locations of tsetse and other non-target agents' monitoring sites. Sites indicated by solid triangles indicate the permanent sites at Zimbiri (Z), Mombo (M), Guai (G), Thapagadi (T), Bobo (B) and Chief's Island (C). Open circles indicate

occasional monitoring sites used at various times during the spraying operations, including Kings Pool (KP), Selinda (S), Vumbura (Va), Vundumxiki (Vi), and Matsibe (Mat). Source: Kgori et al (2006). Xakanaxa Salvinia monitoring sites (XSa), Khwai River (KR) and North

nature; the insecticide deposition remained in the environment only for a short period of

Large-scale trials (1,000 – 4,000 km2) of aerial spraying with endosulfan in Botswana began in 1973 as a pilot programme to assess the feasibility of even bigger operations to eliminate tsetse fly from the whole of the Okavango Delta (Davies, 1979). By the mid 80s, the scale of the annual operations had increased to about 6,000 – 9,000 km2 and very good control was achieved. The distribution limit was reduced significantly from 20,000 km2 to 5,000 km2 and

Gate (NR) – Source: Kurugundla and Serumola (2007).

time and therefore was non-persistent.

targets to be deployed throughout much of the usually inaccessible parts of the Okavango Delta. These gains were to be reversed when good rains returned in 1999/2000, thus again putting the effectiveness of this tsetse control measure at question. With the above average rainfall, the tsetse fly was able to recover and disperse beyond the confines of the Okavango Delta, taking the threat of trypanosomosis once again back to the people and livestock. Cattle deaths resulting from *Nagana* increased during this period (Sharma et al., 2001).

#### **2.5 2000 onwards - aerial spraying; a reversal in strategy**

In the year 2000, the Botswana government initiated a new programme to control tsetse fly and trypanosomosis in and around the Okavango Delta. However, the primary objective remained unchanged; to eliminate tsetse and trypanosomosis from the Okavango Delta and the adjacent Kwando and Linyanti. Tsetse surveys in 2001 showed that the tsetse distribution limits had extended from 5,000 km2 to about 12,000 km2. Some 30,000 cattle within the villages surrounding the Okavango delta were clearly at risk of *Nagana* and they were subsequently treated with prophylactic trypanocides.

Reintroduction of aerial spraying became the cornerstone of the new campaign, but this time around targets were used as protective barriers between successive operations to stop tsetse fly from reinvading treated areas. Two aerial spraying operations were planned and executed in succession (in 2001 and 2002) to cover all the infestation in the Okavango Delta (Kgori et al., 2006; Allsopp & Phillemon-Motsu, 2002). In 2001, about 7,000 km2 (upper box Figure 2) of the northern part of the Delta was aerial sprayed, followed in 2002 by 8,650km2 (Lower Box Figure 2) of all the remaining infestation in the south (Figure 2). The insecticide of choice was deltamethrin (0.35% (w/v) ulv formulation applied at night using four turbo thrush fixed-wing aircraft. The aircraft were all guided by previously unavailable advanced navigation guidance system (Kgori et al., 2009).

### **3. Effectiveness of control measures**

Apart from limited ground spraying in accessible peripheral areas such as Savuti Channel in the Chobe District, aerial spraying using ulv applications of endosulfan was the only method used to control tsetse fly in Botswana in the 1970s and 80s. Unfortunately for Botswana, since tsetse fly control was conducted largely in aquatic and pristine environment, endosulfan concentrations had to be kept lower than usual; it was applied at maximum of 12 g ha-1 compared to operations in other countries where higher levels of 16 to 20 g ha-1 were used to achieve comparatively good results (Douthwaite et al., 1981).

Typically, a series of aerial spray treatments were applied at night-time in winter with stable air and temperature inversion conditions in place, and using Piper Aztec or Cessna 310 aircraft from a height of about 15 m above the tree canopy. Under such inversion conditions, the tiny insecticide droplets would drift through the tsetse habitat in order to effectively kill adult tsetse fly. The insecticide was dispersed through a micronair atomizer set to give droplets in the range of 30–40 microns. This ensured that only small quantities of the insecticide were applied to the tsetse habitat. During these early years of aerial spraying, SAT operations were flown without the benefit of advanced aircraft guiding systems which are available today. Therefore spray distribution relied upon far less sophisticated methods of guidance such as ground marker parties with hand held miniflares positioned at each end of the spray run in order to guide the pilots. Also the formulation was non-residual in

targets to be deployed throughout much of the usually inaccessible parts of the Okavango Delta. These gains were to be reversed when good rains returned in 1999/2000, thus again putting the effectiveness of this tsetse control measure at question. With the above average rainfall, the tsetse fly was able to recover and disperse beyond the confines of the Okavango Delta, taking the threat of trypanosomosis once again back to the people and livestock. Cattle

In the year 2000, the Botswana government initiated a new programme to control tsetse fly and trypanosomosis in and around the Okavango Delta. However, the primary objective remained unchanged; to eliminate tsetse and trypanosomosis from the Okavango Delta and the adjacent Kwando and Linyanti. Tsetse surveys in 2001 showed that the tsetse distribution limits had extended from 5,000 km2 to about 12,000 km2. Some 30,000 cattle within the villages surrounding the Okavango delta were clearly at risk of *Nagana* and they

Reintroduction of aerial spraying became the cornerstone of the new campaign, but this time around targets were used as protective barriers between successive operations to stop tsetse fly from reinvading treated areas. Two aerial spraying operations were planned and executed in succession (in 2001 and 2002) to cover all the infestation in the Okavango Delta (Kgori et al., 2006; Allsopp & Phillemon-Motsu, 2002). In 2001, about 7,000 km2 (upper box Figure 2) of the northern part of the Delta was aerial sprayed, followed in 2002 by 8,650km2 (Lower Box Figure 2) of all the remaining infestation in the south (Figure 2). The insecticide of choice was deltamethrin (0.35% (w/v) ulv formulation applied at night using four turbo thrush fixed-wing aircraft. The aircraft were all guided by previously unavailable advanced

Apart from limited ground spraying in accessible peripheral areas such as Savuti Channel in the Chobe District, aerial spraying using ulv applications of endosulfan was the only method used to control tsetse fly in Botswana in the 1970s and 80s. Unfortunately for Botswana, since tsetse fly control was conducted largely in aquatic and pristine environment, endosulfan concentrations had to be kept lower than usual; it was applied at maximum of 12 g ha-1 compared to operations in other countries where higher levels of 16 to

Typically, a series of aerial spray treatments were applied at night-time in winter with stable air and temperature inversion conditions in place, and using Piper Aztec or Cessna 310 aircraft from a height of about 15 m above the tree canopy. Under such inversion conditions, the tiny insecticide droplets would drift through the tsetse habitat in order to effectively kill adult tsetse fly. The insecticide was dispersed through a micronair atomizer set to give droplets in the range of 30–40 microns. This ensured that only small quantities of the insecticide were applied to the tsetse habitat. During these early years of aerial spraying, SAT operations were flown without the benefit of advanced aircraft guiding systems which are available today. Therefore spray distribution relied upon far less sophisticated methods of guidance such as ground marker parties with hand held miniflares positioned at each end of the spray run in order to guide the pilots. Also the formulation was non-residual in

20 g ha-1 were used to achieve comparatively good results (Douthwaite et al., 1981).

deaths resulting from *Nagana* increased during this period (Sharma et al., 2001).

**2.5 2000 onwards - aerial spraying; a reversal in strategy** 

were subsequently treated with prophylactic trypanocides.

navigation guidance system (Kgori et al., 2009).

**3. Effectiveness of control measures** 

Fig. 2. Operations to control tsetse in the Okavango Delta region of Botswana conducted between 2001 and 2002, and the locations of tsetse and other non-target agents' monitoring sites. Sites indicated by solid triangles indicate the permanent sites at Zimbiri (Z), Mombo (M), Guai (G), Thapagadi (T), Bobo (B) and Chief's Island (C). Open circles indicate occasional monitoring sites used at various times during the spraying operations, including Kings Pool (KP), Selinda (S), Vumbura (Va), Vundumxiki (Vi), and Matsibe (Mat). Source: Kgori et al (2006). Xakanaxa Salvinia monitoring sites (XSa), Khwai River (KR) and North Gate (NR) – Source: Kurugundla and Serumola (2007).

nature; the insecticide deposition remained in the environment only for a short period of time and therefore was non-persistent.

Large-scale trials (1,000 – 4,000 km2) of aerial spraying with endosulfan in Botswana began in 1973 as a pilot programme to assess the feasibility of even bigger operations to eliminate tsetse fly from the whole of the Okavango Delta (Davies, 1979). By the mid 80s, the scale of the annual operations had increased to about 6,000 – 9,000 km2 and very good control was achieved. The distribution limit was reduced significantly from 20,000 km2 to 5,000 km2 and

Management of Tsetse Fly Using Insecticides in Northern Botswana 455

two boom-mounted micronair AU 4000 rotary atomizers operated at cage speeds of 10,800 rpm and flowrate of 7.6-8.6 l/km2. Such technology development used in recent SAT operations had positive implications on the distribution and deposition of formulated insecticide droplets and, ultimately on the efficiency and effectiveness of the spray application. During 2001 and 2002 operations, and later in 2006 at Kwando and Linyanti, deltamethrin insecticide (0.35% (w/w) ulv formulation was applied at 0.26 -0.30 g active ingredient (a.i.) ha-1. The higher dose rate of 0.3 g a.i. ha-1 was used for the first two cycles when tsetse fly

Initially, two successive spray treatments were conducted in 2001 and 2002 to cover approximately 16,000 km2 in the Okavango Delta (Figure 2). A similar and follow up operation took place in 2006 covering an additional 10,000 km2 of the Kwando and Linyanti border area, north of the Okavango Delta which also extended across Namibia's eastern Caprivi border into Southern Angola (Figure 3) in order to guarantee complete removal of all the remaining tsetse fly infestation in northern Botswana. This approach ensured that the northern tsetse infestation along the Kwando and Linyanti Rivers did not re-infest the Okavango Delta.

Fig. 3. Area of deltamethrin spraying in 2006. Sites of bioassay experiments and field monitoring areas to assess the impacts of deltamethrin spray on salvinia weevils and other

aquatic invertebrates in Kwando-Linyanti River System.

population would normally be at its highest density (Saunders, 1962).

the threat of both *Nagana* and human sleeping sickness was effectively controlled due to sequential aerial spraying of insecticide, reduced water levels with less flooded areas and increased drought conditions in Botswana. Sequential spraying was necessary because the tsetse (i.e., young adults) would continue to emerge from the underground pupal stage for several days after each preceding cycle.

However, despite repeated applications complete eradication was not possible. Several possible reasons may have contributed to the failure by early SAT operations to achieve eradication in Botswana - including; low dosages used, lack of boundary protection since odour baited targets were unavailable then, poor navigation and random rather than systematic treatments. The navigation challenges observed often resulted with localized over-spraying which raised environmental concerns, notably fish kill (Merron & Bruton, 1991), but the low dosages used ensured that only limited off-target effects were possible.

## **4. Deltamethrin**

When endosulfan-based tsetse aerial spraying campaign was ended in 1992, targets almost immediately became the next and only preferred option for tsetse fly control in Botswana. At the time, pyrethroid insecticides were also becoming firmly established in the insecticide market and Deltamethrin was the preferred choice for use in the treatment of odour-baited targets (Vale & Torr, 2004). For the next ten years, targets remained the only method used for controlling tsetse in northern Botswana's Okavango Delta, Kwando-Linyanti-Chobe River systems. A new integrated strategy involving the reintroduction of sequential aerial spraying was later introduced in 2001 when targets were proving difficult to effectively implement in Northern Botswana due to accessibility problems.

Prior to commencement of spraying in May 2001, tsetse surveys indicated that tsetse distribution had increased significantly to about 12,000 km2 from the previous 5,000 km2 since the end of the last aerial spraying campaign in the 1980s. Increased spread of tsetse fly could be attributed to a long period of no effective control and above average rainfall coupled with increased flood inflows in the Okavango Delta. Also the use of motorized boats as well as airplanes used to transport tourists in and out of the Delta contributed to the spreading of tsetse flies. This was evidenced by the occasional sighting of the tsetse flies in Maun which became a real concern to authorities, and hence the need for concerted effort to reverse the situation.

A two year aerial spraying operation (2001 and 2002, Figure 2) for the north and south of the Okavango Delta using Turbo Thrush Aircraft was planned and implemented in succession to cover the entire Okavango Delta. At the interface of the two spray blocks a target barrier was deployed to prevent tsetse crossing from one unsprayed area to another sprayed area between the successive spray operations. The revised strategy also included a component of the sterile insect technique (SIT) as technical backstop, should SAT fail to eliminate tsetse as was previously the case in the 1980s (Feldmann, 2004).

Improvement in the reintroduction of aerial spraying in 2001, 2002 and 2006 was the availability of latest navigation systems which could ensure precision placement of spraying material and eliminate overdosing or even under-dosing through erratic track guidance. For instance all aircraft were fitted with GPS-guided SATLOC navigation and spray management equipment accurate to about 1m. The system therefore had control on the distribution of the spray application and automatically cut off the spray if the aircraft wondered out of the spray block and indeed the prescribed flight path. The aircraft insecticide dispersal units involved

the threat of both *Nagana* and human sleeping sickness was effectively controlled due to sequential aerial spraying of insecticide, reduced water levels with less flooded areas and increased drought conditions in Botswana. Sequential spraying was necessary because the tsetse (i.e., young adults) would continue to emerge from the underground pupal stage for

However, despite repeated applications complete eradication was not possible. Several possible reasons may have contributed to the failure by early SAT operations to achieve eradication in Botswana - including; low dosages used, lack of boundary protection since odour baited targets were unavailable then, poor navigation and random rather than systematic treatments. The navigation challenges observed often resulted with localized over-spraying which raised environmental concerns, notably fish kill (Merron & Bruton, 1991), but the low dosages used ensured that only limited off-target effects were possible.

When endosulfan-based tsetse aerial spraying campaign was ended in 1992, targets almost immediately became the next and only preferred option for tsetse fly control in Botswana. At the time, pyrethroid insecticides were also becoming firmly established in the insecticide market and Deltamethrin was the preferred choice for use in the treatment of odour-baited targets (Vale & Torr, 2004). For the next ten years, targets remained the only method used for controlling tsetse in northern Botswana's Okavango Delta, Kwando-Linyanti-Chobe River systems. A new integrated strategy involving the reintroduction of sequential aerial spraying was later introduced in 2001 when targets were proving difficult to effectively

Prior to commencement of spraying in May 2001, tsetse surveys indicated that tsetse distribution had increased significantly to about 12,000 km2 from the previous 5,000 km2 since the end of the last aerial spraying campaign in the 1980s. Increased spread of tsetse fly could be attributed to a long period of no effective control and above average rainfall coupled with increased flood inflows in the Okavango Delta. Also the use of motorized boats as well as airplanes used to transport tourists in and out of the Delta contributed to the spreading of tsetse flies. This was evidenced by the occasional sighting of the tsetse flies in Maun which became a real concern to authorities, and hence the need for concerted effort to

A two year aerial spraying operation (2001 and 2002, Figure 2) for the north and south of the Okavango Delta using Turbo Thrush Aircraft was planned and implemented in succession to cover the entire Okavango Delta. At the interface of the two spray blocks a target barrier was deployed to prevent tsetse crossing from one unsprayed area to another sprayed area between the successive spray operations. The revised strategy also included a component of the sterile insect technique (SIT) as technical backstop, should SAT fail to eliminate tsetse as

Improvement in the reintroduction of aerial spraying in 2001, 2002 and 2006 was the availability of latest navigation systems which could ensure precision placement of spraying material and eliminate overdosing or even under-dosing through erratic track guidance. For instance all aircraft were fitted with GPS-guided SATLOC navigation and spray management equipment accurate to about 1m. The system therefore had control on the distribution of the spray application and automatically cut off the spray if the aircraft wondered out of the spray block and indeed the prescribed flight path. The aircraft insecticide dispersal units involved

implement in Northern Botswana due to accessibility problems.

was previously the case in the 1980s (Feldmann, 2004).

several days after each preceding cycle.

**4. Deltamethrin** 

reverse the situation.

two boom-mounted micronair AU 4000 rotary atomizers operated at cage speeds of 10,800 rpm and flowrate of 7.6-8.6 l/km2. Such technology development used in recent SAT operations had positive implications on the distribution and deposition of formulated insecticide droplets and, ultimately on the efficiency and effectiveness of the spray application. During 2001 and 2002 operations, and later in 2006 at Kwando and Linyanti, deltamethrin insecticide (0.35% (w/w) ulv formulation was applied at 0.26 -0.30 g active ingredient (a.i.) ha-1. The higher dose rate of 0.3 g a.i. ha-1 was used for the first two cycles when tsetse fly population would normally be at its highest density (Saunders, 1962).

Initially, two successive spray treatments were conducted in 2001 and 2002 to cover approximately 16,000 km2 in the Okavango Delta (Figure 2). A similar and follow up operation took place in 2006 covering an additional 10,000 km2 of the Kwando and Linyanti border area, north of the Okavango Delta which also extended across Namibia's eastern Caprivi border into Southern Angola (Figure 3) in order to guarantee complete removal of all the remaining tsetse fly infestation in northern Botswana. This approach ensured that the northern tsetse infestation along the Kwando and Linyanti Rivers did not re-infest the Okavango Delta.

Fig. 3. Area of deltamethrin spraying in 2006. Sites of bioassay experiments and field monitoring areas to assess the impacts of deltamethrin spray on salvinia weevils and other aquatic invertebrates in Kwando-Linyanti River System.

Management of Tsetse Fly Using Insecticides in Northern Botswana 457

Spraying commenced in the south block (8650 km2) on 16 May 2002. Following the start of spraying, the catches declined dramatically (Figure 5). For instance, the mean daily catch from fly-rounds was 101.7 (±0.026, *n*=148) for the period 10 April - 16 May compared to 0.23 (±0.019*, n*=66) for the period 24 May - 3 June, this being the period between the end of 1st spraying cycle and the beginning of the second cycle (Figure 5A) and after the 4th cycle no tsetse were found. The catches from traps showed a similar decline (Figure 5B). During 2006 campaign, the tsetse surveys started on 13th May 2006 (Day 1 = 13 May 2006. Figure 6 shows that the abundance of tsetse was higher than 100 with fluctuations in Kwando-Linyanti prior to spraying and it reduced to zero after 2nd, 3rd, 4th and zero catches after the 5th

1st-Apr 1st-May 1st-Jun 1st-Jul 1st-Aug 1st-Sep

Date 1st-Apr 1st-May 1st-Jun 1st-Jul 1st-Aug 1st-Sep

Fig. 5. Detransformed mean daily catch of tsetse from fly rounds and traps within (Bobo Island, Thapaghadi and Chief's Island) the area sprayed during the 2002 control operation. Vertical grey bars indicate the timing of the five spray cycles. Source: Kgori et al., (2006).

spraying cycle (Kgori et al., 2006, Kgori et al., 2009, VEEU-TCD/DAHP 1998).

Mean daily catch

Mean daily catch

**B. 2002 - Traps**

1

0.0

1

10

**A. 2002 - Flyrounds**

0

0.

Spraying of the block (7000 km2) commenced on 3 June 2001. Figure 4 shows the mean daily catch from the five fly-rounds (Figure 4A) and 18 traps within the spray block (4C), and the fly-rounds outside it (Figure 4B). The results from the fly-rounds within the block show that the mean tsetse catch up to the first day of spraying was 44.6 tsetse day-1. Immediately after each cycle the daily catch declined to zero, but then recovered to peaks of 12.8 tsetse day-1 after the 1st cycle and 20.6 tsetse day-1 after the 2nd cycle. Thereafter recovery was less marked, peaking at 6.3 tsetse day-1 after the 3rd cycle, 1.2 tsetse day-1 after the 4th cycle and finally no tsetse was found after the 5th cycle. By contrast, the daily catches from fly-rounds outside the spray block (Figure 4B) increased significantly over the period of spray operation.

Fig. 4. Detransformed mean daily catch of tsetse from fly rounds and traps within (Mombo, Guai and Zimbiri) or outside (Chief's Island, fly rounds only) the area sprayed during the 2001 control operation. Vertical grey bars indicate the timing of the five spray cycles. Source: Kgori et al., (2006).

Spraying of the block (7000 km2) commenced on 3 June 2001. Figure 4 shows the mean daily catch from the five fly-rounds (Figure 4A) and 18 traps within the spray block (4C), and the fly-rounds outside it (Figure 4B). The results from the fly-rounds within the block show that the mean tsetse catch up to the first day of spraying was 44.6 tsetse day-1. Immediately after each cycle the daily catch declined to zero, but then recovered to peaks of 12.8 tsetse day-1 after the 1st cycle and 20.6 tsetse day-1 after the 2nd cycle. Thereafter recovery was less marked, peaking at 6.3 tsetse day-1 after the 3rd cycle, 1.2 tsetse day-1 after the 4th cycle and finally no tsetse was found after the 5th cycle. By contrast, the daily catches from fly-rounds outside the spray block (Figure 4B) increased significantly over the period of spray

1st Apr 1st May 1st Jun 1st Jul 1st Aug 1st Sep

1st Apr 1st May 1st Jun 1st Jul 1st Aug 1st Sep

Date 1st-Apr 1st-May 1st-Jun 1st-Jul 1st-Aug 1st-Sep

Fig. 4. Detransformed mean daily catch of tsetse from fly rounds and traps within (Mombo, Guai and Zimbiri) or outside (Chief's Island, fly rounds only) the area sprayed during the 2001 control operation. Vertical grey bars indicate the timing of the five spray cycles.

operation.

Mean daily catch

Mean daily catch

Source: Kgori et al., (2006).

Mean daily catch

1

0.

0

1

10

100

1

0

1

10

100

0.0.

**C. 2001 - Traps within spray block**

1

0

1

10

100

**A. 2001 - Fly-rounds within spray block**

**B. 2001 - Fly-rounds outside spray block**

Spraying commenced in the south block (8650 km2) on 16 May 2002. Following the start of spraying, the catches declined dramatically (Figure 5). For instance, the mean daily catch from fly-rounds was 101.7 (±0.026, *n*=148) for the period 10 April - 16 May compared to 0.23 (±0.019*, n*=66) for the period 24 May - 3 June, this being the period between the end of 1st spraying cycle and the beginning of the second cycle (Figure 5A) and after the 4th cycle no tsetse were found. The catches from traps showed a similar decline (Figure 5B). During 2006 campaign, the tsetse surveys started on 13th May 2006 (Day 1 = 13 May 2006. Figure 6 shows that the abundance of tsetse was higher than 100 with fluctuations in Kwando-Linyanti prior to spraying and it reduced to zero after 2nd, 3rd, 4th and zero catches after the 5th spraying cycle (Kgori et al., 2006, Kgori et al., 2009, VEEU-TCD/DAHP 1998).

Fig. 5. Detransformed mean daily catch of tsetse from fly rounds and traps within (Bobo Island, Thapaghadi and Chief's Island) the area sprayed during the 2002 control operation. Vertical grey bars indicate the timing of the five spray cycles. Source: Kgori et al., (2006).

Management of Tsetse Fly Using Insecticides in Northern Botswana 459

one year in soil as residue (Maier-Bode, 1968). It is being phased out globally and a global ban on the manufacture and use of endosulfan was initiated under the Stockholm Convention in April 2011. The ban will take effect in mid 2012 and more than 80 countries became part of the ban (http://en.wikipedia.org/wike/endosulfan). Chemical control methods have been used in anti-tsetse campaigns in at least 20 countries in Africa. Sideeffects on non-target organisms have been studied to a greater or lesser extent in only about

Dieldrin - Ground spray (Mist blower)

Endosulfan ULV Fixed-wing Deltamethrin – Aerial spray

Deltamethrin - Aerial spray

Dieldrin – Ground spray Endosulfan and Decamethrin –

Kenya 1970-72 Dieldrin – Fixed wing Allsopp, 1978

DDT – Ground spray Dieldrin –Ground spray Dieldrin – ULV Helicopter Endosulfan – Helicopter

Dieldrin – Ground spray

Uganda 1963-73 Endosulfan-pyrethroids – Helicopter Wilson, 1972 Zambia 1968 Endosulfan – ULV fixed-wing Magadza, 1979 Zimbabwe 1978 Endosulfan – ULV aerial Cockbill. 1979

oresol), Bush clearing

Namibia 2006 Deltamethrin – Aerial spray Bonyongo & Mazvimavi,

Dieldrin/Endosulfan ULV helicopter DDT/Endosulfan – Ground spray Endosulfan – ULV fixed-wing Pyrethroids –Ground and Helicopter

DNOC (2 methyle-4, 6-dinitro-ortho-

Table 1. Anti-tesetse fly campaigns in Africa and impacts studied on the non-target agents.

Helicopter

spray

Cameroon 1979 Dieldrin – Helicopter Muller et al., 1980 Chad 1972-1974 DDT- Ground spray Tibayrence and Gruvel,

Reference

Graham, 1964

2004; 2004a

2007; 2008

1977

1970 Everts, 1979

1970

2007; 2008

Ali, 1978; Fox et al., 1979 Perkins and Ramberg,

Bonyongo & Mazvimavi,

Koeman and Pennings,

Koeman & Pennings,

Koeman et al., 1971 Koeman et al., 1978 Dortland et al., 1977

Dortland et al., 1977 Koeman et al., 1978 Takken et al., 1976 Smies et al., 1980

Sserunjoji & Tjell 1971 Tarimo & Palloti, 1979

half of the 20 countries (Table 1).

1975-78 2001-02

2006

1968-69

1969-70 1974-76 1977

1976 1976 1977-78

1979

Botswana 1964

Ivory Coat 1979

Niger 1968

Nigeria 1975-76

Tanzania 1961-71

Country Year (s) Type of insecticide + method of application

.

## **5. Environmental implications**

#### **5.1 DDT**

'Silent Spring' (Carson, 1962) described the environmental impacts of the indiscriminate spraying of DDT (Dichloro-diphenyl-trichloroethane) in the world and its effects on ecology or human health. It has high potential to bioaccumualte especially in predatory birds and magnify through food; and still highly effective against disease vectors such as malaria parasite (Thomas, 1981). DDT was banned for agricultural use worldwide under the Stockholm Convention but its limited use in disease vector control still continues in some parts of the world to this day and remains controversial (Connel, 1999). Its persistence ranges from 22 days to 30 years depending on the conditions of the ecosystem. It is insoluble in water, less biodegradable and degrades by means of aerobic, anaerobic and photolysis (Thomas, 1981). No specific monitoring of impacts of DDT spraying in Botswana was ever carried out.

#### **5.2 Dieldrin**

Deldrin was used world-wide to control locusts and tropical disease vectors, such as tsetse fly and mosquitoes prior to the 1970s, and has persistence of 5-25 years. It has similar characteristics to DDT (Hunter & Robinson, 1968). Botswana was one of the first countries in Africa to monitor the side-effects of chemical control of tsetse fly. In 1964 riverine forests were sprayed with Dieldrin against *G. morsitans* in an area near Maun in the Okavango Delta, Botswana. The effects of the motorized spraying (mist blower) on non-target organisms were observed for a period of 10 days following spraying (Allsopp, 1978). Among the animals found dead were many birds, mammals, reptiles and fish (Graham, 1964). Casual observations of the aerial spray showed no difference between the wildlife populations between the sprayed and unsprayed areas (Davies & Bowles, 1979).

#### **5.3 Endosulfan**

Endosulfan degrades more rapidly than DDT and dieldrin; 9% degradation in water between 5-25 days, 15-30 days from vegetation surface, but remains more than 100 days to

Day (1 = 13 May 2006) 0 10 20 30 40 50 60 70 80 90 100110120130

'Silent Spring' (Carson, 1962) described the environmental impacts of the indiscriminate spraying of DDT (Dichloro-diphenyl-trichloroethane) in the world and its effects on ecology or human health. It has high potential to bioaccumualte especially in predatory birds and magnify through food; and still highly effective against disease vectors such as malaria parasite (Thomas, 1981). DDT was banned for agricultural use worldwide under the Stockholm Convention but its limited use in disease vector control still continues in some parts of the world to this day and remains controversial (Connel, 1999). Its persistence ranges from 22 days to 30 years depending on the conditions of the ecosystem. It is insoluble in water, less biodegradable and degrades by means of aerobic, anaerobic and photolysis (Thomas, 1981). No specific monitoring of impacts of DDT spraying in Botswana was ever

Deldrin was used world-wide to control locusts and tropical disease vectors, such as tsetse fly and mosquitoes prior to the 1970s, and has persistence of 5-25 years. It has similar characteristics to DDT (Hunter & Robinson, 1968). Botswana was one of the first countries in Africa to monitor the side-effects of chemical control of tsetse fly. In 1964 riverine forests were sprayed with Dieldrin against *G. morsitans* in an area near Maun in the Okavango Delta, Botswana. The effects of the motorized spraying (mist blower) on non-target organisms were observed for a period of 10 days following spraying (Allsopp, 1978). Among the animals found dead were many birds, mammals, reptiles and fish (Graham, 1964). Casual observations of the aerial spray showed no difference between the wildlife

Endosulfan degrades more rapidly than DDT and dieldrin; 9% degradation in water between 5-25 days, 15-30 days from vegetation surface, but remains more than 100 days to

populations between the sprayed and unsprayed areas (Davies & Bowles, 1979).

Fig. 6. Tsetse survey results at Kwando/Linyanti 2006 in five spray cycles.

.

Tsetse/day

**5. Environmental implications** 

**5.1 DDT** 

carried out.

**5.2 Dieldrin** 

**5.3 Endosulfan** 

one year in soil as residue (Maier-Bode, 1968). It is being phased out globally and a global ban on the manufacture and use of endosulfan was initiated under the Stockholm Convention in April 2011. The ban will take effect in mid 2012 and more than 80 countries became part of the ban (http://en.wikipedia.org/wike/endosulfan). Chemical control methods have been used in anti-tsetse campaigns in at least 20 countries in Africa. Sideeffects on non-target organisms have been studied to a greater or lesser extent in only about half of the 20 countries (Table 1).


Table 1. Anti-tesetse fly campaigns in Africa and impacts studied on the non-target agents.

Management of Tsetse Fly Using Insecticides in Northern Botswana 461

relationship was however, not apparent in the study because the very fatty insectivore *Marcusenius macrolepidotus* was never found dead after spraying as they accumulated higher residue concentration of 1.002 µg g-1 wet weight in its viscera lipid. So the bioaccumulated insecticide can do little damage to vital metabolic processes. Indeed, the laboratory experiments with *Hepsetus* suggest that the fatty lean fish is susceptible and succumb most readily to poisoning to endosulfan and thus fish weight is probably the dominant factor determining the survival, i.e. fry and small individuals are at greater risk. Tests with the predators of fish such as crocodile *Crocodilus niloticus*, fish eagle *Heliaeetus vocifer*, Kingfisher *Ceryle rudis* accumulated endosulfan residues, although these never exceeded 0.2 µg g-1 wet weight, except in the visceral fat of the crocodile with 0.783 µg g-1 wet weight suggesting again that fatty organs tended to accumulate the highest residue concentrations

During ultra-low-volume aerial applications of endosulfan at 14g ha-1 for control of tsetse fly in savanna woodland, Zimbabwe, no deleterious effect of the insecticide was demonstrated, other than fish (*Tilapia* spp.) in shallow water (Cockbill, 1979). In general, endosulfan killed small fish first, (*Alestes lateralis)* although almost all species were ultimately affected by the 10 g ha-1 spray. An endosulfan spray done in the Okavango Delta in 1978 showed that the fish mortality extended over a period of 3 days after spraying in four cycles (Table 3). Fish species affected included *Tilapia, Hemichromis, Haplochromis, Pseudocrenilabrus, Serranochromis, Clarias, Barbus, Schilbe and Hepsetus.* Among them, *Tilapia* was the dominant species affected with 65% poisoning followed by *Pseudocrenilabrus* with 13% (Douthwaite et

Cycle 1 Cycle 2 Cycle 3 Cycle 4

12 g ha-1 12 g ha-1 9 g ha-2 6 g ha-2

Table 3. Fish mortality resulting from endosulfan spray in Khwai River in the Okavango Delta. (SE ± = sd/√*n*). Figures in the parantheses indicate minimum and maximum

The general symptoms of endosulfan or other pesticides' poisoning in fish are epithelial lifting (Bucke et al., 1996, Choudary et al., 2003), Hyperplasia (Munshi et al., 1996), muscle hypertrophy and aedema, liver necrosis (Bucher & Hofer, 1993). Samples of liver and brain were tested from *Hepsetus odoe, Tilapia sparrmanii, T. rendalli and Sarotherodon andersoni* throughout the 1978 spraying and during the ensuing 7 months. Whereas liver samples alone were collected from *Clarias ngamensis and C. gariepinus*. In general endosulfan spray induced liver focal (peripheral) necrosis, brain aedema leading to encephalitis, lining in cephalic tissue during the spray periods as well as 15 days after cycle 6 in the species tested. However, the fish showed healing areas of focal necrosis in liver, absence of aedema in brain in the post spray periods after 7 months. Among the species, *Hepsetus odoe* was tolerant to endosulfan showing only slight change in the fatty liver few days after the spray

672.1 ±336.5 (0-2531)

2.0 ±1.6 (0-16)

544.9 ±266.5 (0-2344)

(Douthwaite et al., 1981).

Fish mortality per hectare (*n* = 9 sites)

mortality (Source: Douthwaite et al., 1981).

**Mortality** 

al., 1981).

542.7 ±339.6 (0-3250)

(Douthwaite et al., 1981).

## **Deposition**

The insecticide deposition in the various habitats in the Okavango Delta is presented in Table 2. However, the range and frequency of individual values are insignificant between them (Douthwaite et al., 1981). Endosulfan concentration in different types of aquatic habitats nine hours after spraying at 9.5 g ha-1 varied with depths and the values were insignificant between the various habitats: In marshes ≤ 0.5 m = 0.81 µg l-1; in main river between 1-2 m depth = 1.16 µg l-1 and in open pool at ≤ 0.5 m depth = 1.54 µg l-1 where as persistence of the insecticide in pools of water declined from 1.42 µg l-1 nine hours after spraying and undetectable levels within five days (Douthwaite et al., 1981). Its half life in aquatic environment is between one and five days (Moulton, 1973).


Table 2. Quantities of endosulfan (µg) drift found on aluminum sheets placed in different habitats and analyzed one hour after spraying 9.5 g ha-1 (4 µg per sheet = 1 g ha-1). Source: Douthwaite et al., (1981).

## **5.3.1 Fish**

## **Bioaccumulation**

The major route of uptake in aquatic invertebrates is probably via the digestive system (Roberts, 1975), whereas fish absorb most endosulfan directly from the surrounding water injuring gills, thus reducing the oxygen consumption and disrupting the osmoregulatory function of aquatic organisms (Saravana & Geraldine, 2000). Dortland et al., (1977) have shown that endosulfan applied at 900 g ha-1 for tsetse control near West African Rivers produced residue levels of 1.4 and 37.3 µg g-1 fish muscle and liver respectively. Experimental applications of endosulafan to fish in paddy fields (Moulton, 1973) showed that gouramis, *Tricho gaster pectoralis* could accumulate the pesticide in abdominal organs to concentrations over 1000 times those in surrounding water. It was found that the absorbed endosulfan in fish was rapidly metabolized to the endosulfan sulphate (Ali, 1978).

The edible fish species tested for bioaccumulation during the endosulfan spraying in Botswana include species of *Clarias, Serranochromis, Schilbe, Haplochromis, Tilapia, Marcusenius*. Endosulfan mean residue concentration with respect to percentage lipid 5 days after the spraying was 0.19 µg g-1 wet tissue in muscle, while the maximum found in whole dead small fish was 1.5 µg g-1 wet tissue. These values refer to the combined concentration of alpha+beta + endosulfan sulfate - all three compounds are equally toxic to the fish (Anon, 1973). The USA has set a tolerance limit for endosulfan in meat of 0.2 µg g-1 fresh weight; this therefore means muscle tissue from living fish in the Okavango would be considered safe (Douthwaite et al., 1981).

Fatty species tend to accumulate the most endosulfan (Douthwaite et al., 1981), and one might perhaps expect these groups would show greater mortality in the field. Such relationship was however, not apparent in the study because the very fatty insectivore *Marcusenius macrolepidotus* was never found dead after spraying as they accumulated higher residue concentration of 1.002 µg g-1 wet weight in its viscera lipid. So the bioaccumulated insecticide can do little damage to vital metabolic processes. Indeed, the laboratory experiments with *Hepsetus* suggest that the fatty lean fish is susceptible and succumb most readily to poisoning to endosulfan and thus fish weight is probably the dominant factor determining the survival, i.e. fry and small individuals are at greater risk. Tests with the predators of fish such as crocodile *Crocodilus niloticus*, fish eagle *Heliaeetus vocifer*, Kingfisher *Ceryle rudis* accumulated endosulfan residues, although these never exceeded 0.2 µg g-1 wet weight, except in the visceral fat of the crocodile with 0.783 µg g-1 wet weight suggesting again that fatty organs tended to accumulate the highest residue concentrations (Douthwaite et al., 1981).

## **Mortality**

460 Insecticides – Pest Engineering

The insecticide deposition in the various habitats in the Okavango Delta is presented in Table 2. However, the range and frequency of individual values are insignificant between them (Douthwaite et al., 1981). Endosulfan concentration in different types of aquatic habitats nine hours after spraying at 9.5 g ha-1 varied with depths and the values were insignificant between the various habitats: In marshes ≤ 0.5 m = 0.81 µg l-1; in main river between 1-2 m depth = 1.16 µg l-1 and in open pool at ≤ 0.5 m depth = 1.54 µg l-1 where as persistence of the insecticide in pools of water declined from 1.42 µg l-1 nine hours after spraying and undetectable levels within five days (Douthwaite et al., 1981). Its half life in

> 17.7 (3.6-45.9) 193.8

Table 2. Quantities of endosulfan (µg) drift found on aluminum sheets placed in different habitats and analyzed one hour after spraying 9.5 g ha-1 (4 µg per sheet = 1 g ha-1). Source:

The major route of uptake in aquatic invertebrates is probably via the digestive system (Roberts, 1975), whereas fish absorb most endosulfan directly from the surrounding water injuring gills, thus reducing the oxygen consumption and disrupting the osmoregulatory function of aquatic organisms (Saravana & Geraldine, 2000). Dortland et al., (1977) have shown that endosulfan applied at 900 g ha-1 for tsetse control near West African Rivers produced residue levels of 1.4 and 37.3 µg g-1 fish muscle and liver respectively. Experimental applications of endosulafan to fish in paddy fields (Moulton, 1973) showed that gouramis, *Tricho gaster pectoralis* could accumulate the pesticide in abdominal organs to concentrations over 1000 times those in surrounding water. It was found that the absorbed

The edible fish species tested for bioaccumulation during the endosulfan spraying in Botswana include species of *Clarias, Serranochromis, Schilbe, Haplochromis, Tilapia, Marcusenius*. Endosulfan mean residue concentration with respect to percentage lipid 5 days after the spraying was 0.19 µg g-1 wet tissue in muscle, while the maximum found in whole dead small fish was 1.5 µg g-1 wet tissue. These values refer to the combined concentration of alpha+beta + endosulfan sulfate - all three compounds are equally toxic to the fish (Anon, 1973). The USA has set a tolerance limit for endosulfan in meat of 0.2 µg g-1 fresh weight; this therefore means muscle tissue from living fish in the Okavango would be considered

Fatty species tend to accumulate the most endosulfan (Douthwaite et al., 1981), and one might perhaps expect these groups would show greater mortality in the field. Such

endosulfan in fish was rapidly metabolized to the endosulfan sulphate (Ali, 1978).

swamp Open grassland Grassland under

21.1 (4.5-43.8) 188.3

tree canopy

14.3 (2.4-24.5) 54.2

aquatic environment is between one and five days (Moulton, 1973).

20.7 (1.4-42.9) 116.1

Open water Water in grass

**Deposition** 

Mean (Range) Variance

Douthwaite et al., (1981).

safe (Douthwaite et al., 1981).

**5.3.1 Fish** 

**Bioaccumulation** 

During ultra-low-volume aerial applications of endosulfan at 14g ha-1 for control of tsetse fly in savanna woodland, Zimbabwe, no deleterious effect of the insecticide was demonstrated, other than fish (*Tilapia* spp.) in shallow water (Cockbill, 1979). In general, endosulfan killed small fish first, (*Alestes lateralis)* although almost all species were ultimately affected by the 10 g ha-1 spray. An endosulfan spray done in the Okavango Delta in 1978 showed that the fish mortality extended over a period of 3 days after spraying in four cycles (Table 3). Fish species affected included *Tilapia, Hemichromis, Haplochromis, Pseudocrenilabrus, Serranochromis, Clarias, Barbus, Schilbe and Hepsetus.* Among them, *Tilapia* was the dominant species affected with 65% poisoning followed by *Pseudocrenilabrus* with 13% (Douthwaite et al., 1981).


Table 3. Fish mortality resulting from endosulfan spray in Khwai River in the Okavango Delta. (SE ± = sd/√*n*). Figures in the parantheses indicate minimum and maximum mortality (Source: Douthwaite et al., 1981).

The general symptoms of endosulfan or other pesticides' poisoning in fish are epithelial lifting (Bucke et al., 1996, Choudary et al., 2003), Hyperplasia (Munshi et al., 1996), muscle hypertrophy and aedema, liver necrosis (Bucher & Hofer, 1993). Samples of liver and brain were tested from *Hepsetus odoe, Tilapia sparrmanii, T. rendalli and Sarotherodon andersoni* throughout the 1978 spraying and during the ensuing 7 months. Whereas liver samples alone were collected from *Clarias ngamensis and C. gariepinus*. In general endosulfan spray induced liver focal (peripheral) necrosis, brain aedema leading to encephalitis, lining in cephalic tissue during the spray periods as well as 15 days after cycle 6 in the species tested. However, the fish showed healing areas of focal necrosis in liver, absence of aedema in brain in the post spray periods after 7 months. Among the species, *Hepsetus odoe* was tolerant to endosulfan showing only slight change in the fatty liver few days after the spray (Douthwaite et al., 1981).

Management of Tsetse Fly Using Insecticides in Northern Botswana 463

periods, but no species disappeared from both sprayed and unsprayed transects. A comparison of 39 species was grouped by diet, and by site and method of feeding. To determine the source of the heterogeneity comparisons were made within groups of insectivores, frugivores and granivores and birds with mixed diets. Significant heterogeneity occurred only in the granivores such as *Lamprotornis* starlings that increased in the unsprayed transects but declined in the sprayed transects, and the Grey Hornbill *Tockus nasutus* and Sengegal Coucal *Centropus senegalensis*, which did the opposite. The other insectivore species showed divergent occurrences include Meyer's Parrot *Poicephalus meyeri,* a granivore, Brubru *Nilaus afer* and Red-billed Wood-hoopoe *Phoeniculus purpureus*. These changes in occurrence could, in view of the varied habitats of the species concerned, be as well explained by rainfall, vegetation re-growth between sprayed and unsprayed transects

The major diet for kingfisher (*Ceryle rudis*) is fish ranging from 28-112 mm and 0.2 to19.1 g in weight. The diet consists mostly of *Tilapia, Haplochromis, Barbus and Pseudocrenilabrus*. These species accumulated the endosulfan in liver and brain and the feeding of these fish by kingfisher may have some concentrations of endosulfan. The study showed the total concentration of alpha and beta endosulfan and endosulfan sulfate in the pooled samples from three kingfisher birds as 0.012 µg g-1 wet weight in the liver and 0.205 µg g-1 weight in the brain. These concentrations are no higher than the levels found in fish and agree with

Deltamethrin is a broad spectrum insecticide, relatively stable but less persistent in the environment than the organochlorine pesticides (Grant & Crick, 1987). It has been in wide use in various crops, in gardens, indoors, outdoors for controlling pests such as mites, ants, weevils, beetles, leafhoppers the world over (Tomlin, 2006). Its persistence ranges from 8 to 48 hours (Erstfield, 1999) in water and 5.7 to 209 days (EFASP, 1999) in terrestrial habitats. Reported concentrations in water are rarely greater than 20 *n*g l-1 (Amweg et al., 2006) and were only found to be toxic to honey bees (*Apis* sp.) (Tomlin, 2006). The 2001 deltamethrin spraying over the upper Okavango Delta was done with very little systematic monitoring of the impacts/effects on the non-target organisms (Perkins & Ramberg, 2004). However, the subsequent spraying in 2002 period was done with adequate regular monitoring from the 1st to the 5th cycle. The analysis of data has

i. Sampling was carried out just before each cycle spraying event and in the following day after the spraying. This assumed that significant changes in abundance of individuals in

ii. Analysis of trends in numbers of any taxa is compared from cycle 1 to 5 to show the

In 2002, the spray deposition as determined by rotating slides was between 23 and 867 drops cm-2 and varied considerably within and between sites (Wolski & Huntsman-Mapila, 2002), which could be due to habitat variation, wind, temperature and distance from the flight lines. The wool strands that were exposed to the spraying absorbed the insecticide and revealed that for tsetse fly lethal concentrations were likely to have lasted for four to five days after the spray event (Perkins & Ramberg, 2004). However deltamethrin deposition determined in water, sediments and soil in 2002 and 2006 yielded almost insignificant results except one sediment sample that had low levels of the insecticide (Bonyongo &

as by the effect of spraying.

**5.4 Deltamethrin** 

earlier observations (Maier-Bode, 1968).

been assessed by two methods.

tolerance and susceptibility.

any taxa are likely to be caused by the spraying.

## **5.3.2 Aquatic invertebrates**

Among the Oligochaeta, Chironomidae, Trichoptera and Ephemeroptera groups the numbers of Chironomid larvae reduced significantly (P≥ 0.001), Oligochaets and Ephemeroptera reduced in abundance while Trichoptera showed fluctuations during the 1978 spray. The decrease in Chironomid larvae in the sprayed lagoon was unexpected as the reported LC50 values for the larvae were considerably higher than the residue levels recorded in the study indicating a normal seasonal change. The Ephemeroptera, Oligochaeta and Chironomidae remained almost constant in pre spray, mid spray while in post spray they increased significantly reflecting positive response to the falling water levels rather than related to the endosulfan effects. With the exception of *Hexarthra* sp. the species namely *Filina, Brachionus, Keratella* and *Polyarthra* perecentage representation was considerably lower in the lagoons following the spraying season (Douthwaite et al., 1981).

## **5.3.3 Terrestrial invertebrates**

The abundance of terrestrial invertebrates in floodplains, grassland and in dry land under tree canopies, *Colophospermamum mopane* canopies in the sprayed and unsprayed areas was determined in the Okavango Delta. The major groups in the studies included Chironomidae, Cicadellidae, Diptera, Hymenoptera, Orthoptera, Araneae, Formicidae, Coleoptera. Only in the case of adult *Chironomids* was there a large reduction in numbers in the sprayed site. However their abundance was almost identical in the post spray periods with the 'control sample' attributing doubts over the spray effects. Three of the major groups namely Formicidae, Tenebrionidae and Diptera showed reduction in activity and the most affected genus was the *Pheidole* in Formicidae following the spray. There was a significant decrease in numbers (P≥ 0.05) of spiders in the sprayed site which correspond to the significant increase (P≥ 0.001) in the sprayed site in the 3rd cycle (Douthwaite et al., 1981).

### **5.3.4 Flying insects**

The behaviour of nocturnal insects during the spraying in the Okavango Delta was caught in the water trays in the grassland along the margins of the permanent swamps before and after each spray application in both sprayed and unsprayed areas. In the first spray cycle a sharp peak in mortality was observed in the adult *Chironomid* insects trapped in the sprayed site. However similar results were not obtained during subsequent spray cycles. This could be due to the emergence of adults from the larvae as the adults have short life-span. Among other groups, only Culicidae, some *Nematocera* and *Diptera spp.* were abundant in water traps and fluctuated in numbers from night to night in both sprayed and control sites, thus showing that there was no consistent evidence for either decreases or increases following each spray cycle (Douthwaite et al., 1981).

## **5.3.5 Birds**

Endosulfan is neither toxic nor cumulative in birds and therefore unlikely to be lethal to them. However, there could be indirect effects on the insectivorous and piscivorous birds through disruption of their food supplies. The occurrence of birds in *Acacia* woodland was monitored before and after spraying in 1978 using transects. Besides similarities in the species in four transects, each transect lost and gained some species between sampling periods, but no species disappeared from both sprayed and unsprayed transects. A comparison of 39 species was grouped by diet, and by site and method of feeding. To determine the source of the heterogeneity comparisons were made within groups of insectivores, frugivores and granivores and birds with mixed diets. Significant heterogeneity occurred only in the granivores such as *Lamprotornis* starlings that increased in the unsprayed transects but declined in the sprayed transects, and the Grey Hornbill *Tockus nasutus* and Sengegal Coucal *Centropus senegalensis*, which did the opposite. The other insectivore species showed divergent occurrences include Meyer's Parrot *Poicephalus meyeri,* a granivore, Brubru *Nilaus afer* and Red-billed Wood-hoopoe *Phoeniculus purpureus*. These changes in occurrence could, in view of the varied habitats of the species concerned, be as well explained by rainfall, vegetation re-growth between sprayed and unsprayed transects as by the effect of spraying.

The major diet for kingfisher (*Ceryle rudis*) is fish ranging from 28-112 mm and 0.2 to19.1 g in weight. The diet consists mostly of *Tilapia, Haplochromis, Barbus and Pseudocrenilabrus*. These species accumulated the endosulfan in liver and brain and the feeding of these fish by kingfisher may have some concentrations of endosulfan. The study showed the total concentration of alpha and beta endosulfan and endosulfan sulfate in the pooled samples from three kingfisher birds as 0.012 µg g-1 wet weight in the liver and 0.205 µg g-1 weight in the brain. These concentrations are no higher than the levels found in fish and agree with earlier observations (Maier-Bode, 1968).

#### **5.4 Deltamethrin**

462 Insecticides – Pest Engineering

Among the Oligochaeta, Chironomidae, Trichoptera and Ephemeroptera groups the numbers of Chironomid larvae reduced significantly (P≥ 0.001), Oligochaets and Ephemeroptera reduced in abundance while Trichoptera showed fluctuations during the 1978 spray. The decrease in Chironomid larvae in the sprayed lagoon was unexpected as the reported LC50 values for the larvae were considerably higher than the residue levels recorded in the study indicating a normal seasonal change. The Ephemeroptera, Oligochaeta and Chironomidae remained almost constant in pre spray, mid spray while in post spray they increased significantly reflecting positive response to the falling water levels rather than related to the endosulfan effects. With the exception of *Hexarthra* sp. the species namely *Filina, Brachionus, Keratella* and *Polyarthra* perecentage representation was considerably lower in the lagoons following the spraying season (Douthwaite et al.,

The abundance of terrestrial invertebrates in floodplains, grassland and in dry land under tree canopies, *Colophospermamum mopane* canopies in the sprayed and unsprayed areas was determined in the Okavango Delta. The major groups in the studies included Chironomidae, Cicadellidae, Diptera, Hymenoptera, Orthoptera, Araneae, Formicidae, Coleoptera. Only in the case of adult *Chironomids* was there a large reduction in numbers in the sprayed site. However their abundance was almost identical in the post spray periods with the 'control sample' attributing doubts over the spray effects. Three of the major groups namely Formicidae, Tenebrionidae and Diptera showed reduction in activity and the most affected genus was the *Pheidole* in Formicidae following the spray. There was a significant decrease in numbers (P≥ 0.05) of spiders in the sprayed site which correspond to the significant increase (P≥ 0.001) in the sprayed site in the 3rd cycle (Douthwaite et al., 1981).

The behaviour of nocturnal insects during the spraying in the Okavango Delta was caught in the water trays in the grassland along the margins of the permanent swamps before and after each spray application in both sprayed and unsprayed areas. In the first spray cycle a sharp peak in mortality was observed in the adult *Chironomid* insects trapped in the sprayed site. However similar results were not obtained during subsequent spray cycles. This could be due to the emergence of adults from the larvae as the adults have short life-span. Among other groups, only Culicidae, some *Nematocera* and *Diptera spp.* were abundant in water traps and fluctuated in numbers from night to night in both sprayed and control sites, thus showing that there was no consistent evidence for either decreases or increases following

Endosulfan is neither toxic nor cumulative in birds and therefore unlikely to be lethal to them. However, there could be indirect effects on the insectivorous and piscivorous birds through disruption of their food supplies. The occurrence of birds in *Acacia* woodland was monitored before and after spraying in 1978 using transects. Besides similarities in the species in four transects, each transect lost and gained some species between sampling

**5.3.2 Aquatic invertebrates** 

**5.3.3 Terrestrial invertebrates** 

**5.3.4 Flying insects** 

**5.3.5 Birds** 

each spray cycle (Douthwaite et al., 1981).

1981).

Deltamethrin is a broad spectrum insecticide, relatively stable but less persistent in the environment than the organochlorine pesticides (Grant & Crick, 1987). It has been in wide use in various crops, in gardens, indoors, outdoors for controlling pests such as mites, ants, weevils, beetles, leafhoppers the world over (Tomlin, 2006). Its persistence ranges from 8 to 48 hours (Erstfield, 1999) in water and 5.7 to 209 days (EFASP, 1999) in terrestrial habitats. Reported concentrations in water are rarely greater than 20 *n*g l-1 (Amweg et al., 2006) and were only found to be toxic to honey bees (*Apis* sp.) (Tomlin, 2006). The 2001 deltamethrin spraying over the upper Okavango Delta was done with very little systematic monitoring of the impacts/effects on the non-target organisms (Perkins & Ramberg, 2004). However, the subsequent spraying in 2002 period was done with adequate regular monitoring from the 1st to the 5th cycle. The analysis of data has been assessed by two methods.


In 2002, the spray deposition as determined by rotating slides was between 23 and 867 drops cm-2 and varied considerably within and between sites (Wolski & Huntsman-Mapila, 2002), which could be due to habitat variation, wind, temperature and distance from the flight lines. The wool strands that were exposed to the spraying absorbed the insecticide and revealed that for tsetse fly lethal concentrations were likely to have lasted for four to five days after the spray event (Perkins & Ramberg, 2004). However deltamethrin deposition determined in water, sediments and soil in 2002 and 2006 yielded almost insignificant results except one sediment sample that had low levels of the insecticide (Bonyongo &

Management of Tsetse Fly Using Insecticides in Northern Botswana 465

60% in lagoons. The spraying caused significant reductions in abundance of many sensitive taxa often specific for respective habitats like lagoons and channels. What remained in both habitats was a common group of resistant species. The distinct feature is that the reappearance of some of the sensitive taxa that was affected in 2002 spraying was found again during the 2003 recovery studies. At the family level, the most negatively affected families were Atyidae (shrimps) which are characteristic of areas of permanent flow, and Pleidae (pygmy backswimmers), that are found in more seasonal areas. The abundance of Naucoridae (creeping water bugs) was significantly lower than the benchmark as well. Out of 39 identified morphospecies four (10%) were classified as sensitive as they did not reappear during the 2003 recovery study. Three species belonged to the family Notonectidae (Backswimmers) and one to Naucoridae (creeping water bugs), which might reflect a loss from the system. It is however difficult to assign this to persistent post-spray impacts of deltamethrin as the natural variation and abundance in aquatic invertebrates from year to

Samples were collected at 21 sites in the main Kwando- Linyanti Rivers, Zibadianja and Dumatau lagoons, floodplains and on the vegetation of *Phragmites*, papyrus (*Cyperus papyrus*), hippo grass (*Vossia cuspidata*) and water lilies, (*Nymphaea* spp.) using kick nets (Picker et al 2002). The abundance of macro-invertebrates reduced between 10 and 90% in all the sample sites and affected marginally in the rivers, lagoons and the floodplains. About 70% macro-invertebrates were reduced in numbers from the vegetation types. Abundance of the invertebrate taxa reduced between 40 and 90% except *Chironomids* which appeared to increase during the spraying period. Twenty-four families of macro invertebrates were knocked down almost completely when compared between pre-spray and at the end of spray. About 11 of the total families survived the five spraying cycles (Masundire &

An average of about 25% of the macroinvertebrates in 24 taxa (Table 4) that were present before spraying did not reappear one year after spraying. Perkins & Ramberg (2004a) reported 26% loss in taxa following spraying in the Okavango Delta in 2002. The mean abundance of all macro-invertebrates was only 11% in Kwando River while more than 100% in Linyanti River, Floodplains, Zibadianja and Dumatau lagoon areas. The *Phragmites* – and hippo grass dominated habitats had still below 50% of pre-spray levels of abundance and in other sites the invertebrates recovered well. Families such as Atydae and Dytiscidae were still well below pre-spray levels while Corixidae, Hirudinea and Chironomidae exceeded their pre spray levels of abundance. Planorbinae appear to have been little affected by the

In 2002, sampling was done by collecting the terrestrial invertebrates that had been knocked down by the aerial spray, under tree crowns of *Kigelia Africana, Lonchocarpus capassa* and *Combretum imberbe* on plastic sheets with an area of 3 m2 in the riparian zone and under *Colophospermum mopane* in the drier land (Dangerfield, 2002). In 2006, similar studies were

year in the Delta is unknown (Palmer & Davies-Coleman, 2003)

**Impact – 2006** 

Mosepele, 2006). **Recovery – 2007** 

spraying (Masundire, 2007).

**Impact – 2002 and 2006** 

**5.4.2 Terrestrial invertebrates** 

Mazvimavi, 2007). Nevertheless, the insecticide deposition as captured by spreading aluminium foil sheets during the monitoring of salvinia weevils gave between 0.2 µg m-2 and 6.9 µg m-2 in 2002 and 2006 spraying occasions (Kurugundla & Serumola. 2007; Kurugundla et al., 2010).

## **5.4.1 Aquatic Invertebrates**

## **Impacts - 2002**

It is well known that flowing and still water usually differ in biotype composition and hence aquatic macro invertebrate studies were undertaken in still water of pools and flowing channels in Xakanaxa. The results were compared with those of the control site of Khwai River at North Gate. A total of 695 macro invertebrate samples and 200 zooplankton samples were collected, and 64 macro invertebrate families were identified. In channels, abundance declined by 46% after five spray cycles (927 individuals to 520) and in lagoons it declined by 25% (1230 individuals to 917). However, in the reference control site in Khwai River at North Gate, the abundance increased slightly over five spray cycles (789 individuals to 839) (Palmer, 2002).

Samples fixed in 75% ethanol were identified to family in the field using Gerber & Gabrial, (2002) and Davies & Day, (1998). A total of 47 taxa were recorded in channels and 49 taxa in lagoons and in total 65 taxa were identified, of which 23 taxa commonly occurred consistently in samples before and after the spraying. Out of these common taxa 6 showed distinct rapid declines after the first spray cycle and had disappeared completely after the fifth cycle. This corresponds to a loss of 26% of common taxa and was likely caused by the deltamethrin spray deposition. Whereas at North Gate on the Khwai River control site, the number of taxa increased from 29 to 30 after five spray cycles.

It has been shown that the mortality differs between the channels and lagoons and also between the taxonomic groups. Among 64 taxa, more than 24 have been found to be susceptible while few taxa (11) were found to be resistant to the insecticide spraying. Mollusks are probably more physiologically resistant to spraying than insects for obvious shell protection. The other survivor insect families include Chironomidae (non-biting midges), Ceratopogonidae (biting midges), Libellulidae (hairy dragon flies) and Caenidae (crawling mayflies). All these entire insect families live in the sediment, which may function as a protection against the insecticide spray, which is less bioavailable as the pyrethroids could be partitioned and adsorbed to various organic sediments (Muir et al. 1985). On the other hand, the elimination rate recorded for the Hemiptera (water bugs) Carduliidae (dragonflies), for most Ephemeroptera (mayflies) families and some other families could be understood by their active behavior in free water surfaces, sediment and vegetation surfaces. In particular the air breathing behaviour exhibited by most Hemiptera and Coleoptera (beetles) force them to come into contact with deltamethrin and oil-based carrier, paraffin that accumulate on water surface as a thin film (Perkins & Ramberg, 2004; Bonyongo & Mazvimavi, 2007).

#### **Recovery - 2003**

The recovery monitoring was measured in 2003 at the same periods and similar sites to those used in 2002 in order to assess recovery at community, family and morphospecies levels. The total abundance after a year was still significantly lower: 39% in channels and 60% in lagoons. The spraying caused significant reductions in abundance of many sensitive taxa often specific for respective habitats like lagoons and channels. What remained in both habitats was a common group of resistant species. The distinct feature is that the reappearance of some of the sensitive taxa that was affected in 2002 spraying was found again during the 2003 recovery studies. At the family level, the most negatively affected families were Atyidae (shrimps) which are characteristic of areas of permanent flow, and Pleidae (pygmy backswimmers), that are found in more seasonal areas. The abundance of Naucoridae (creeping water bugs) was significantly lower than the benchmark as well. Out of 39 identified morphospecies four (10%) were classified as sensitive as they did not reappear during the 2003 recovery study. Three species belonged to the family Notonectidae (Backswimmers) and one to Naucoridae (creeping water bugs), which might reflect a loss from the system. It is however difficult to assign this to persistent post-spray impacts of deltamethrin as the natural variation and abundance in aquatic invertebrates from year to year in the Delta is unknown (Palmer & Davies-Coleman, 2003)

## **Impact – 2006**

464 Insecticides – Pest Engineering

Mazvimavi, 2007). Nevertheless, the insecticide deposition as captured by spreading aluminium foil sheets during the monitoring of salvinia weevils gave between 0.2 µg m-2 and 6.9 µg m-2 in 2002 and 2006 spraying occasions (Kurugundla & Serumola. 2007;

It is well known that flowing and still water usually differ in biotype composition and hence aquatic macro invertebrate studies were undertaken in still water of pools and flowing channels in Xakanaxa. The results were compared with those of the control site of Khwai River at North Gate. A total of 695 macro invertebrate samples and 200 zooplankton samples were collected, and 64 macro invertebrate families were identified. In channels, abundance declined by 46% after five spray cycles (927 individuals to 520) and in lagoons it declined by 25% (1230 individuals to 917). However, in the reference control site in Khwai River at North Gate, the abundance increased slightly over five spray cycles (789 individuals

Samples fixed in 75% ethanol were identified to family in the field using Gerber & Gabrial, (2002) and Davies & Day, (1998). A total of 47 taxa were recorded in channels and 49 taxa in lagoons and in total 65 taxa were identified, of which 23 taxa commonly occurred consistently in samples before and after the spraying. Out of these common taxa 6 showed distinct rapid declines after the first spray cycle and had disappeared completely after the fifth cycle. This corresponds to a loss of 26% of common taxa and was likely caused by the deltamethrin spray deposition. Whereas at North Gate on the Khwai River control site, the

It has been shown that the mortality differs between the channels and lagoons and also between the taxonomic groups. Among 64 taxa, more than 24 have been found to be susceptible while few taxa (11) were found to be resistant to the insecticide spraying. Mollusks are probably more physiologically resistant to spraying than insects for obvious shell protection. The other survivor insect families include Chironomidae (non-biting midges), Ceratopogonidae (biting midges), Libellulidae (hairy dragon flies) and Caenidae (crawling mayflies). All these entire insect families live in the sediment, which may function as a protection against the insecticide spray, which is less bioavailable as the pyrethroids could be partitioned and adsorbed to various organic sediments (Muir et al. 1985). On the other hand, the elimination rate recorded for the Hemiptera (water bugs) Carduliidae (dragonflies), for most Ephemeroptera (mayflies) families and some other families could be understood by their active behavior in free water surfaces, sediment and vegetation surfaces. In particular the air breathing behaviour exhibited by most Hemiptera and Coleoptera (beetles) force them to come into contact with deltamethrin and oil-based carrier, paraffin that accumulate on water surface as a thin film (Perkins & Ramberg, 2004;

The recovery monitoring was measured in 2003 at the same periods and similar sites to those used in 2002 in order to assess recovery at community, family and morphospecies levels. The total abundance after a year was still significantly lower: 39% in channels and

number of taxa increased from 29 to 30 after five spray cycles.

Kurugundla et al., 2010).

to 839) (Palmer, 2002).

Bonyongo & Mazvimavi, 2007).

**Recovery - 2003** 

**Impacts - 2002** 

**5.4.1 Aquatic Invertebrates** 

Samples were collected at 21 sites in the main Kwando- Linyanti Rivers, Zibadianja and Dumatau lagoons, floodplains and on the vegetation of *Phragmites*, papyrus (*Cyperus papyrus*), hippo grass (*Vossia cuspidata*) and water lilies, (*Nymphaea* spp.) using kick nets (Picker et al 2002). The abundance of macro-invertebrates reduced between 10 and 90% in all the sample sites and affected marginally in the rivers, lagoons and the floodplains. About 70% macro-invertebrates were reduced in numbers from the vegetation types. Abundance of the invertebrate taxa reduced between 40 and 90% except *Chironomids* which appeared to increase during the spraying period. Twenty-four families of macro invertebrates were knocked down almost completely when compared between pre-spray and at the end of spray. About 11 of the total families survived the five spraying cycles (Masundire & Mosepele, 2006).

## **Recovery – 2007**

An average of about 25% of the macroinvertebrates in 24 taxa (Table 4) that were present before spraying did not reappear one year after spraying. Perkins & Ramberg (2004a) reported 26% loss in taxa following spraying in the Okavango Delta in 2002. The mean abundance of all macro-invertebrates was only 11% in Kwando River while more than 100% in Linyanti River, Floodplains, Zibadianja and Dumatau lagoon areas. The *Phragmites* – and hippo grass dominated habitats had still below 50% of pre-spray levels of abundance and in other sites the invertebrates recovered well. Families such as Atydae and Dytiscidae were still well below pre-spray levels while Corixidae, Hirudinea and Chironomidae exceeded their pre spray levels of abundance. Planorbinae appear to have been little affected by the spraying (Masundire, 2007).

## **5.4.2 Terrestrial invertebrates**

## **Impact – 2002 and 2006**

In 2002, sampling was done by collecting the terrestrial invertebrates that had been knocked down by the aerial spray, under tree crowns of *Kigelia Africana, Lonchocarpus capassa* and *Combretum imberbe* on plastic sheets with an area of 3 m2 in the riparian zone and under *Colophospermum mopane* in the drier land (Dangerfield, 2002). In 2006, similar studies were

Management of Tsetse Fly Using Insecticides in Northern Botswana 467

was no change in fly or ant abundance between 2002 and 2003 while Hemeptera abundance was significantly greater in 2003 than before the spray events. Fifty-seven morphospecies were found for the first time in 2003 indicating that the species that were lost from 2002 had started to recover in 2003. This shows that there is great variation between years in the composition of invertebrates (Dangerfield & McCulloch, 2003). There was sharp increase in the flies and mosquitoes in Kwando/Linyanti areas in the recovery periods of 2007. Beetle abundance was highest at above 60% and wasps and ants increased more than 100% a year after the spraying. However crickets, *Gryllus bimaculatus* were not detected in 2007 where as Grasshoppers *Lithdiopsis carinatus, Truxaloides sp., Ailopus thalassinus* and *Eucoptacara exguae* that had disappeared from the riparian grasslands started to recover in 2007 (Chikwenhere,

About 7,500 individuals, mostly flies, were sampled in 2002. Within each cycle catches were lower in the days after the spray event but increased between the spray cycles and much greater in the subsequent pre-cycle catches. Increase in the abundance of flies over the cycles was most likely due to increasing temperatures and the arrival of the annual flood in the

Using gill nets, the abundance of fish in the Kwando and Linyanti Rivers were studied. In general, a comparison of relative abundance before and after the five spray cycles remained stable. Relative abundance in fish in a given water body reflects the seasonal events, water levels, feeding and breeding behaviour (Mosepele, 2006). However there was decrease in species diversity from the pre spray period until cycle 2 and diversity remained stable after cycle 5. Being an opportunistic predator (Teferra et al., 2003, Mosepele et al., 2005) slight decrease in *Schilbe intermedius* is caused by feeding of knocked down terrestrial and aquatic invertebrates. Increased competition for food between the species does occur. *Hepsetus odoe* and *S. intermedius* were pscivorous predators (Merron & Bruton, 1991) and deltamethrin would have increased inter-specific competition for food between these two species as the feeding rate of *S. intermedius* progressively decreased from the pre spray periods until cycle 5. *Brycinus lateralis* and *B. poechii* feed on small aquatic invertebrates and therefore it was possible that their abundance decreased in the river systems due to decreased food supply caused by deltamethrin spray. The lack of significant change in the feeding behavior of *Marcusenius macrolepidotus* during the spraying suggests that its dominant prey item

Using circular point counts for monitoring forest birds, transects for *Acacia* thorn-veld species, and boat surveys for water dependent species, sampling was carried out on three occasions in the Okavango Delta. In summary, there were some local changes in bird populations during the spraying, but these changes could not be attributed to effects of deltamethrin (Pendleton, 2002; Perkins & Ramberg, 2004). The spraying of deltamethrin over the Kwando-Linyanti areas in 2006 did not have discernible negative effects on any of the bird species monitored (Slaty Egret, Arnot's Chat, raptors and

(Chironomids) were not significantly affected by deltamethrin.

2007)

**5.4.3 Flying insects** 

sampled areas.

**5.4.4 Fish** 

**5.4.5 Birds** 

conducted in Kwando during the spraying in the woodland dominated by *Croton megalobotrys, Colophospermum mopane, Combretum imberbe, Lonchocarpus nelsii* and in open vegetation dominated by *Pechuel-loeshea leubnitziae* and *Cynodon dactylon* (riparian grass land).The results were compared to the control sites in unsprayed Khwai River at North Gate. Percent declines in terrestrial invertebrate abundance in combined woodland types in 2002 (Dangerfield, 2002) and 2006 were 64% and 63% respectively. All other common taxa showed significant declines as well with the exception of Flies (Diptera Families, Chironomidae and Ceratopogonidae) that have their larval stages in water and therefore produced mass swarming during spring and early summer.

During 2006, 21 taxa groups belonging to 14 families were identified to species level compared to 31 families of flies identified to morphospecies during 2002 spraying. There was marked decrease (50%) in arthropod species richness in the spray block among the woodlands as compared to pre-spray and after spray cycle 5. Significant difference in the composition of families of flies was recorded between the 2002 and 2006 monitoring. The most common families of flies during the 2002 and 2006 monitoring were Tabanidae, Tipulidae, Muscidae, Calliphoridae, Anthomyiidae, Syrphidae, Tephritidae. Spider families sampled in 2002 and 2006 were the same with the exception of family Theraphosidae which was first recorded during 2006. Family Heteropodidae was recorded in 2006 but not in 2002. Family Oxyopidae disappeared after 5th cycle of spraying in both 2002 and 2006. The crickets, particularly *Gryllus bimaculatus* was lost after the 1st cycle and was not captured throughout the remaining spraying cycles. Grasshoppers, *Lithdiopsis carinatus, Truxaloides* sp., *Ailopus thalassinus* and *Eucoptacara exguae* disappeared from the riparian grasslands (Chikwenhere & Shereni, 2006)


Table 4. Effects of deltamethrin on terrestrial arthropods dwelling in woodland types after spray cycle 2 in 2002 and 2006 (Source: Bonyongo and Mazvimavi, 2007).

In 2006, mosquitoes suffered the highest reduction of 80% followed by ants with 74%, the wasps had 65% reduction, while flies reduced by 60%. The rest of the groups listed declined below 50% and the least reduced was spiders at 24%. Conversely, ants were the least abundant while maximum reductions were found in beetles at 84% followed by mosquitoes with 71% reduction in 2002 (Table 4).

#### **Recovery - 2003 and 2007**

In May and August 2003 and 2007 sampling of terrestrial invertebrates in the selected woodland tree types yielded interesting results in the Okavango and Kwando respectively. In the key insect groups, the spider abundance recovered in all cases to pre-spray levels; beetle abundance had not recovered on *K. africana*, but recovered on other tree species; there was no change in fly or ant abundance between 2002 and 2003 while Hemeptera abundance was significantly greater in 2003 than before the spray events. Fifty-seven morphospecies were found for the first time in 2003 indicating that the species that were lost from 2002 had started to recover in 2003. This shows that there is great variation between years in the composition of invertebrates (Dangerfield & McCulloch, 2003). There was sharp increase in the flies and mosquitoes in Kwando/Linyanti areas in the recovery periods of 2007. Beetle abundance was highest at above 60% and wasps and ants increased more than 100% a year after the spraying. However crickets, *Gryllus bimaculatus* were not detected in 2007 where as Grasshoppers *Lithdiopsis carinatus, Truxaloides sp., Ailopus thalassinus* and *Eucoptacara exguae* that had disappeared from the riparian grasslands started to recover in 2007 (Chikwenhere, 2007)

## **5.4.3 Flying insects**

About 7,500 individuals, mostly flies, were sampled in 2002. Within each cycle catches were lower in the days after the spray event but increased between the spray cycles and much greater in the subsequent pre-cycle catches. Increase in the abundance of flies over the cycles was most likely due to increasing temperatures and the arrival of the annual flood in the sampled areas.

## **5.4.4 Fish**

466 Insecticides – Pest Engineering

conducted in Kwando during the spraying in the woodland dominated by *Croton megalobotrys, Colophospermum mopane, Combretum imberbe, Lonchocarpus nelsii* and in open vegetation dominated by *Pechuel-loeshea leubnitziae* and *Cynodon dactylon* (riparian grass land).The results were compared to the control sites in unsprayed Khwai River at North Gate. Percent declines in terrestrial invertebrate abundance in combined woodland types in 2002 (Dangerfield, 2002) and 2006 were 64% and 63% respectively. All other common taxa showed significant declines as well with the exception of Flies (Diptera Families, Chironomidae and Ceratopogonidae) that have their larval stages in water and therefore

During 2006, 21 taxa groups belonging to 14 families were identified to species level compared to 31 families of flies identified to morphospecies during 2002 spraying. There was marked decrease (50%) in arthropod species richness in the spray block among the woodlands as compared to pre-spray and after spray cycle 5. Significant difference in the composition of families of flies was recorded between the 2002 and 2006 monitoring. The most common families of flies during the 2002 and 2006 monitoring were Tabanidae, Tipulidae, Muscidae, Calliphoridae, Anthomyiidae, Syrphidae, Tephritidae. Spider families sampled in 2002 and 2006 were the same with the exception of family Theraphosidae which was first recorded during 2006. Family Heteropodidae was recorded in 2006 but not in 2002. Family Oxyopidae disappeared after 5th cycle of spraying in both 2002 and 2006. The crickets, particularly *Gryllus bimaculatus* was lost after the 1st cycle and was not captured throughout the remaining spraying cycles. Grasshoppers, *Lithdiopsis carinatus, Truxaloides* sp., *Ailopus thalassinus* and *Eucoptacara exguae* disappeared from the riparian grasslands

Common name Taxa order % Reduction – 2002 % Reduction – 2006

Table 4. Effects of deltamethrin on terrestrial arthropods dwelling in woodland types after

In 2006, mosquitoes suffered the highest reduction of 80% followed by ants with 74%, the wasps had 65% reduction, while flies reduced by 60%. The rest of the groups listed declined below 50% and the least reduced was spiders at 24%. Conversely, ants were the least abundant while maximum reductions were found in beetles at 84% followed by mosquitoes

In May and August 2003 and 2007 sampling of terrestrial invertebrates in the selected woodland tree types yielded interesting results in the Okavango and Kwando respectively. In the key insect groups, the spider abundance recovered in all cases to pre-spray levels; beetle abundance had not recovered on *K. africana*, but recovered on other tree species; there

Ants Hymenoptera 11 74 Flies Diptera 41 60 Mosquitoes Diptera 71 80 Beetles Coleoptera 84 43 Wasps Hymenoptera 23 65 Leaf hoppers & bugs Hemiptera 46 45 Spiders Arachnida 30 24

spray cycle 2 in 2002 and 2006 (Source: Bonyongo and Mazvimavi, 2007).

produced mass swarming during spring and early summer.

(Chikwenhere & Shereni, 2006)

with 71% reduction in 2002 (Table 4).

**Recovery - 2003 and 2007** 

Using gill nets, the abundance of fish in the Kwando and Linyanti Rivers were studied. In general, a comparison of relative abundance before and after the five spray cycles remained stable. Relative abundance in fish in a given water body reflects the seasonal events, water levels, feeding and breeding behaviour (Mosepele, 2006). However there was decrease in species diversity from the pre spray period until cycle 2 and diversity remained stable after cycle 5. Being an opportunistic predator (Teferra et al., 2003, Mosepele et al., 2005) slight decrease in *Schilbe intermedius* is caused by feeding of knocked down terrestrial and aquatic invertebrates. Increased competition for food between the species does occur. *Hepsetus odoe* and *S. intermedius* were pscivorous predators (Merron & Bruton, 1991) and deltamethrin would have increased inter-specific competition for food between these two species as the feeding rate of *S. intermedius* progressively decreased from the pre spray periods until cycle 5. *Brycinus lateralis* and *B. poechii* feed on small aquatic invertebrates and therefore it was possible that their abundance decreased in the river systems due to decreased food supply caused by deltamethrin spray. The lack of significant change in the feeding behavior of *Marcusenius macrolepidotus* during the spraying suggests that its dominant prey item (Chironomids) were not significantly affected by deltamethrin.

#### **5.4.5 Birds**

Using circular point counts for monitoring forest birds, transects for *Acacia* thorn-veld species, and boat surveys for water dependent species, sampling was carried out on three occasions in the Okavango Delta. In summary, there were some local changes in bird populations during the spraying, but these changes could not be attributed to effects of deltamethrin (Pendleton, 2002; Perkins & Ramberg, 2004). The spraying of deltamethrin over the Kwando-Linyanti areas in 2006 did not have discernible negative effects on any of the bird species monitored (Slaty Egret, Arnot's Chat, raptors and

Management of Tsetse Fly Using Insecticides in Northern Botswana 469

the weevils normally hide in buds, roots and beneath the leaves. They deposit their eggs in buds and underneath leaves, and emerging larvae normally feed inside the rhizome (Forno et al 1983). Therefore adults and larvae would often be protected from contact with the

Okavango Delta - Basins – 2002 Kwando-Linyanti - Cages- 2006

\*25.0 ±1.0 (47.0%

38.8 ±1.3 (19.9%)

31.0 ±1.4 (32.6%

37.0 ±1.4 (21.3%

\*32.2 ±1.5 (29.1%)

Weevil mortality (%) Deltamethrin

Before After Before After Before After Before After Before After Cy cle 1 Cycle 2 Cycle 3 Cy cle 4 Cy cle 5

Fig. 7. Number of weevils in 20 plants by standard plant method before and after the spray

Average weevil mortalities are typically 26.5% in 2002 and 29.7% in 2006 at 2.7% and 4.1% deltamethrin respectively (Kurugundla& Serumola 2007; Kurugundla et al., 2010). It was also observed that Paradise pool and Lebala pool were completely covered with the salvinia two months after the end of spraying, yet the weevils controlled the infestation after 7 to 8 months. Aerial spraying of deltamethrin for controlling tsetse fly in any given area is not a continuous process and it is applied only in winter, when the breeding rate of the weevils

Shummamorei Hamokata Lebala Selinda

Okavango Delta- mean of 5 cycles Kwando-Linyanti - Mean of 5 cycles

Average 2.7 ±0.5 26.5 ±1.0 4.1 ±0.8 29.7 ±5.1 Table 5. Mean survival of 50 weevils (SE = sd√n) in the Okavango Delta and Kwando-Linyanti in response to deltamethrin spray deposition (% m-2). Figures in parentheses indicate corrected percent mortality with respect to controls in each cycle. \* Probability ≤0.05

Control 12 hours 36 hours 60 hours Control 12 hours 36 hours 60 hours

47.0 ±1.6 (06.0%)

46.5 ±0.6 (07.0%)

45.5 ±0.6 (09.0%)

46.3 ±0.9 (07.1%)

46.3 ±1.3 (07.4%)

deposition (% m-2)

39.3 ±2.4 (16.4%)

39.1 ±2.5 (16.0%)

37.7 ±3.3 (17.2%)

\*35.9 ±3.1 (22.5%)

\*36.1 ±1.7 (22.0%)

37.4 ±4.5 (20.4)

\*35.2 ±3.3 (24.3%)

\*34.4 1.4 (24.6%)

\*33.7 ±1.7 (27.2%)

36.7 ±1.8 (20.7%)

Weevil mortality (%)

35.7 ±1.8 (24.0%)

\*35.8 ±2.0 (23.0%)

\*35.0 ±1.9 (23.1%)

\*22.0 ±1.8 (52.5%)

\*34.3 ±3.1 (26.0%)

insecticide (Schlettwein & Giliomee, 1990).

\*32.0 ±1.3 (32.3%)

31.7 ±0.9 (34.5%)

42.3 ±0.6 (08.1%)

37.0 ±1.1 (21.3%)

\*30.3 ±0.7 (33.3%)

\*30.5 ±1.4 (35.4%)

38.2 ±1.0 (21.1%)

33.5 ±0.9 (27.2%)

44.7 ±1.3 (04.9%)

\*30.5 ±0.9 (32.8%)

Cycle

1 47.2 ±1.2 (5.6%)

2 48.4 ±0.5

3 46.0 ±0.6

4 47.0 ±0.6

5 45.4 ±0.5

(03.2%)

(08.0%)

(06.0%)

(09.2%)

with reference to controls.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

in five cycles.

Weevil No./20 Std. plants

Deltamethrin deposition (% m-2)

vultures). The only significant effect recorded was increased feeding success of Slaty Egrets during spraying due to the negative effect of the deltamethrin on its prey. This was short-lived and the temporarily depressed fish stocks did not cause the Slaty Egrets to vacate the area.

## **6. Aquatic plants (***Salvinia molesta***) and biological control weevil**  *Cyrtobagous salviniae*

The distribution of salvinia and weevil populations varies widely under field conditions. Therefore, using field and static short-term toxicity bioassay methods (Reish & Cshida, 1987) in the Okavango (Figure 2) and iron cages representing open conditions in Kwando (Figure 3), the impacts of the insecticide on the weevil were determined during the five sequential aerial spray cycles. The controls were maintained in unsprayed area of Khwai River at North Gate. Assessments of the impacts were carried out at 12, 36 and 60 hours after the spray in each cycle from basins and cages as well as in field conditions before and after each spraying cycle (Kurugundla & Serumola, 2007, Kurugundla et al., 2010).

Mean survival of the 50 adult salvinia weevils extracted using Berlese funnels (Boland & Room, 1983) in the controls was generally in the range of 45.5 (mortality = 09.0%) to 48.4 (mortality = 03.2%) in number. The range of weevils' survival in closed basins exposed to deltamethrin in 2002 was between 25.0 (mortality = 47.0%) and 44.7 (mortality = 04.9%) while it was 33.7 (mortality = 27.2%) and 39.3 (mortality = 16.4%) in cages in 2006 with respect to controls (Table 5). However, in the 4th cycle of 2006 spray, the weevil mortality was 52.5% (live weevils = 22.0 ±1.8) (Table 5), which was obviously due to the formation of ice crystals and the cold conditions in the weevil extraction cups (≤ 50C) increasing the weevil mortality significantly (P ≤0.05), but not necessarily as a result of deltamethrin spray drift. Cesida (1980) found that pyrethroid toxicity increased at low temperatures.

The percentage deltamethrin collected on the aluminium foil sheets was in the range of 0.2% to 6.9% of the applied rate and the insecticide drift is varied depending on the inversion, wind direction and temperature (Perkins & Ramberg, 2002; Bonyongo & Mazvimavi, 2007). Significant difference in weevil mortality was observed between 2.3% and 6.9% (Table 5) deltamethrin deposition and did not show significant mortality at less than 2% deposition as determined from the aluminium foils (Kurugundla & Serumola, 2007; Kurugundla et al., 2010). The fluctuations in the abundance of weevils in the field Salvinia infestations at the sites of Xakanaxa (Figure 2) and Kwando (Figure 3) after praying was due to the spatial distribution of weevils and to less breeding during winter (Forno et al., 1983; Naidu et al., 2000) despite some spray effects. In all five cycles the abundance of weevils in 20 standard plants was higher than one (Forno, 1987) showing that weevils maintained their equilibrium at Shummamori, Hamokata, Lebala and Selinda (Figure 3) during the spray programme.

Although deltamethrin deposition caused significant weevil mortalities, the insecticide did not affect the weevil ability to control salvinia. It might be difficult to relate the declines of weevils in the field to the deltamethrin toxicity, as toxicity is influenced by factors such as temperature, season of spraying, habitat conditions and a protective mechanism possessed by the life stages of weevils (Schlettwein & Giliomee, 1990). In aquatic habitat conditions deltamethrin aerosols could be diluted, partitioned and adsorbed onto various organic sediments (Muir et al., 1985), which would reduce the toxicity not only to the weevils but several aquatic invertebrates. It is also suggested that the vegetation might act as a limiting factor for insecticide deposition on target surfaces, unlike in the open areas. On cold nights

vultures). The only significant effect recorded was increased feeding success of Slaty Egrets during spraying due to the negative effect of the deltamethrin on its prey. This was short-lived and the temporarily depressed fish stocks did not cause the Slaty Egrets

The distribution of salvinia and weevil populations varies widely under field conditions. Therefore, using field and static short-term toxicity bioassay methods (Reish & Cshida, 1987) in the Okavango (Figure 2) and iron cages representing open conditions in Kwando (Figure 3), the impacts of the insecticide on the weevil were determined during the five sequential aerial spray cycles. The controls were maintained in unsprayed area of Khwai River at North Gate. Assessments of the impacts were carried out at 12, 36 and 60 hours after the spray in each cycle from basins and cages as well as in field conditions before and after each

Mean survival of the 50 adult salvinia weevils extracted using Berlese funnels (Boland & Room, 1983) in the controls was generally in the range of 45.5 (mortality = 09.0%) to 48.4 (mortality = 03.2%) in number. The range of weevils' survival in closed basins exposed to deltamethrin in 2002 was between 25.0 (mortality = 47.0%) and 44.7 (mortality = 04.9%) while it was 33.7 (mortality = 27.2%) and 39.3 (mortality = 16.4%) in cages in 2006 with respect to controls (Table 5). However, in the 4th cycle of 2006 spray, the weevil mortality was 52.5% (live weevils = 22.0 ±1.8) (Table 5), which was obviously due to the formation of ice crystals and the cold conditions in the weevil extraction cups (≤ 50C) increasing the weevil mortality significantly (P ≤0.05), but not necessarily as a result of deltamethrin spray

The percentage deltamethrin collected on the aluminium foil sheets was in the range of 0.2% to 6.9% of the applied rate and the insecticide drift is varied depending on the inversion, wind direction and temperature (Perkins & Ramberg, 2002; Bonyongo & Mazvimavi, 2007). Significant difference in weevil mortality was observed between 2.3% and 6.9% (Table 5) deltamethrin deposition and did not show significant mortality at less than 2% deposition as determined from the aluminium foils (Kurugundla & Serumola, 2007; Kurugundla et al., 2010). The fluctuations in the abundance of weevils in the field Salvinia infestations at the sites of Xakanaxa (Figure 2) and Kwando (Figure 3) after praying was due to the spatial distribution of weevils and to less breeding during winter (Forno et al., 1983; Naidu et al., 2000) despite some spray effects. In all five cycles the abundance of weevils in 20 standard plants was higher than one (Forno, 1987) showing that weevils maintained their equilibrium at Shummamori, Hamokata, Lebala and Selinda (Figure 3) during the spray programme. Although deltamethrin deposition caused significant weevil mortalities, the insecticide did not affect the weevil ability to control salvinia. It might be difficult to relate the declines of weevils in the field to the deltamethrin toxicity, as toxicity is influenced by factors such as temperature, season of spraying, habitat conditions and a protective mechanism possessed by the life stages of weevils (Schlettwein & Giliomee, 1990). In aquatic habitat conditions deltamethrin aerosols could be diluted, partitioned and adsorbed onto various organic sediments (Muir et al., 1985), which would reduce the toxicity not only to the weevils but several aquatic invertebrates. It is also suggested that the vegetation might act as a limiting factor for insecticide deposition on target surfaces, unlike in the open areas. On cold nights

**6. Aquatic plants (***Salvinia molesta***) and biological control weevil** 

spraying cycle (Kurugundla & Serumola, 2007, Kurugundla et al., 2010).

drift. Cesida (1980) found that pyrethroid toxicity increased at low temperatures.

to vacate the area.

*Cyrtobagous salviniae* 

the weevils normally hide in buds, roots and beneath the leaves. They deposit their eggs in buds and underneath leaves, and emerging larvae normally feed inside the rhizome (Forno et al 1983). Therefore adults and larvae would often be protected from contact with the insecticide (Schlettwein & Giliomee, 1990).


Table 5. Mean survival of 50 weevils (SE = sd√n) in the Okavango Delta and Kwando-Linyanti in response to deltamethrin spray deposition (% m-2). Figures in parentheses indicate corrected percent mortality with respect to controls in each cycle. \* Probability ≤0.05 with reference to controls.

Fig. 7. Number of weevils in 20 plants by standard plant method before and after the spray in five cycles.

Average weevil mortalities are typically 26.5% in 2002 and 29.7% in 2006 at 2.7% and 4.1% deltamethrin respectively (Kurugundla& Serumola 2007; Kurugundla et al., 2010). It was also observed that Paradise pool and Lebala pool were completely covered with the salvinia two months after the end of spraying, yet the weevils controlled the infestation after 7 to 8 months. Aerial spraying of deltamethrin for controlling tsetse fly in any given area is not a continuous process and it is applied only in winter, when the breeding rate of the weevils

Management of Tsetse Fly Using Insecticides in Northern Botswana 471

the spraying season. Endosulfan had possible influence on migration of fish and *Tilapia rendalli* abundance declines in shallow vegetated areas. Residue of endosulfan was highest in *Schilbe mystus* at 0.04 ppm in muscle and 0.28 ppm in viscera in gram wet weight. The spraying influenced the feeding in king fisher due to behavioural changes. However, physiological studies in fish showed that surviving fish became significantly debilitated although recovery followed cessation of spraying. However, several groups of invertebrates, especially arthropods, are susceptible to the deltamethrin and deltamethrin spraying caused significant reductions in abundance of sensitive aquatic and terrestrial taxa. The results indicate that the surface dwelling arthorpods were affected in great deal rather than the groups such as leaches, snails, pond damsels and others that live in sediments. High elimination rate recorded for the order Hemiptera (water fly), Ephemeroptera (may flies) and Coleoptera (beetles) as they are active in free water and on vegetation surfaces. However deltamethrin did not affect the fish and birds as the result of deltamethrin

Ali, S. (1978). Degradation and environmental fate of endosulfan isomers and endosulfan

Allsopp, R. (1978). The effect of Dieldrin, sprayed by aerial application for tsetse control on

Allsopp R & Phillemon-Motsu, T.K. (2002). Tsetse Control in Botswana – a reversal in

Amweg, E. L. Weston, D.P., You, J. & Lydy, M. J. (2006). Pyrethroid Insecticides and

Anon (1973) FAO/Who Evaluation of Endosulfan Residues in Food. FAO/AGP: CP/103,

Bendsen, H., Mmopelwa, G. & Motsholapheko, M. (2006). Socio-economic issues. *In*:

Berlese Funnels. *Journal of Australian Entomological Society* 22: 353-354. Bonyongo, M. C. & Mazvimavi.D (2007). Environmental Monitoring of May-August 2006

Mazvimavi (Eds.)-2008, HOORC, University of Botswana, 105pp

Bucher, F. &Hofer, R. (1993). The effects of treated domestic sewage on three organs (gills, kidney, liver) of brown trout *(Salmo trutta*), *Water Research* 27: 255-261

game animals. *Journal of Applied Ecology* 15: 117–127.

strategy. *Pesticide Outlook* 13: (2) 73-76

*Technol.* 40 (5), 1700-1706.

Sulphate in mouse, insect and laboratory model ecosystems. *Diss. Abstr. Int*. *B* 39:

Sediment Toxicity in Urban Creeks fromCalifornia and Tennessee. *Environ. Sci.* 

Environmental Monitoring of May-August Aerial Spraying of Deltamethrin for Tsetse Fly Eradication in the Kwando-Linyanti and Caprivi Region – 2007. M. C. Bonyongo & D. Mazvimavi (Eds.). HOORC, University of Botswana, pp 224 – 283 Boland, N. P & Room, P.M. (1983). Estimating population density of *Cyrtobagous singularis*

Hustache (Coleoptera: Curculionidae) on the floating weed *Salvinia molesta* using

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spraying.

**9. References** 

2117

Rome

generally low. The two important monitoring studies conducted in 2002 and 2006 confirm that, although *C. salviniae* was affected negatively by the aerial spraying of deltamethrin, it recovered thereafter as shown by the subsequent effective control of salvinia in Paradise and Lebala pools in the Okavango delta and Kwando River respectively.

## **7. Socio-economic implications**

No side effects on human health was reported and people expressed their appreciation about the programme. However, there were sporadic reports of irritation to eyes during the spraying as reported by the humans. During the spraying campaign, people continued utilizing crops, fish and wild veld products. No short-term land use changes were observed and no disturbances to domestic live stock (Bendsen et al., 2006). People who moved from the core of tsetse infested areas during 1960s and 1970s have now settled permanently in Caprivi region. The changes in land use did not become apparent during the spraying and in the post spray periods. Botswana is one of the prime wilderness tourism destinations and there were no direct or indirect impacts on the tourism inflow as the result of spraying. The successful eradication of the flies has created an enabling environment for livestock development. No stock losses due to *nagana* have occurred after the spray of 2001, 2002 and 2006 and the carrying capacity of the rangelands has increased. Eighty-two commercial livestock farms, at a size of 2000 ha each, have already been sanctioned by the Namibian Government in the western section of the Spray Block area in Caprivi. Only 8% of the lodges were against the spraying and 92% of the tour operators appreciated the tsetse eradication. The successful eradication of tsetse fly would save the Botswana Government the recurrent costs that were invested annually for the control of flies through the maintenance of 10,000 odour baited targets in the tsetse dominated areas (Bonyongo & Mazvimavi, 2008).

## **8. Conclusions**

The incidence of *nagana* in northern Botswana as a result of tsetse fly spread increased between 1950 and 1960. Besides large scale clearing of bush and vegetation, ground sprays using insecticides such as DDT, Dieldrin, endosulfan and deltamethrin have been used to control the spread of tsetse fly. Application of non-residual spraying of endosulfan in the Okavango Delta, coupled with odour bait technique in the northern wetlands in 1992, reduced the tsetse fly distribution from 20,000 km2 to 5000 km2. By exploiting the improved aerial spraying techniques (fitted with GPS-guided spray equipment fixed to the aircraft), Botswana Government sprayed deltamthrin in 2001 and 2002 in the Okavango Delta and in 2006 in the Kwando-Linyanti systems. Almost 10 years following the end of spraying in the Okavango Delta, tsetse fly have still not been found and the threat of cattle trypanosomosis has been quelled. Furthermore, the tsetse frontiers involving the northern tsetse fly distribution along the Kwando and Linyanti Rivers bordering Caprivi region in Namibia – which is part of the continental common tsetse fly-belt has been effectively pushed back into Southern Angola. As such, the threat of reintroduction of tsetse fly back into northern Botswana has been greatly reduced.

Endosulfan aerial spraying did not produce serious harm to terrestrial invertebrates and no significant difference between seasonal and spraying effects was found in aquatic invertebrates at 12 g ha-1 of endosulfan applications. Possible exceptions included adult Chironomidae and Hymenoptera other than ants, both of which showed some declines in the spraying season. Endosulfan had possible influence on migration of fish and *Tilapia rendalli* abundance declines in shallow vegetated areas. Residue of endosulfan was highest in *Schilbe mystus* at 0.04 ppm in muscle and 0.28 ppm in viscera in gram wet weight. The spraying influenced the feeding in king fisher due to behavioural changes. However, physiological studies in fish showed that surviving fish became significantly debilitated although recovery followed cessation of spraying. However, several groups of invertebrates, especially arthropods, are susceptible to the deltamethrin and deltamethrin spraying caused significant reductions in abundance of sensitive aquatic and terrestrial taxa. The results indicate that the surface dwelling arthorpods were affected in great deal rather than the groups such as leaches, snails, pond damsels and others that live in sediments. High elimination rate recorded for the order Hemiptera (water fly), Ephemeroptera (may flies) and Coleoptera (beetles) as they are active in free water and on vegetation surfaces. However deltamethrin did not affect the fish and birds as the result of deltamethrin spraying.

### **9. References**

470 Insecticides – Pest Engineering

generally low. The two important monitoring studies conducted in 2002 and 2006 confirm that, although *C. salviniae* was affected negatively by the aerial spraying of deltamethrin, it recovered thereafter as shown by the subsequent effective control of salvinia in Paradise and

No side effects on human health was reported and people expressed their appreciation about the programme. However, there were sporadic reports of irritation to eyes during the spraying as reported by the humans. During the spraying campaign, people continued utilizing crops, fish and wild veld products. No short-term land use changes were observed and no disturbances to domestic live stock (Bendsen et al., 2006). People who moved from the core of tsetse infested areas during 1960s and 1970s have now settled permanently in Caprivi region. The changes in land use did not become apparent during the spraying and in the post spray periods. Botswana is one of the prime wilderness tourism destinations and there were no direct or indirect impacts on the tourism inflow as the result of spraying. The successful eradication of the flies has created an enabling environment for livestock development. No stock losses due to *nagana* have occurred after the spray of 2001, 2002 and 2006 and the carrying capacity of the rangelands has increased. Eighty-two commercial livestock farms, at a size of 2000 ha each, have already been sanctioned by the Namibian Government in the western section of the Spray Block area in Caprivi. Only 8% of the lodges were against the spraying and 92% of the tour operators appreciated the tsetse eradication. The successful eradication of tsetse fly would save the Botswana Government the recurrent costs that were invested annually for the control of flies through the maintenance of 10,000

odour baited targets in the tsetse dominated areas (Bonyongo & Mazvimavi, 2008).

The incidence of *nagana* in northern Botswana as a result of tsetse fly spread increased between 1950 and 1960. Besides large scale clearing of bush and vegetation, ground sprays using insecticides such as DDT, Dieldrin, endosulfan and deltamethrin have been used to control the spread of tsetse fly. Application of non-residual spraying of endosulfan in the Okavango Delta, coupled with odour bait technique in the northern wetlands in 1992, reduced the tsetse fly distribution from 20,000 km2 to 5000 km2. By exploiting the improved aerial spraying techniques (fitted with GPS-guided spray equipment fixed to the aircraft), Botswana Government sprayed deltamthrin in 2001 and 2002 in the Okavango Delta and in 2006 in the Kwando-Linyanti systems. Almost 10 years following the end of spraying in the Okavango Delta, tsetse fly have still not been found and the threat of cattle trypanosomosis has been quelled. Furthermore, the tsetse frontiers involving the northern tsetse fly distribution along the Kwando and Linyanti Rivers bordering Caprivi region in Namibia – which is part of the continental common tsetse fly-belt has been effectively pushed back into Southern Angola. As such, the threat of reintroduction of tsetse fly back into northern

Endosulfan aerial spraying did not produce serious harm to terrestrial invertebrates and no significant difference between seasonal and spraying effects was found in aquatic invertebrates at 12 g ha-1 of endosulfan applications. Possible exceptions included adult Chironomidae and Hymenoptera other than ants, both of which showed some declines in

Lebala pools in the Okavango delta and Kwando River respectively.

**7. Socio-economic implications** 

**8. Conclusions** 

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Monitoring of May-August Aerial Spraying of Deltamethrin for Tsetse Fly Eradication in the Kwando-Linyanti and Caprivi Region – 2007. M. C. Bonyongo &

Spraying of Deltamethrin for Tsetse fly Eradication in the Kwando-Linyanti and Caprivi Region - 2008. Department of Animal Health and Production. M. C. Bonyongo and D. Mazvimavi (Eds.), HOORC, University of Botswana, pp 46-73 Matthiessen, P. & Douthwaite, R. J. (1985). The impact of tsetse fly control campaigns on

spraying with insecticides on the fish stocks of the Okavango Delta, Botswana.

Silver catfish (Schilbe intermedius Ruppal 1832) in a seasonal floodplain of the

Deltamethrin for Tsetse Fly Eradication in the Kwando-Linyanti and Caprivi Region – 2007. M. C. Bonyongo & D. Mazvimavi (Eds.). HOORC, University of

cypermethrin, deltamethrin, fenvalerate and permethrin by *Chironomus tentans* larvae in sediment and water. *Environmental Toxicological Chemistry* 4: 51-61 Müller, P., Nagel, P. & Flacke, W. (1980). Ecological side effects of Dieldrine application

against tsetse flies in Adamaoua, Cameroon. Report presented at Expert


**Part 4** 

**Toxicological Profile of Insecticides** 


## **Part 4**

**Toxicological Profile of Insecticides** 

476 Insecticides – Pest Engineering

Smies, M., Evers, R.H.J., Piejnenburg, F.H.M. & Koeman, J. H. (1980) Environmental

Sserunjoji, S.J.M. & Tjell, T.C. (1971). Insecticide Residues following Tsetse Control in Uganda. Paper No. 13 of a joint FAO/IAEA panel, Vienna, October 1971. Takken, W., Bruijckere, F. L. G. & Koeman J. H. (1976). Environmental impact studies

Tarimo, C.S. & Pallotti, E. (1979). Residues of aerially sprayed DNOC in biological materials. OAU/STRC, 15th meeting ISCTR 1977: 572–578 (TTIQ abstract No. 776). Teffera, G., Feledi, B. & Motlhabane , D. (2003). The effects of rainfall on the composition

Thomas D. R. (1981). *DDT: Scientists, Citizens, and Public Policy*. New Jersey: Princeton

Vale, G.A. and Torr, S.J. (2004). Development of bait technology to control tsetse. In The *Trypanosomiases* (Maudlin, I. *et al.,* (Eds.) pp. 533-546, CABI Publishing. VEEU-TCD/DAHP (1998) Socio-economics of Tsetse Fly Control in Northern Botswana. In

Wilson, Vivian, J. 1972 Observations on the effect of Dieldrin on wildlife during tsetse fly

Wolski, P and Huntsman-Mapila, P. (2002). Mateorology and spray deposition. *In*:

*Glossina morsitans* control operations in Eastern Zambia. *Arnoldia* (Rhodesia) 5: 1-12

Environmental Monitoring of Tsetse Fly Spraying Impacts in the Okavango Delta-. Perkins J.S and Ramberg L (Eds.) - 2004. Okavango Report Series No. 2. HOORC,

collaboration with DFID UK. 92- pp. Gaborone, Botswana

University of Botswana, Botswana. pp 17-27

*Ecotoxicology, Environment and Safe* 4: 114-128

Wageningen, the Netherlands.

*Vét. Pays trop*. 30: 31–39.

University Press

aspects of field trials with pyrethroids to eradicate tsetse fly in Nigeria,

concerning the fixed-wing aircraft endosulfan applications in the Southern Guinea Savanna zone in Nigeria. Report Dept. Toxicology Agriculture University.

and quality of food ingested by two species of fish *Schilbe intermedius* and *Oreochromis massambicus* in Gaborone Dam. *Botswana Notes and Records* 53: 179-186 Tibayrenc, R. & J. Gruvel (1977) La campagne de lutte contre les glossines dans le bassin du

> Lac Tchad II. Contrôle de l'assainissement glossinaire. Critique technique et financière de l'ensemble de la campagne. Conclusions générales. *Rev. Elev. Méd.*

**22** 

*2IRSP/ Ouidah,* 

*1,5Burkina Faso* 

*2,8Bénin 3UK 4,6,7France* 

**Trends in Insecticide Resistance in Natural** 

K. R. Dabiré1, A. Diabaté1, M. Namountougou1, L. Djogbenou2,

**West Africa: 10 Years' Surveys** 

T. Martin6, M. Weill7 and T. Baldet8 *1IRSS/Centre Muraz, BP 390 Bobo-Dioulasso,* 

*5IRD/IRSS, Bobo-Dioulasso UMR MIGEVEC,* 

*7ISEM, CNRS/Université Montpellier 2, Montpellier,* 

*3Liverpool School of Tropical Medicine, 4LIN/Montpellier UMR MIGEVEC* 

*6CIRAD, UR HORTSYS, Montpellier,* 

*8IRD/CIRAD/CREC, Cotonou,* 

C. Wondji3, F. Chandre4, F. Simard5, J-B. Ouédraogo1,

**Populations of Malaria Vectors in Burkina Faso,** 

Malaria is a major threat in endemic regions causing at least 1 million deaths each year affecting poor and underserved populations living in tropical and sub-tropical regions. Diseases control ideally entails prevention and treatment of human infections. However, few vaccines are currently available and many pathogens are now resistant to anti-parasitic drugs. Additionally populations from endemic countries have less access to treatments due to economical impediments. Thus, the control of vectors in many instances is the only affordable measure (Beier *et al*., 2008). Mosquito control is mainly achieved by using insecticides and secondarily bio-larvicides (*Bt*-H14, *B. sphaericus*), predators (fish or copepod predators) or parasitic load (fungi), and/or by modifying the physical environment (WHO, 2006). Insecticides target a vital physiological function, leading to mosquito death. Unfortunately, due to their extremely large numbers and short generation span, mosquito populations evolve very rapidly and become resistant to insecticides, leading to repeated field control failures. Resistance results from the selection of mutant individuals able to survive and reproduce in presence of insecticide, the insecticide failing to disrupt the function of its target. In 2007, more than 100 mosquito species were resistant to at least one insecticide, some species being resistant to several compounds (Whalon *et al*., 2008). Very few classes of synthetic insecticides are available today for vector control, the most recent

**1. Introduction** 

## **Trends in Insecticide Resistance in Natural Populations of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys**

K. R. Dabiré1, A. Diabaté1, M. Namountougou1, L. Djogbenou2, C. Wondji3, F. Chandre4, F. Simard5, J-B. Ouédraogo1, T. Martin6, M. Weill7 and T. Baldet8 *1IRSS/Centre Muraz, BP 390 Bobo-Dioulasso, 2IRSP/ Ouidah, 3Liverpool School of Tropical Medicine, 4LIN/Montpellier UMR MIGEVEC 5IRD/IRSS, Bobo-Dioulasso UMR MIGEVEC, 6CIRAD, UR HORTSYS, Montpellier, 7ISEM, CNRS/Université Montpellier 2, Montpellier, 8IRD/CIRAD/CREC, Cotonou, 1,5Burkina Faso 2,8Bénin 3UK 4,6,7France* 

#### **1. Introduction**

Malaria is a major threat in endemic regions causing at least 1 million deaths each year affecting poor and underserved populations living in tropical and sub-tropical regions. Diseases control ideally entails prevention and treatment of human infections. However, few vaccines are currently available and many pathogens are now resistant to anti-parasitic drugs. Additionally populations from endemic countries have less access to treatments due to economical impediments. Thus, the control of vectors in many instances is the only affordable measure (Beier *et al*., 2008). Mosquito control is mainly achieved by using insecticides and secondarily bio-larvicides (*Bt*-H14, *B. sphaericus*), predators (fish or copepod predators) or parasitic load (fungi), and/or by modifying the physical environment (WHO, 2006). Insecticides target a vital physiological function, leading to mosquito death. Unfortunately, due to their extremely large numbers and short generation span, mosquito populations evolve very rapidly and become resistant to insecticides, leading to repeated field control failures. Resistance results from the selection of mutant individuals able to survive and reproduce in presence of insecticide, the insecticide failing to disrupt the function of its target. In 2007, more than 100 mosquito species were resistant to at least one insecticide, some species being resistant to several compounds (Whalon *et al*., 2008). Very few classes of synthetic insecticides are available today for vector control, the most recent

Trends in Insecticide Resistance in Natural Populations

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 481

resistance status to DDT and pyrethroids and the distribution of L1014F *kdr* among *An. gambiae s.l.* into different agro climatic zones (table 1). We were also interested more recently from 2007 to perform bioassays with some insecticides among OP and CX and also to detect the *ace-1R* mutation 2-3 days aged females of *An*. *gambiae s.l*. issued from wild larvae were exposed to several molecules such as DDT 4%, permethrin 0.75%, deltamethrin 0.05%, bendiocarb 0.1%, chlorpyriphos methyl (CM) 0.04%, carbosulfan 0.04% and fenithrotion 0.04% according to the WHO tube protocol (WHO, 1998). These active molecules were chosen as they represented each family of classic insecticides commonly used in public health. Some of these molecules such as permethrin, deltamethrin and bendiocarb are now

**Study sites:** Burkina Faso covers three ecological zones, the Sudan savannah zone in the south and west, the arid savannah zone (Sudan-sahelian) which extends throughout much of the central part of the country and the arid land (Sahel) in the north. The northern part of the country experiences a dry season of 6-8 months with less than 500 mm of rainfall per year. Rainfall is heaviest in the south-west (5-6 months) with a relatively short dry season. The varied ecological conditions are reflected in the different agricultural systems practiced throughout the country, from arable to pastoral lands. The western region constitutes the main cotton belt extending to the south where some new cotton areas have been cultivated. All ecological zones support the existence of *Anopheles* species that vector malaria and the

Fig. 1. Localities of the study through the three agro-climatic zones of Burkina Faso.

in use in Burkina Faso through impregnated bednets and IRS application.

disease is widespread throughout the country (Figure 1).

has been introduced 20 years ago and none are expected in the near future (Nauen, 2007). The low availability of insecticides due to resistance is further reduced in many countries by the removal from the market of compounds for public health because for their toxicity for humans or the lack of specificity in non-target species (Rogan & Chen, 2005).

Resistance is a genetic adaptation to the modification of the environment induced by insecticides. It usually appears locally, sometimes independently in different places, but may spread rapidly through migration (Brogdon *&* McAllister, 1998; Weill *et al*., 2003). However, mosquito resistance is not only due to the insecticides used for mosquito control, but also to the many pesticide pollutions present in their environment which are generated by a large variety of human activities including insect control for agriculture and other house-hold protections. These pollutions may dramatically affect resistance genes dynamics and threaten vector control strategies. The overall pesticides pressure that select resistance in mosquitoes need to be clarified, both in terms of insecticides usage and quantity.

*An. gambiae* is a complex, with seven sibling species that are closely related and morphologically indistinguishable from each other by routine taxonomic methods (Gillies & Coetzee, 1987). These sibling species are however different with respect to ecological and behavioral characteristics and to vectorial competence. In West Africa, *An. gambiae s.s*. and *An. arabiensis* are the two main species of the complex that transmit malaria, with the former being the most efficient vector due to its high anthropophily (White, 1974, Lemasson *et al*., 1997). Previous study carried out on the species composition in Burkina Faso indicated that *An. gambiae s.l*. was found to be a mixture of *An. gambiae s.s.* and *An. arabiensis* across the Sudan (98.3% *vs.* 1.7%), Sudan-sahelian (78.6% *vs.* 21.4%) and the Sahel (91.5% *vs.* 8.5%) ecotypes (Dabiré *et al.*, 2009a). *An. gambiae s.s.* contains two molecular forms, M and S, which co-exist in West Africa (della Torre *et al*., 2005). The M form was predominant in permanent breeding sites such as rice fields, whereas the S form was predominant in temporary habitats notably rain-filled puddles which are productive during the wet season. In Burkina Faso, genes conferring resistance to insecticides display large frequency differences in M and S forms of *An. gambiae s.s.* and *An. arabiensis*. Resistance of *An. gambiae s.l.* to DDT and pyrethroids (PYR) is especially conferred in West Africa by mutation of the sodium channel target site, the L1014F *kdr* (Chandre *et al*., 1999; Diabaté *et al*., 2002; Awolola *et al*., 2005; Nguessan *et al*., 2007). Burkina Faso is composed of three agro-climatic zones and the use of insecticides to control agricultural and human health pests varies considerably in the different zones particularly as the main cotton cropping areas are found in the south west of the country. In this last region, the intensive use of insecticides most notably for fighting the cotton *Gossypium hirsutum* L. pest is thought to have selected insecticide resistance genes in mosquitoes whose breeding sites are exposed to pesticide runoff (Diabaté *et al*., 2002; Dabiré *et al*., 2009a & b). The goal of this chapter is to summarise the resistance to insecticides status mainly in *An. gambiae s.l.* populations throughout these different agro-climatic areas and to discuss how it could limit the efficacy of malaria vector control strategies in short and long terms at the country scale. Such information is vital to determine the suitability of pyrethroids used for bednet impregnation and CX or OP based-combinations for indoor residual spraying (IRS).

#### **2. Materials and methods**

In Burkina Faso country-wide surveys associating bioassays and molecular investigations were carried out from 2000 to 2010 through 26 localities and they allow updating the

has been introduced 20 years ago and none are expected in the near future (Nauen, 2007). The low availability of insecticides due to resistance is further reduced in many countries by the removal from the market of compounds for public health because for their toxicity for

Resistance is a genetic adaptation to the modification of the environment induced by insecticides. It usually appears locally, sometimes independently in different places, but may spread rapidly through migration (Brogdon *&* McAllister, 1998; Weill *et al*., 2003). However, mosquito resistance is not only due to the insecticides used for mosquito control, but also to the many pesticide pollutions present in their environment which are generated by a large variety of human activities including insect control for agriculture and other house-hold protections. These pollutions may dramatically affect resistance genes dynamics and threaten vector control strategies. The overall pesticides pressure that select resistance

*An. gambiae* is a complex, with seven sibling species that are closely related and morphologically indistinguishable from each other by routine taxonomic methods (Gillies & Coetzee, 1987). These sibling species are however different with respect to ecological and behavioral characteristics and to vectorial competence. In West Africa, *An. gambiae s.s*. and *An. arabiensis* are the two main species of the complex that transmit malaria, with the former being the most efficient vector due to its high anthropophily (White, 1974, Lemasson *et al*., 1997). Previous study carried out on the species composition in Burkina Faso indicated that *An. gambiae s.l*. was found to be a mixture of *An. gambiae s.s.* and *An. arabiensis* across the Sudan (98.3% *vs.* 1.7%), Sudan-sahelian (78.6% *vs.* 21.4%) and the Sahel (91.5% *vs.* 8.5%) ecotypes (Dabiré *et al.*, 2009a). *An. gambiae s.s.* contains two molecular forms, M and S, which co-exist in West Africa (della Torre *et al*., 2005). The M form was predominant in permanent breeding sites such as rice fields, whereas the S form was predominant in temporary habitats notably rain-filled puddles which are productive during the wet season. In Burkina Faso, genes conferring resistance to insecticides display large frequency differences in M and S forms of *An. gambiae s.s.* and *An. arabiensis*. Resistance of *An. gambiae s.l.* to DDT and pyrethroids (PYR) is especially conferred in West Africa by mutation of the sodium channel target site, the L1014F *kdr* (Chandre *et al*., 1999; Diabaté *et al*., 2002; Awolola *et al*., 2005; Nguessan *et al*., 2007). Burkina Faso is composed of three agro-climatic zones and the use of insecticides to control agricultural and human health pests varies considerably in the different zones particularly as the main cotton cropping areas are found in the south west of the country. In this last region, the intensive use of insecticides most notably for fighting the cotton *Gossypium hirsutum* L. pest is thought to have selected insecticide resistance genes in mosquitoes whose breeding sites are exposed to pesticide runoff (Diabaté *et al*., 2002; Dabiré *et al*., 2009a & b). The goal of this chapter is to summarise the resistance to insecticides status mainly in *An. gambiae s.l.* populations throughout these different agro-climatic areas and to discuss how it could limit the efficacy of malaria vector control strategies in short and long terms at the country scale. Such information is vital to determine the suitability of pyrethroids used for bednet impregnation and CX or OP based-combinations for indoor

In Burkina Faso country-wide surveys associating bioassays and molecular investigations were carried out from 2000 to 2010 through 26 localities and they allow updating the

in mosquitoes need to be clarified, both in terms of insecticides usage and quantity.

residual spraying (IRS).

**2. Materials and methods** 

humans or the lack of specificity in non-target species (Rogan & Chen, 2005).

resistance status to DDT and pyrethroids and the distribution of L1014F *kdr* among *An. gambiae s.l.* into different agro climatic zones (table 1). We were also interested more recently from 2007 to perform bioassays with some insecticides among OP and CX and also to detect the *ace-1R* mutation 2-3 days aged females of *An*. *gambiae s.l*. issued from wild larvae were exposed to several molecules such as DDT 4%, permethrin 0.75%, deltamethrin 0.05%, bendiocarb 0.1%, chlorpyriphos methyl (CM) 0.04%, carbosulfan 0.04% and fenithrotion 0.04% according to the WHO tube protocol (WHO, 1998). These active molecules were chosen as they represented each family of classic insecticides commonly used in public health. Some of these molecules such as permethrin, deltamethrin and bendiocarb are now in use in Burkina Faso through impregnated bednets and IRS application.

**Study sites:** Burkina Faso covers three ecological zones, the Sudan savannah zone in the south and west, the arid savannah zone (Sudan-sahelian) which extends throughout much of the central part of the country and the arid land (Sahel) in the north. The northern part of the country experiences a dry season of 6-8 months with less than 500 mm of rainfall per year. Rainfall is heaviest in the south-west (5-6 months) with a relatively short dry season. The varied ecological conditions are reflected in the different agricultural systems practiced throughout the country, from arable to pastoral lands. The western region constitutes the main cotton belt extending to the south where some new cotton areas have been cultivated. All ecological zones support the existence of *Anopheles* species that vector malaria and the disease is widespread throughout the country (Figure 1).

Fig. 1. Localities of the study through the three agro-climatic zones of Burkina Faso.

Trends in Insecticide Resistance in Natural Populations

specific PCR as described by Martinez-Torres *et al*. (1998).

ethidium bromide and visualized under UV light.

(ver.3.4) software (Raymond & Rousset 1995)

**3. Resistance to pyrethroids and organochlorine** 

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 483

mosquitoes were transferred into insecticide free tubes and maintained on sucrose solution. Final mortality was recorded 24 h after exposure. The threshold of susceptibility was fixed at 98% for the four active molecules according to the protocol of WHO (1998). Are considered as susceptible, suspected resistant and resistant populations with respectively 100 to 98%, 98 to 80 %and under 80% of mortality rates. Dead and survivor mosquitoes were

**Molecular analysis:** DNA extraction and PCR identification of the *An. gambiae* M and S and *An. arabienis*: Genomic DNA was extracted from individual mosquitoes according to a slightly modified version of the procedure described by Collins *et al*. (1987). After quantification of the extracted DNA, adults of *An. gambiae s.l.* tested in bioassay were processed by PCR for molecular identification of species of the *An. gambiae* complex and molecular forms respectively (Scott *et al*., 1993; Favia *et al*., 2001). Those survived or dead in bioassay were after processed in other PCR analysis for the detection of *kdr* and *ace-1*<sup>R</sup> mutations. For *kdr* detection, a sub-sample of 30 mosquitoes per site of the permethrin/deltamethrin-tested specimens and those collected by indoor spraying were processed by PCR for prior species identification and molecular characterisation of M and S forms of *An. gambiae s.s.* according to Scott *et al.* (1993) and Favia *et al*. (2001) respectively. The frequency of the L1014F mutation in the same samples was determined by allele-

A*ce-1R* mutation was detected using the PCR-RFLP assay described by Weill *et al*. (2004) with minor modifications. Specific primers, *Ex3AGdir* (GATCGTGGACACCGTGTTCG) and *Ex3AGrev* (AGGATGGCCCGCTGGAACAG) were used in PCR reactions (25µl) containing 2.5µl of 10X *Taq* DNA polymerase buffer, 200µM of each desoxynucleoside triphosphate (dNTP), 0.1U of *Taq* DNA polymerase (Qiagen, France), 10pmol of each primer and approximately 1 to 10ng of DNA. PCR conditions included an initial denaturation step at 94°C for 5min followed by thirty five cycles of 94°C for 30s, 54°C for 30s and 72°C for 30s, with a final extension at 72°C for 5min. Fifteen microlitres of PCR product was digested with 5U of *Alu*I restriction enzyme (Promega, France) in a final volume of 25 µl at 37°C for 3 hours. Products were then analysed by electrophoresis on a 2% agarose gel stained with

**Statistical analysis:** The proportion of each species and molecular forms were compared between the study sites. The frequencies of *kdr* and *ace-1R* mutations were calculated according to the formula *p*= 2AA+Aa/2n where AA was the number of homozygotes, Aa the number of heterozygotes and n the size of analyzed sample. It was compared between sites and between *An. gambiae* M and S molecular forms and *An. arabiensis* by chi square tests. The genotypic frequencies of ace-1R in mosquito populations were compared to Hardy-Weinberg expectations using the exact test procedures implemented in GenePOP

Reports of resistance in mosquito vector populations in Burkina Faso appeared as early as the 1960 s, when *An*. *funestus* and *An*. *gambiae s.l*. populations that showed resistance to dieldrin and DDT, were described (Hamon *et al*., 1968a; Hamon *et al.*, 1968b). More recent studies have confirmed that resistance to DDT4% is still prevailing with highest level in *An*. *gambiae s.l*. populations in Burkina Faso where also resistance to certain pyrethroids was increasingly reported (Diabaté *et al*., 2002, 2004a; Dabiré *et al*., 2009a). Indeed *An*. *gambiae s.l.*

grouped separately and stored on silicagel at -20°C for subsequent PCR analysis.


Table 1. Main study sites across the country from where natural populations of *An. gambiae s.l.* were collected for susceptibility tests to insecticides in Burkina Faso.

**Mosquitoes sampling:** To evaluate the status of resistance of *An*. *gambiae s.l*. to insecticides in the three ecological zones of Burkina Faso, anopheline larvae were sampled in countrywide collections during the rainy season, from September to October. Larvae were collected at each locality from breeding sites such as gutters, tires, swallow wells and pools of standing water. Larvae were brought back to the insectary and reared to adulthood. When it was not possible to collect larvae because of the distance between the sampling site and the insectary or due to sampling constraints at the site such as excessive rainfall or flooding, alternative collections of adult mosquitoes were made using indoor aerosol insecticide spraying. *An. gambiae s.l.* were identified morphologically using standard identification keys of Gillies & Coetzee (1987). The results presented here summarized those of transversal studies in whole country 2000 and 2009 with particular focus on the period from September to October 2009.

Insecticide susceptibility test: Susceptibility test was performed on 2-3-day-old *An. gambiae s.l.* females provided by larva collections using the WHO standard vertical tube protocol. Three insecticide-impregnated papers were used: DDT 4%, permethrin 0.75% (cis:trans = 25:75), deltamethrin 0.05%, bendiocarb 0.1%, CM 0.04%, carbofuran 0.04% and fenithrotion 0.04%. Mosquitoes were tested against "Kisumu" a fully susceptible reference laboratory strain. Mortality controls were carried out by exposing both the "Kisumu" strain and wild populations from each site to non-insecticidal impregnated paper. After 1 h exposure,

**references Climatic areas Environment Agricultural** 

Batié 9°80'N; 2°90'W Sudanian rural cereals, cotton 20/08/09 Gaoua 10°40'N; 3°15'W Sudanian sub-urban cereals, cotton 20/08/09 Diébougou 10°95'N; 3°24'W Sudanian sub-urban cereals, cotton 20/08/09 Dano 11°10'N; 3°05'W Sudanian rural cereals, cotton 20/08/09 Banfora 10°60'N; 4°70'W Sudanian sub-urban cereals, cotton 15/08/09 Sidéradougou 10°60'N; 4°25'W Sudanian rural cereals, cotton 15/08/09 Tiéfora 10°50'N; 4°50'W Sudanian rural cereals, cotton 15/08/09 Orodara 11°00'N; 4°91'W Sudanian rural fruits, cotton 15/08/09 Dioulassoba 11°22'N; 4°30'W Sudanian traditionnal-urban swamp 15/08/09 Soumousso 11°01'N; 4°02'W Sudanian rural cotton 15/08/09 VK7 11°41'N; 4°44'W Sudanian rural rice, cotton 08/08/09 VK5 11°24'N ; 4°23'W Sudanian rural rice 08/08/09 Pô 11°20'N; 1°10'W Sudanian sub-urban cereals, cotton 28/08/09 Houndé 11°50'N; 3°55'W Sudanian sub-urban cotton 10/08/09 Boromo 11°75'N; 2°92'W Sudan-sahelian sub-urban cotton 16/08/09 Solenzo 12°37'N; 3°55'W Sudan-sahelian rural cotton 16/08/09 Dedougou 12°50'N; 3°45'W Sudan-sahelian sub-urban cotton 16/08/09 Nouna 12°70'N; 3°90'W Sudan-sahelian sub-urban cotton 16/08/09 Koubri 12°35'N; 1°50'W Sudan-sahelian rural vegetables 28/08/09 Kombissiri 12°05'N; 1°35'W Sudan-sahelian rural vegetables, cotton 28/08/09 Manga 11°66'N; 1°05'W Sudan-sahelian sub-urban cereals, cotton 28/08/09 Koupela 12°20'N; 0°40'W Sudan-sahelian sub-urban cotton 30/08/09 Fada 12°05'N; 3°55'E Sudan-sahelian sub-urban cotton 30/08/09 Kompienga 11°30'N; 0°40E Sudan-sahelian rural vegetables, cotton 30/09/09 Komiyenga 11°70'N; 0°60E Sudan-sahelian rural cotton 30/09/09 Yamtenga 12°21'N ;1°31'W Sudan-sahelian peri-urban swamp 28/08/09 Table 1. Main study sites across the country from where natural populations of *An. gambiae*

*s.l.* were collected for susceptibility tests to insecticides in Burkina Faso.

**Mosquitoes sampling:** To evaluate the status of resistance of *An*. *gambiae s.l*. to insecticides in the three ecological zones of Burkina Faso, anopheline larvae were sampled in countrywide collections during the rainy season, from September to October. Larvae were collected at each locality from breeding sites such as gutters, tires, swallow wells and pools of standing water. Larvae were brought back to the insectary and reared to adulthood. When it was not possible to collect larvae because of the distance between the sampling site and the insectary or due to sampling constraints at the site such as excessive rainfall or flooding, alternative collections of adult mosquitoes were made using indoor aerosol insecticide spraying. *An. gambiae s.l.* were identified morphologically using standard identification keys of Gillies & Coetzee (1987). The results presented here summarized those of transversal studies in whole country 2000 and 2009 with particular focus on the period

Insecticide susceptibility test: Susceptibility test was performed on 2-3-day-old *An. gambiae s.l.* females provided by larva collections using the WHO standard vertical tube protocol. Three insecticide-impregnated papers were used: DDT 4%, permethrin 0.75% (cis:trans = 25:75), deltamethrin 0.05%, bendiocarb 0.1%, CM 0.04%, carbofuran 0.04% and fenithrotion 0.04%. Mosquitoes were tested against "Kisumu" a fully susceptible reference laboratory strain. Mortality controls were carried out by exposing both the "Kisumu" strain and wild populations from each site to non-insecticidal impregnated paper. After 1 h exposure,

**practices** 

**Recent date of collection** 

**Study sites Geographic** 

from September to October 2009.

mosquitoes were transferred into insecticide free tubes and maintained on sucrose solution. Final mortality was recorded 24 h after exposure. The threshold of susceptibility was fixed at 98% for the four active molecules according to the protocol of WHO (1998). Are considered as susceptible, suspected resistant and resistant populations with respectively 100 to 98%, 98 to 80 %and under 80% of mortality rates. Dead and survivor mosquitoes were grouped separately and stored on silicagel at -20°C for subsequent PCR analysis.

**Molecular analysis:** DNA extraction and PCR identification of the *An. gambiae* M and S and *An. arabienis*: Genomic DNA was extracted from individual mosquitoes according to a slightly modified version of the procedure described by Collins *et al*. (1987). After quantification of the extracted DNA, adults of *An. gambiae s.l.* tested in bioassay were processed by PCR for molecular identification of species of the *An. gambiae* complex and molecular forms respectively (Scott *et al*., 1993; Favia *et al*., 2001). Those survived or dead in bioassay were after processed in other PCR analysis for the detection of *kdr* and *ace-1*<sup>R</sup> mutations. For *kdr* detection, a sub-sample of 30 mosquitoes per site of the permethrin/deltamethrin-tested specimens and those collected by indoor spraying were processed by PCR for prior species identification and molecular characterisation of M and S forms of *An. gambiae s.s.* according to Scott *et al.* (1993) and Favia *et al*. (2001) respectively. The frequency of the L1014F mutation in the same samples was determined by allelespecific PCR as described by Martinez-Torres *et al*. (1998).

A*ce-1R* mutation was detected using the PCR-RFLP assay described by Weill *et al*. (2004) with minor modifications. Specific primers, *Ex3AGdir* (GATCGTGGACACCGTGTTCG) and *Ex3AGrev* (AGGATGGCCCGCTGGAACAG) were used in PCR reactions (25µl) containing 2.5µl of 10X *Taq* DNA polymerase buffer, 200µM of each desoxynucleoside triphosphate (dNTP), 0.1U of *Taq* DNA polymerase (Qiagen, France), 10pmol of each primer and approximately 1 to 10ng of DNA. PCR conditions included an initial denaturation step at 94°C for 5min followed by thirty five cycles of 94°C for 30s, 54°C for 30s and 72°C for 30s, with a final extension at 72°C for 5min. Fifteen microlitres of PCR product was digested with 5U of *Alu*I restriction enzyme (Promega, France) in a final volume of 25 µl at 37°C for 3 hours. Products were then analysed by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV light.

**Statistical analysis:** The proportion of each species and molecular forms were compared between the study sites. The frequencies of *kdr* and *ace-1R* mutations were calculated according to the formula *p*= 2AA+Aa/2n where AA was the number of homozygotes, Aa the number of heterozygotes and n the size of analyzed sample. It was compared between sites and between *An. gambiae* M and S molecular forms and *An. arabiensis* by chi square tests. The genotypic frequencies of ace-1R in mosquito populations were compared to Hardy-Weinberg expectations using the exact test procedures implemented in GenePOP (ver.3.4) software (Raymond & Rousset 1995)

## **3. Resistance to pyrethroids and organochlorine**

Reports of resistance in mosquito vector populations in Burkina Faso appeared as early as the 1960 s, when *An*. *funestus* and *An*. *gambiae s.l*. populations that showed resistance to dieldrin and DDT, were described (Hamon *et al*., 1968a; Hamon *et al.*, 1968b). More recent studies have confirmed that resistance to DDT4% is still prevailing with highest level in *An*. *gambiae s.l*. populations in Burkina Faso where also resistance to certain pyrethroids was increasingly reported (Diabaté *et al*., 2002, 2004a; Dabiré *et al*., 2009a). Indeed *An*. *gambiae s.l.*

Trends in Insecticide Resistance in Natural Populations

**Localities**

Orodara Houndé Boromo Dédougou Solenzo VK7 VK5 Soumousso Diébougou Dano Batié Gaoua Sidéradougou Pô Koubri Manga Nouna

**Localities**

Kombissiri Manga

Kompienga

Orodara

Sudan-sahelian and sahelian areas in Burkina Faso.

Houndé Dédougou

Boromo

Nouna

Solenzo

VK7

VK5

Dioulassoba

Diébougou Banfora

Soumousso Dano

Yamtenga Koupéla

Sideradougou Fada

Sudan-sahelian and sahelian areas in Burkina Faso.

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 485

**Permethrin 0.75%**

**Suspected resistant Resistant Susceptible** 

0 10 20 30 40 50 60 70 80 90 100

Fig. 3. Mortality rates of *An. gambiae s.l*. populations to permethrin 0.75 % from Sudan,

**Mortality %**

**Deltamethrin 0.05%**

**Susceptible Suspected resistant Resistant**

**Mortality %**

0 10 20 30 40 50 60 70 80 90 100

Fig. 4. Mortality rates of *An. gambiae s.l*. populations to deltamethrin 0.05 % from Sudan,

populations were resistant to DDT4% in every part of the country and mortality rates below 60 % were observed at the country scale (Fig. 2). They were found also resistant to permethrin 0.75% in the Sudan climatic zone in the western region and also in several sites in the central part of Burkina Faso (Fig. 3). Surprisingly, except for the areas with a very long history of cotton cropping, the tested populations of *An. gambiae* remained susceptible to deltamethrin 0.05%, although decreased mortality values lead to suspect an emergence of resistance in the ongoing years (Dabiré *et al.*, 2009a). However, this result should be interpreted with caution as the resistance is a progressive process and recent data recorded in 2009-2010 showed 5 sites mostly located in the central region remained susceptible foci (Fig. 4).

Fig. 2. Mortality rates of *Anopheles gambiae s.l.* populations to DDT 4% from Sudan, Sudansahelian and sahelian areas in Burkina Faso.

Resistance is the result of a limited number of physiological mechanisms; it is often monogenic and due to point mutations in a structural gene, gene amplification or changes in transcriptional regulation (Hollingworth *&* Dong, 2008). It results from the selection of mutant individuals able to survive and reproduce in presence of insecticide, the insecticide failing to disrupt the function of its target (Whalon *et al*., 2008). The resistance phenotype to pyrethroids and DDT 4% observed in natural populations of *An. gambiae s.l.* was already attributed to a *kdr* mutation as it is the major mechanism involved in cross resistance to pyrethroids and DDT4% in West Africa (Chandre *et al*., 1999; Diabaté *et al*., 2002). Until recently, it was assumed that this mutation was the L1014F substitution in West Africa while the L1014S substitution was found in the East (Ranson *et al*., 2000). However, we now know that both mutations coexist in some countries and are widely distributed throughout sub-Saharan continent and also in Benin and Burkina Faso (Verhaeghen *et al*., 2006; Etang *et al*., 2006; Djegbe *et al*., 2011; Dabiré *et al*., unpublished).

populations were resistant to DDT4% in every part of the country and mortality rates below 60 % were observed at the country scale (Fig. 2). They were found also resistant to permethrin 0.75% in the Sudan climatic zone in the western region and also in several sites in the central part of Burkina Faso (Fig. 3). Surprisingly, except for the areas with a very long history of cotton cropping, the tested populations of *An. gambiae* remained susceptible to deltamethrin 0.05%, although decreased mortality values lead to suspect an emergence of resistance in the ongoing years (Dabiré *et al.*, 2009a). However, this result should be interpreted with caution as the resistance is a progressive process and recent data recorded in 2009-2010 showed 5 sites mostly located in the central region remained susceptible foci

**DDT4%**

**Localities Suspected resistant Resistant**

0 10 20 30 40 50 60 70 80 90 100

**Mortality %**

Fig. 2. Mortality rates of *Anopheles gambiae s.l.* populations to DDT 4% from Sudan, Sudan-

Resistance is the result of a limited number of physiological mechanisms; it is often monogenic and due to point mutations in a structural gene, gene amplification or changes in transcriptional regulation (Hollingworth *&* Dong, 2008). It results from the selection of mutant individuals able to survive and reproduce in presence of insecticide, the insecticide failing to disrupt the function of its target (Whalon *et al*., 2008). The resistance phenotype to pyrethroids and DDT 4% observed in natural populations of *An. gambiae s.l.* was already attributed to a *kdr* mutation as it is the major mechanism involved in cross resistance to pyrethroids and DDT4% in West Africa (Chandre *et al*., 1999; Diabaté *et al*., 2002). Until recently, it was assumed that this mutation was the L1014F substitution in West Africa while the L1014S substitution was found in the East (Ranson *et al*., 2000). However, we now know that both mutations coexist in some countries and are widely distributed throughout sub-Saharan continent and also in Benin and Burkina Faso (Verhaeghen *et al*., 2006; Etang *et al*.,

(Fig. 4).

Sidéradougou

Pô Koubri

Orodara Houndé Boromo Dédougou Solenzo VK7 VK5 Soumousso Diébougou Dano Batié Gaoua

sahelian and sahelian areas in Burkina Faso.

2006; Djegbe *et al*., 2011; Dabiré *et al*., unpublished).

Fig. 3. Mortality rates of *An. gambiae s.l*. populations to permethrin 0.75 % from Sudan, Sudan-sahelian and sahelian areas in Burkina Faso.

Fig. 4. Mortality rates of *An. gambiae s.l*. populations to deltamethrin 0.05 % from Sudan, Sudan-sahelian and sahelian areas in Burkina Faso.

Trends in Insecticide Resistance in Natural Populations

Frequency of kdr L1014F

0

form, A: *An. arabiensis*].

% of survivors

0

permethrin 0.75%.

10

20

30

40

50

20

40

60

80

100

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 487

Fig. 6. Evolution of allelic frequencies (in percentages) of L1014F *kdr* in natural populations of *An. gambiae s.l.* from 1999 to 2009 in Burkina Faso[S: *An. gambiae* S form; M: *An. gambiae* M

y = 0.39x + 0.90, r = 0,91

*kdr* allelic frequency (%)

0 20 40 60 80 100

Fig. 7. Correlation between *kdr* frequency and mortality rates of *An. gambiae s.l*. tested with

mean frequency of the *kdr* allele was the highest in populations from the Sudanian zone where the lowest mortality rates to pyrethroids and DDT 4% were seen in bioassays. By contrast, the *kdr* allele frequency was lower in *An*. *gambiae s.s*. populations is central and eastern sites where cotton cultivation is recent. A majority of susceptible phenotypes were observed in wild populations of *An*. *gambiae* from these areas. In addition, the results of this

Freq *kdr*(S) Freq *kdr* (M) Freq *kdr* (A)

Years

In Burkina Faso, the frequency of the L1014F *kdr* mutation was first described in the S form of *An. gambiae s.s.* in high frequencies especially in the Western part of the country where the use of insecticides is intensive in agriculture (Chandre *et al*., 1999, Diabaté *et al.,* 2002). But few years later it had been also found within the M form and was suspected to be the result of an introgression from the S form *An. gambiae s.s*. (Weill *et al.*, 2000; Diabaté *et al*., 2003). Up to day the distribution of this mutation at the country scale is variable, ranging from 0.5 to 0.97 for the S form in the Sudanian region and decreasing in the Sudano-sahelian and Sahelian areas with averaged values fluctuating between 0.1 and 0.6. Compared to 2000 data (Diabaté *et al*., 2004a), the frequency of L1014F *kdr* mutation increased notably from 2004 to 2006 before getting stable around the fixation level in some localities (Fig. 5). As mentioned above, no *kdr* was detected in 1999 in the M form (Chandre *et al*., 1999). But early in 2000, the L1014F mutation was identified from few specimens of M form from rice growing area, peaking maximally at 0.04 (Diabaté *et al*., 2003). Nowadays the L1014F *kdr* has increased drastically in the M form with varying frequencies between climatic areas, and reaching high frequencies (0.93) in cotton growing belts with a geographic expansion to the sudanosahelian region where it was formerly absent (Dabiré *et al*., 2009a). It has also increased in *An. arabiensis* (0.28) where it was formerly reported only from one specimen in 2002 (Diabaté *et al*., 2004b) (Fig. 6).

Fig. 5. Geographic distribution of L1014F *kdr* allele in *An. gambiae s.l*. populations inducing pyrethroids and DDT resistance profile in Burkina Faso in 2009 [numbers in bracket represent frequency of L1014F *kdr* allele frequencies].

Globally the distribution of DDT and pyrethroids resistance in regions of intensive cotton cultivation suggests that indirect selection pressures from the agricultural use of insecticides may be responsible for the development of resistance in *An*. *gambiae s.l*. populations. The

In Burkina Faso, the frequency of the L1014F *kdr* mutation was first described in the S form of *An. gambiae s.s.* in high frequencies especially in the Western part of the country where the use of insecticides is intensive in agriculture (Chandre *et al*., 1999, Diabaté *et al.,* 2002). But few years later it had been also found within the M form and was suspected to be the result of an introgression from the S form *An. gambiae s.s*. (Weill *et al.*, 2000; Diabaté *et al*., 2003). Up to day the distribution of this mutation at the country scale is variable, ranging from 0.5 to 0.97 for the S form in the Sudanian region and decreasing in the Sudano-sahelian and Sahelian areas with averaged values fluctuating between 0.1 and 0.6. Compared to 2000 data (Diabaté *et al*., 2004a), the frequency of L1014F *kdr* mutation increased notably from 2004 to 2006 before getting stable around the fixation level in some localities (Fig. 5). As mentioned above, no *kdr* was detected in 1999 in the M form (Chandre *et al*., 1999). But early in 2000, the L1014F mutation was identified from few specimens of M form from rice growing area, peaking maximally at 0.04 (Diabaté *et al*., 2003). Nowadays the L1014F *kdr* has increased drastically in the M form with varying frequencies between climatic areas, and reaching high frequencies (0.93) in cotton growing belts with a geographic expansion to the sudanosahelian region where it was formerly absent (Dabiré *et al*., 2009a). It has also increased in *An. arabiensis* (0.28) where it was formerly reported only from one specimen in 2002 (Diabaté

**GHANA TOGO**

Manga Kombissiri

**[70][93][50]**

*An. gambiae* forme S *An. gambiae* forme M *An. arabiensis*

[50] frequency of *kdr* in M-form [30] frequency of *kdr* in *An. arabiensis*

Fig. 5. Geographic distribution of L1014F *kdr* allele in *An. gambiae s.l*. populations inducing pyrethroids and DDT resistance profile in Burkina Faso in 2009 [numbers in bracket

Globally the distribution of DDT and pyrethroids resistance in regions of intensive cotton cultivation suggests that indirect selection pressures from the agricultural use of insecticides may be responsible for the development of resistance in *An*. *gambiae s.l*. populations. The

Koubri

**[30][50][5]**

**Dori**

**[12] [30]**

**[93][94][13]**

**[20]**

**Kompienga [88][50]**

**Climatic zones Study sites(21)**

Cotton belt since 1960 [73] frequency of *kdr* in S-form

**NIGER**

**[91][14]**

Sahlian Sudan-sahelian Sudaniann

Rice growing area since 1972

Koupela Fada

**[94] Komiyanga**

**[60][22]**

**[0]**

**BENIN**

*et al*., 2004b) (Fig. 6).

**COTE D'IVOIRE**

Orodara VK5 VK7

Banfora

**[79][31] Sidéra [98]**

**[99][0]**

[98][85]

**[98]**

Soumousso

Tiefora

represent frequency of L1014F *kdr* allele frequencies].

Echelle:0 <sup>50</sup> 100 km

Gaoua

**[97]**

Diébougou

**[83][60]**

Batié

Houndé

**Dano [96][71]**

**[90]**

Dédougou

**[30]**

**[83][67][0]**

Nanoro

**[0][0][0]**

Yamtenga

**[37][50]**

**Po**

**[98] [97]**

**[67]**

**Boromo**

**[93][71][40]**

Nouna

**[96][93][71]**

Solenzo **[63][28][28]**

**[35]**

**[38]**

**MALI**

Fig. 6. Evolution of allelic frequencies (in percentages) of L1014F *kdr* in natural populations of *An. gambiae s.l.* from 1999 to 2009 in Burkina Faso[S: *An. gambiae* S form; M: *An. gambiae* M form, A: *An. arabiensis*].

Fig. 7. Correlation between *kdr* frequency and mortality rates of *An. gambiae s.l*. tested with permethrin 0.75%.

mean frequency of the *kdr* allele was the highest in populations from the Sudanian zone where the lowest mortality rates to pyrethroids and DDT 4% were seen in bioassays. By contrast, the *kdr* allele frequency was lower in *An*. *gambiae s.s*. populations is central and eastern sites where cotton cultivation is recent. A majority of susceptible phenotypes were observed in wild populations of *An*. *gambiae* from these areas. In addition, the results of this

Trends in Insecticide Resistance in Natural Populations

0

Number of mosquitoes tested

GST in the VK7 sample.

5

10

Number 15

of mosquitoes

20

 tested 25

30

Number of mosquitoes tested

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 489

**Kisumu VK7**

**Kisumu Vk7**

kisumu vk7

**C**

**B**

**A**

<21 21-40 41-60 61-80 81-100 101-120 121-140 141-160 **Class of esterases activities** (nmol a-naph,/min/mg protein)

> **class of oxydases concentration** (pmol P450/mg protein)

<11 11-20 21-30 31-40 41-50

Fig. 8. Activity of detoxifying enzymes such as esterases (A), oxydases (B), and GST (C) in natural populations of *An. gambiae* from Vallée du Kou (VK7) compared to that of *An. gambiae* "Kisumu" (susceptible reference strain). Note the over-expression of oxydases and

GST activities classes (mmol GSH conj,/min/mg protein)

<10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 101-110

study suggest that the domestic use of insecticides may also exert a selection on *An*. *gambiae*  populations that is secondary to that from the agricultural insecticides. Indeed all collections made in cities located outside the cotton belt showed high mortality rates and a relative low frequency of *kdr* compared to those of cotton belt. The correlation between the high frequencies of L1014F *kdr* mutation and the proportion of surviving individuals after DDT/pyrethroids exposures (figure 7) suggests that this mutation is the main mechanism of resistance to these insecticides.

## **4. Metabolic resistance**

Most studies conducted in Burkina Faso have focused on the modification of target sites by mutation and did investigate the occurrence and the role of metabolic resistance in the observed resistance of *An. gambiae s.l.* Recent tests performed in Dioulassoba (an old central district of Bobo-Dioulasso crossed by the Houet river) showed that *An. arabiensis* from urban polluted breeding sites was resistant to DDT 4% but fully susceptible to pyrethroids and OP/CX, suggesting an existence of metabolic resistance probably GST which is more specific to DDT acting as the main resistance mechanism (Dabiré *et al*., unpubl.). Even more recently, preliminary results gathered only in VK7 (a sample from a rice growing area surrounded by cotton fields) showed an overexpression of detoxifying enzymes such as glutation-S-transferases, cytochrome P450 oxygenases in populations of *An. gambiae s.s.* with high *kdr* frequencies suggesting the existence of multi-resistance mechanisms to pyrethroids (Fig. 8A,B&C). But more investigations are needed to better address the role of metabolic components on the expression of resistance phenotypes observed in natural populations of *An. gambiae s.l.* especially in areas where insecticide pressure is high.

## **5. Resistance to organophosphates (OP) and carbamates (CX) and geographic distribution of** *ace-1R* **mutations and duplicated** *ace-1<sup>D</sup>***allele**

In Burkina Faso the resistance to OP/CX has been monitored since 2002 only in few sites of the Western areas of the country, and lately extended to the country scale since 2006. Although fenithrotion 0.4%, chlorpyriphos methyl (CM) 0.4%, carbosulfan 0.4% and bendiocarb 0.1 % were tested, the monitoring was well sustained only with bendiocarb 0.1% which was expected to be used in Burkina Faso indoor residual spraying to supplement the efficacy of ITNs especially in localities where *An. gambiae* is resistant to PY. Except for CM 0.4% for which *An. gambiae* populations were fully susceptible irrespectively of the locality, the other OP/CX mentioned above showed mortality rates ranging from 5% to 100% (Fig. 9). The lowest mortality rates were obtained with carbosulfan 0.4% (5%) and bendiocarb 0.1% (20%) especially in areas located in cotton belt such as Houndé, Orodara, Tiefora and Banfora. The susceptibility to bendiocarb 0.1% was also recorded in the central areas where cotton growing is recent (Fig. 10). From 2005 on, the detection of *ace-1R* mutation involved in OP/CX resistance allowed to evaluate the distribution of this allele in field populations of *An. gambiae s.l*. The characterisation of this allele was based on a PCR-RFLP diagnostic (Weill *et al.*, 2004) that allow the identification of the amino-acid substitution, from a glycine to a serine at the position 119, in the AChE1 catalytic site (G119S). In *Culex pipiens*, there is direct and indirect evidence that the resistance allele (*ace-1R*) entails a large fitness cost, probably due to the mutated AChE1 having a much lower level of activity. Homozygous *ace-1R* mosquitoes survive in the presence of insecticide, but are rapidly outcompeted in the

study suggest that the domestic use of insecticides may also exert a selection on *An*. *gambiae*  populations that is secondary to that from the agricultural insecticides. Indeed all collections made in cities located outside the cotton belt showed high mortality rates and a relative low frequency of *kdr* compared to those of cotton belt. The correlation between the high frequencies of L1014F *kdr* mutation and the proportion of surviving individuals after DDT/pyrethroids exposures (figure 7) suggests that this mutation is the main mechanism of

Most studies conducted in Burkina Faso have focused on the modification of target sites by mutation and did investigate the occurrence and the role of metabolic resistance in the observed resistance of *An. gambiae s.l.* Recent tests performed in Dioulassoba (an old central district of Bobo-Dioulasso crossed by the Houet river) showed that *An. arabiensis* from urban polluted breeding sites was resistant to DDT 4% but fully susceptible to pyrethroids and OP/CX, suggesting an existence of metabolic resistance probably GST which is more specific to DDT acting as the main resistance mechanism (Dabiré *et al*., unpubl.). Even more recently, preliminary results gathered only in VK7 (a sample from a rice growing area surrounded by cotton fields) showed an overexpression of detoxifying enzymes such as glutation-S-transferases, cytochrome P450 oxygenases in populations of *An. gambiae s.s.* with high *kdr* frequencies suggesting the existence of multi-resistance mechanisms to pyrethroids (Fig. 8A,B&C). But more investigations are needed to better address the role of metabolic components on the expression of resistance phenotypes observed in natural populations of

*An. gambiae s.l.* especially in areas where insecticide pressure is high.

**5. Resistance to organophosphates (OP) and carbamates (CX) and geographic distribution of** *ace-1R* **mutations and duplicated** *ace-1<sup>D</sup>***allele** 

In Burkina Faso the resistance to OP/CX has been monitored since 2002 only in few sites of the Western areas of the country, and lately extended to the country scale since 2006. Although fenithrotion 0.4%, chlorpyriphos methyl (CM) 0.4%, carbosulfan 0.4% and bendiocarb 0.1 % were tested, the monitoring was well sustained only with bendiocarb 0.1% which was expected to be used in Burkina Faso indoor residual spraying to supplement the efficacy of ITNs especially in localities where *An. gambiae* is resistant to PY. Except for CM 0.4% for which *An. gambiae* populations were fully susceptible irrespectively of the locality, the other OP/CX mentioned above showed mortality rates ranging from 5% to 100% (Fig. 9). The lowest mortality rates were obtained with carbosulfan 0.4% (5%) and bendiocarb 0.1% (20%) especially in areas located in cotton belt such as Houndé, Orodara, Tiefora and Banfora. The susceptibility to bendiocarb 0.1% was also recorded in the central areas where cotton growing is recent (Fig. 10). From 2005 on, the detection of *ace-1R* mutation involved in OP/CX resistance allowed to evaluate the distribution of this allele in field populations of *An. gambiae s.l*. The characterisation of this allele was based on a PCR-RFLP diagnostic (Weill *et al.*, 2004) that allow the identification of the amino-acid substitution, from a glycine to a serine at the position 119, in the AChE1 catalytic site (G119S). In *Culex pipiens*, there is direct and indirect evidence that the resistance allele (*ace-1R*) entails a large fitness cost, probably due to the mutated AChE1 having a much lower level of activity. Homozygous *ace-1R* mosquitoes survive in the presence of insecticide, but are rapidly outcompeted in the

resistance to these insecticides.

**4. Metabolic resistance** 

Fig. 8. Activity of detoxifying enzymes such as esterases (A), oxydases (B), and GST (C) in natural populations of *An. gambiae* from Vallée du Kou (VK7) compared to that of *An. gambiae* "Kisumu" (susceptible reference strain). Note the over-expression of oxydases and GST in the VK7 sample.

Trends in Insecticide Resistance in Natural Populations

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 491

absence of insecticide. There are evidences that the same phenomenon exists also in *An. gambiae s.s* (Djogbenou *et al*., 2010). Even though the results of bioassays were more recent

This mutation was distributed throughout the Sudan and Sudan-Sahelian localities reaching relative high frequencies (0.6) in the South-West, moderate frequencies (<50%) in the central region, and being absent in the Sahel. It was far more frequent in the S form than in the sympatric M mosquitoes (averaging in mean 0.32 for the S form *vs.* 0.036 for the M form) (Djogbenou *et al*., 2008a). Even though the *ace-1R* mutation was spread across two climatic zones, it was recorded mostly in the cotton growing areas (Dabiré *et al*., 2009b). Although the *ace-1R* mutation was less spread within the *An. gambiae* s.s. M form, the highest frequency (0.63) was recorded in this form at Houndé located just on the limit of the Sudan region in 2008 (Fig. 11). The observed genotypic frequencies were not significantly different from Hardy-Weinberg expectations at the 95% confidence level in populations from any site except in the *An. gambiae* s.s. S form population from Orodara, where an excess of heterozygotes was observed. S-form samples from a number of other sites also showed a higher than expected number of heterozygous genotypes including Banfora (expected 10, observed 13), Diebougou (expected 11, observed 16) and VK7 (expected 12, observed 17) although Hardy–Weinberg equilibrium was not rejected. These results suggest that a fitness cost is associated to this mutation (Labbé *et al.*, 2007), but see the next paragraph. No *An.* 

(2009) we presented only the *ace-1R* frequencies from 2006 to 2008 samples.

*arabiensis* was detected up today carrying the *ace-1R* mutation.

**GHANA**

Fig. 11. Geographic distribution of *ace-1R* allele in *An. gambiae s.l*. populations inducing OP/CX resistance profile in Burkina Faso in 2008 [numbers in bracket represent percentage of *ace-1R* allele frequencies]. No *An. arabiensis* was found carrying ace-1R allele so we did not

*An. gambiae S* form *An. gambiae M* form *An. arabiensis*

**Manga Kombissiri [0.03][0]**

[43] frequency of *ace.1R* in S form

[63] frequency of *ace.1R* in M form

**Boromo**

**[36][0]**

**[39]**

**[15]**

**Ouagadougou Kaya**

**[0]**[0]

**[0]**[0]

**Gaoua**

**[39]**

**[24]**[0]

**Batié**

**Houndé**

**Diébougou**

**Soumousso**

**[42]**

**[3,1] [63]**

**Nanoro Nou**

**[17][0]**

**[35]**

**[1]**

**Dédougou**

**[43]**

**na**

**TOGO**

**Dori**

**[0]**

**Koubri Koupela**

**[0] [0] [0]**

**[0.9]**[0]

**Climatic regions Study sites (21)**

**NIGER**

**BENIN**

Sahelian Sudan- sahelian Sudanian

**Fada**

**[-]**

Rice areas since 1972

Cotton areas since 1968

**COTE D'IVOIRE**

represent its frequency for this species.

**Orodara**

**VK5 VK7** 

**[40]**

**Banfora**

**[35]**

[9] **[37]**

**[38]**

**[20]**

**Niangoloko**

Echelle: 0 50 100 km

**MALI**

Fig. 9. Mortality rates of *An. gambiae s.l*. populations exposed to Chlorpyriphos methyl (CM) 0.04 %, carbofuran 0.04% and fenithrotion 0.04% from four sites located on the cotton belt in South west of Burkina Faso [100-98%=susceptible; 98-80%= suspected resistance; <80%=resistant].

Fig. 10. Mortality rates of *An. gambiae s.l.* populations exposed to bendiocarb 0.1% from Sudan, Sudan-sahelian and sahelian areas in Burkina Faso.

Fenithrotion 0.4% Carbosulfan 0.4% CM 0.4%

Mortality **%**

Fig. 9. Mortality rates of *An. gambiae s.l*. populations exposed to Chlorpyriphos methyl (CM) 0.04 %, carbofuran 0.04% and fenithrotion 0.04% from four sites located on the cotton belt in

**Bendiocarb 0.1%**

Localities **Susceptible Suspected resistant Resistant**

0 10 20 30 40 50 60 70 80 90 100

Mortality **%**

Fig. 10. Mortality rates of *An. gambiae s.l.* populations exposed to bendiocarb 0.1% from

South west of Burkina Faso [100-98%=susceptible; 98-80%= suspected resistance;

Orodara

Orodara

Houndé Boromo Dédougou Nouna Solenzo VK7 VK5 Dioulassobâ Soumousso Diébougou Dano Banfora Sidéradougou Yamtenga Fada Koupéla Kompienga Kombissiri Manga

Sudan, Sudan-sahelian and sahelian areas in Burkina Faso.

<80%=resistant].

Hounde

Soumousso

Diebougou **Localities**

0 20 40 60 80 100

absence of insecticide. There are evidences that the same phenomenon exists also in *An. gambiae s.s* (Djogbenou *et al*., 2010). Even though the results of bioassays were more recent (2009) we presented only the *ace-1R* frequencies from 2006 to 2008 samples.

This mutation was distributed throughout the Sudan and Sudan-Sahelian localities reaching relative high frequencies (0.6) in the South-West, moderate frequencies (<50%) in the central region, and being absent in the Sahel. It was far more frequent in the S form than in the sympatric M mosquitoes (averaging in mean 0.32 for the S form *vs.* 0.036 for the M form) (Djogbenou *et al*., 2008a). Even though the *ace-1R* mutation was spread across two climatic zones, it was recorded mostly in the cotton growing areas (Dabiré *et al*., 2009b). Although the *ace-1R* mutation was less spread within the *An. gambiae* s.s. M form, the highest frequency (0.63) was recorded in this form at Houndé located just on the limit of the Sudan region in 2008 (Fig. 11). The observed genotypic frequencies were not significantly different from Hardy-Weinberg expectations at the 95% confidence level in populations from any site except in the *An. gambiae* s.s. S form population from Orodara, where an excess of heterozygotes was observed. S-form samples from a number of other sites also showed a higher than expected number of heterozygous genotypes including Banfora (expected 10, observed 13), Diebougou (expected 11, observed 16) and VK7 (expected 12, observed 17) although Hardy–Weinberg equilibrium was not rejected. These results suggest that a fitness cost is associated to this mutation (Labbé *et al.*, 2007), but see the next paragraph. No *An. arabiensis* was detected up today carrying the *ace-1R* mutation.

Fig. 11. Geographic distribution of *ace-1R* allele in *An. gambiae s.l*. populations inducing OP/CX resistance profile in Burkina Faso in 2008 [numbers in bracket represent percentage of *ace-1R* allele frequencies]. No *An. arabiensis* was found carrying ace-1R allele so we did not represent its frequency for this species.

Trends in Insecticide Resistance in Natural Populations

duplication modifies the fitness of its carriers.

populations.

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 493

observed on the littoral of Ivory Coast with high frequencies in the M form, and may be

If, as suspected by Labbé *et al.* (2007), *ace-1D* allele has a lower fitness cost than *ace-1R*, it would increase dramatically the diffusion of OP and CX resistance in *An. gambiae s.s.* natural

Presently, there is no simple test to characterized the duplicated allele *ace-1D* as diagnostic tests do not discriminate between heterozygotes *ace-1S/ace-1R* and genotypes with the *ace-1D* allele. Formal identification of *ace-1D* in a field female thus necessitate to cross this female with a susceptible male, and screen its offspring for CX resistance (see Labbé *et al*. 2007 for detailed procedure). This is clearly not possible on a large number of specimens and to better address the role and the impact of the *ace-1* duplication in resistance schemes, it appears urgent to build an *ace-1D* homozygous laboratory strain to investigate how the

Fig. 13. Ag- *ace-1D* frequency in Western Africa. The frequency is given for each *An. gambiae* molecular form: S (red) and M blue). Samples in which *ace-1D* was detected by molecular analysis are bolded and underlined. Significant presence of the duplicated allele (before Bonferroni correction) is given with \* for *P* < 0.05, \*\* for *P* < 0.01 and \*\*\* for *P* < 0.001.

(Figure from Djogbenou *et al*., 2009 in Malaria Journal)

present at a low frequency in Benin and Togo (Djogbenou *et al*., 2010) (Fig. 13).

Fig. 12. Evolution of allelic frequencies of *ace-1R* in natural populations of *An. gambiae s.s.* in Sudan area (cotton belt) from 2006 to 2008 in Burkina Faso.

The frequency evolution of this allele during the two years is not regular (fig.12), but considering only the Sudan area, it seems to decrease within the S form and increase slightly in the M form from 2006 to 2008 (Fig. 12). As no data existed before 2006, we did not know the trait of evolution of this allele in the past. That should greatly contribute to explain the inverse tendency of this allele within the two forms because the resistance pattern is complex in this area where excess of heterozygous for *ace-1R* allele should probably co-exist with the duplication allele Ag-*ace-1D (*see the next paragraph). However the allele frequencies in the two forms need to be compared statistically from a solid sample sizes. Regular monitoring of the same localities with the same protocols should give a better insight of the evolution of the G119S mutation of the *ace-1* gene. Data will have to be analyzed in relation with the possible coexistence of other resistance mechanisms such as *kdr* mutation or metabolic based resistance as well as with the existence of the duplicated allele Ag-*ace-1D* which may decrease the fitness cost of this mutation (Berticat *et al*., 2008).

#### **6. Duplication of** *ace-1<sup>D</sup>*  **allele in** *An. gambiae s.s.* **from Burkina Faso**

The G119S mutation conferring resistance to organophosphates and carbamates was distributed throughout the Sudan and Sudan-sahelian correlated with the cotton growing areas. This mutation has been identified in the *ace-1R* allele, and recently in a "duplicated allele" (*ace-1D*), putting in tandem a susceptible and a resistant copy of *ace-1* on the same chromosome. The ace*-1D* has been recorded in field populations of *An. gambiae* M and S forms and was shown to have come from the same duplication event in both forms (Djogbenou *et al*., 2008b). A unique *ace-1D* allele has been observed in Côte d'Ivoire and Burkina Faso, with an estimated frequency >50% in some populations (Djogbenou *et al.*, 2009).

In Burkina Faso, the *ace-1D* allele frequency could reach 50% and is mainly present in the S form principally in the old cotton belt in the South West. The duplicated allele was also

*An. gambiae* S-form *An. gambiae* M-form

2006 2008 **Years**

 **allele in** *An. gambiae s.s.* **from Burkina Faso** 

Fig. 12. Evolution of allelic frequencies of *ace-1R* in natural populations of *An. gambiae s.s.* in

The frequency evolution of this allele during the two years is not regular (fig.12), but considering only the Sudan area, it seems to decrease within the S form and increase slightly in the M form from 2006 to 2008 (Fig. 12). As no data existed before 2006, we did not know the trait of evolution of this allele in the past. That should greatly contribute to explain the inverse tendency of this allele within the two forms because the resistance pattern is complex in this area where excess of heterozygous for *ace-1R* allele should probably co-exist with the duplication allele Ag-*ace-1D (*see the next paragraph). However the allele frequencies in the two forms need to be compared statistically from a solid sample sizes. Regular monitoring of the same localities with the same protocols should give a better insight of the evolution of the G119S mutation of the *ace-1* gene. Data will have to be analyzed in relation with the possible coexistence of other resistance mechanisms such as *kdr* mutation or metabolic based resistance as well as with the existence of the duplicated allele

Ag-*ace-1D* which may decrease the fitness cost of this mutation (Berticat *et al*., 2008).

The G119S mutation conferring resistance to organophosphates and carbamates was distributed throughout the Sudan and Sudan-sahelian correlated with the cotton growing areas. This mutation has been identified in the *ace-1R* allele, and recently in a "duplicated allele" (*ace-1D*), putting in tandem a susceptible and a resistant copy of *ace-1* on the same chromosome. The ace*-1D* has been recorded in field populations of *An. gambiae* M and S forms and was shown to have come from the same duplication event in both forms (Djogbenou *et al*., 2008b). A unique *ace-1D* allele has been observed in Côte d'Ivoire and Burkina Faso, with an estimated frequency >50% in some populations (Djogbenou *et al.*,

In Burkina Faso, the *ace-1D* allele frequency could reach 50% and is mainly present in the S form principally in the old cotton belt in the South West. The duplicated allele was also

0

Sudan area (cotton belt) from 2006 to 2008 in Burkina Faso.

5

10

15

Frequency

**6. Duplication of** *ace-1<sup>D</sup>*

2009).

 of *aer-1R*

20

25

30

35

observed on the littoral of Ivory Coast with high frequencies in the M form, and may be present at a low frequency in Benin and Togo (Djogbenou *et al*., 2010) (Fig. 13).

If, as suspected by Labbé *et al.* (2007), *ace-1D* allele has a lower fitness cost than *ace-1R*, it would increase dramatically the diffusion of OP and CX resistance in *An. gambiae s.s.* natural populations.

Presently, there is no simple test to characterized the duplicated allele *ace-1D* as diagnostic tests do not discriminate between heterozygotes *ace-1S/ace-1R* and genotypes with the *ace-1D* allele. Formal identification of *ace-1D* in a field female thus necessitate to cross this female with a susceptible male, and screen its offspring for CX resistance (see Labbé *et al*. 2007 for detailed procedure). This is clearly not possible on a large number of specimens and to better address the role and the impact of the *ace-1* duplication in resistance schemes, it appears urgent to build an *ace-1D* homozygous laboratory strain to investigate how the duplication modifies the fitness of its carriers.

Fig. 13. Ag- *ace-1D* frequency in Western Africa. The frequency is given for each *An. gambiae* molecular form: S (red) and M blue). Samples in which *ace-1D* was detected by molecular analysis are bolded and underlined. Significant presence of the duplicated allele (before Bonferroni correction) is given with \* for *P* < 0.05, \*\* for *P* < 0.01 and \*\*\* for *P* < 0.001. (Figure from Djogbenou *et al*., 2009 in Malaria Journal)

Trends in Insecticide Resistance in Natural Populations

field populations needs to be more investigated.

populations.

Africa is required.

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 495

*An. funestus* like other malaria vectors is controlled by the use of insecticides such as insecticide treated materials or as indoor residual spraying (IRS). Unfortunately, *An. funestus* is increasingly developing resistance across Africa to different classes of insecticides used in public health, such as PYR, CX and DDT (Brooke *et al*., 2001; Casimiro *et al*., 2006; Cuamba *et al.,* 2010; Morgan *et al*., 2010). There are alternative agrochemicals, such as fipronil that could be introduced but the potential for cross-resistance from existing mechanisms segregating in

The insecticide resistance in *An. funestus* populations was early recorded from Burkina Faso, where resistance was found to dieldrin, a cyclodiene abundantly used in Africa in the 1960s for cotton crop protection but also for malaria vector control (Hamon *et al*., 1968b). Dieldrin resistance was also reported in *An. funestus* from Cameroon, Benin, Nigeria and Mali (Service, 1960; Toure, 1982; Brown, 1986). Recent studies have shown that *An. funestus* remains fully susceptible to all tested insecticides (DDT, PYR, OP/CX) except to dieldrin for which resistance remains high despite the fact that cyclodienes are no longer used in public health control programs (Dabire *et al*., 2007). But the distribution of this resistance across the rest of the continent is unknown and need to be clarified. The understanding of factors explaining the persistence of high levels of resistance against cyclodienes in *An. funestus* as well as the geographical distribution of this resistance across the continent has been recently addressed by Wondji *et al*., 2011. These studies indicated that *RdlR* mutation extensively reported in West and Central Africa should sustain dieldrin resistance in such *An. funestus*

**9. Pesticide pressures on disease vectors are from multiple origins** 

The question that remains to be clearly identified is the origin of insecticide pressures that select the resistances observed in mosquitoes from sub-Saharan Africa. In Burkina Faso, the emergence of the *ace-1R* mutation in *An. gambiae s.s.* populations is also associated with the insecticide treatment history with OP and CX of cotton. Since the mid 1990s and until recently a pest management strategy including four windows of treatment per cropping cycle using pyrethroids, OP/CX (such as chlorpyrifos, profenofos and trizophos) and organochlorines had been adopted in order to manage the pyrethroid-resistance of *Helicoverpa armigera* and *Bemisia tabaci* that emerged throughout the cotton belt. Some bioassays performed in 2003 on *An. gambiae* populations from four sites located in the cotton belt of western Burkina Faso revealed early resistance against CX and OP insecticides pre-empting the discovery of the genetic resistance mechanism revealed in further studies. In a previous study the agricultural use of insecticides was already implicated in the development of resistance to pyrethroids in *An. gambiae s.l.* populations. Then the geographical distribution of resistance decreased in *An. gambiae s.l.* populations from the Sudan savannah to Sahelian areas and the highest levels of resistance were found in sites of cotton cultivation. The areas under cotton cultivation have expanded dramatically in the last ten years (210,000 ha in 1996 to more than 520,000 ha in 2005). A corresponding increase in the level of insecticide use has also been reported reaching more than 3x106 litres of pesticide per cropping campaign. Furthermore, a clear knowledge concerning the practices of populations regarding the uses of insecticides in

#### **7. Multiresistance status in natural populations of** *An. gambiae s.l***.: the coexistence of** *kdr* **and** *ace-1<sup>R</sup>*  **mutations**

It was in 2005 when the detection of *ace-1R* (G119S) mutation was systematically performed for the first time in *An. gambiae s.s.* natural populations from Burkina Faso (Dabiré *et al*., 2008). This mutation was found together with *kdr* mutation within the same populations. That suggests the existence of multiresistance mechanisms occurring in the same populations. The same individuals were found carrying the two genes but the *kdr* always appeared as homozygous (*kdr*R/*kdr*R). The functional links of the two genes needed to be further investigated (Dabire *et al*., 2008). Indeed as indicated by the results of bioassays, the occurrence of such multiresistance mechanisms should explain why *An. gambiae s.s.* populations are becoming resistant to all classes of insecticides especially in the South west parts of the country. The individuals carrying the two genes appeared to be phenotypically more resistant to pyrethroids and bendiocarb than those carrying only *kdr* or *ace-1R*, respectively. Such a synergy between *kdr* and *ace-1R* has been observed in *Culex pipiens* (Berticat *et al*., 2008). Assuming that the *ace-1R* is associated to a high fitness cost (Djogbenou *et al*., 2010), it should be interesting to investigate how the *kdr* mutation influences the fitness cost related to *ace-1R*. Also, it was previously shown in *Culex pipiens* that mosquitoes carrying both *kdr* and *ace-1R* mutations suffer less cost than the one carrying only *ace-1R* (Berticat *et al*., 2008). Both this synergy between pyrethroids and OP/CX resistance mechanisms and the spread of the *ace-1D* allele in natural populations of *An. gambiae s.s.* could largely hamper the expected results of using OP/CX as alternative insecticides to the PYR becoming ineffective by the presence of *kdr*. It is crucial to build laboratory colonies carrying the two mutations from which the benefit of changing insecticides could be properly tested. More recently, in 2011, we also recorded metabolic based resistance in *An. gambiae* from Burkina Faso. Even though these results are preliminary, they further complicate the pattern of resistance in this country, and may represent a dramatic threat for malaria vector control in the near future.

In conclusion, all these aspects need to be properly addressed by fine fundamental research to decrypt the link between the resistance schemes and to give sense in vector control point of view.

## **8. Other malaria vectors**

*Anopheles funestus* belongs to a group of no less than nine species that are difficult to distinguish based solely on morphological characters of a single life stage (Gillies and Coetzee 1987, Harbach 1994). Species identification difficulties have been recently addressed by molecular techniques based on the polymerase chain reaction (PCR) by using a cocktail of species-specific primers permitting the identification of the six most common species of the group (Koekemoer *et al*., 2002). Recent analyses of rDNA sequences (Cohuet *et al*., 2003) revealed the occurrence, in West and Central Africa, of a new taxon morphologically related to *An. rivulorum* Leeson*,* which is provisionally named *An. rivulorum*-like, thereby enlarging the number of members of the *An. funestus* group to 10 species. Among all the members of the *funestus* group, *An. funestus* s.s. is the most anthropophilic species, and it is considered as the only major malaria vector (Coetzee & Fontenille, 2004), although in a Tanzanian village the circumsporozoite protein of *Plasmodium falciparum* was detected by immunological techniques in some *An. rivulorum* specimens (Wilkes *et al*. 1996).

It was in 2005 when the detection of *ace-1R* (G119S) mutation was systematically performed for the first time in *An. gambiae s.s.* natural populations from Burkina Faso (Dabiré *et al*., 2008). This mutation was found together with *kdr* mutation within the same populations. That suggests the existence of multiresistance mechanisms occurring in the same populations. The same individuals were found carrying the two genes but the *kdr* always appeared as homozygous (*kdr*R/*kdr*R). The functional links of the two genes needed to be further investigated (Dabire *et al*., 2008). Indeed as indicated by the results of bioassays, the occurrence of such multiresistance mechanisms should explain why *An. gambiae s.s.* populations are becoming resistant to all classes of insecticides especially in the South west parts of the country. The individuals carrying the two genes appeared to be phenotypically more resistant to pyrethroids and bendiocarb than those carrying only *kdr* or *ace-1R*, respectively. Such a synergy between *kdr* and *ace-1R* has been observed in *Culex pipiens* (Berticat *et al*., 2008). Assuming that the *ace-1R* is associated to a high fitness cost (Djogbenou *et al*., 2010), it should be interesting to investigate how the *kdr* mutation influences the fitness cost related to *ace-1R*. Also, it was previously shown in *Culex pipiens* that mosquitoes carrying both *kdr* and *ace-1R* mutations suffer less cost than the one carrying only *ace-1R* (Berticat *et al*., 2008). Both this synergy between pyrethroids and OP/CX resistance mechanisms and the spread of the *ace-1D* allele in natural populations of *An. gambiae s.s.* could largely hamper the expected results of using OP/CX as alternative insecticides to the PYR becoming ineffective by the presence of *kdr*. It is crucial to build laboratory colonies carrying the two mutations from which the benefit of changing insecticides could be properly tested. More recently, in 2011, we also recorded metabolic based resistance in *An. gambiae* from Burkina Faso. Even though these results are preliminary, they further complicate the pattern of resistance in this country, and may represent a dramatic threat for

In conclusion, all these aspects need to be properly addressed by fine fundamental research to decrypt the link between the resistance schemes and to give sense in vector control point

*Anopheles funestus* belongs to a group of no less than nine species that are difficult to distinguish based solely on morphological characters of a single life stage (Gillies and Coetzee 1987, Harbach 1994). Species identification difficulties have been recently addressed by molecular techniques based on the polymerase chain reaction (PCR) by using a cocktail of species-specific primers permitting the identification of the six most common species of the group (Koekemoer *et al*., 2002). Recent analyses of rDNA sequences (Cohuet *et al*., 2003) revealed the occurrence, in West and Central Africa, of a new taxon morphologically related to *An. rivulorum* Leeson*,* which is provisionally named *An. rivulorum*-like, thereby enlarging the number of members of the *An. funestus* group to 10 species. Among all the members of the *funestus* group, *An. funestus* s.s. is the most anthropophilic species, and it is considered as the only major malaria vector (Coetzee & Fontenille, 2004), although in a Tanzanian village the circumsporozoite protein of *Plasmodium falciparum* was detected by immunological

techniques in some *An. rivulorum* specimens (Wilkes *et al*. 1996).

**7. Multiresistance status in natural populations of** *An. gambiae s.l***.: the** 

 **mutations** 

**coexistence of** *kdr* **and** *ace-1<sup>R</sup>*

malaria vector control in the near future.

**8. Other malaria vectors** 

of view.

*An. funestus* like other malaria vectors is controlled by the use of insecticides such as insecticide treated materials or as indoor residual spraying (IRS). Unfortunately, *An. funestus* is increasingly developing resistance across Africa to different classes of insecticides used in public health, such as PYR, CX and DDT (Brooke *et al*., 2001; Casimiro *et al*., 2006; Cuamba *et al.,* 2010; Morgan *et al*., 2010). There are alternative agrochemicals, such as fipronil that could be introduced but the potential for cross-resistance from existing mechanisms segregating in field populations needs to be more investigated.

The insecticide resistance in *An. funestus* populations was early recorded from Burkina Faso, where resistance was found to dieldrin, a cyclodiene abundantly used in Africa in the 1960s for cotton crop protection but also for malaria vector control (Hamon *et al*., 1968b). Dieldrin resistance was also reported in *An. funestus* from Cameroon, Benin, Nigeria and Mali (Service, 1960; Toure, 1982; Brown, 1986). Recent studies have shown that *An. funestus* remains fully susceptible to all tested insecticides (DDT, PYR, OP/CX) except to dieldrin for which resistance remains high despite the fact that cyclodienes are no longer used in public health control programs (Dabire *et al*., 2007). But the distribution of this resistance across the rest of the continent is unknown and need to be clarified. The understanding of factors explaining the persistence of high levels of resistance against cyclodienes in *An. funestus* as well as the geographical distribution of this resistance across the continent has been recently addressed by Wondji *et al*., 2011. These studies indicated that *RdlR* mutation extensively reported in West and Central Africa should sustain dieldrin resistance in such *An. funestus* populations.

## **9. Pesticide pressures on disease vectors are from multiple origins**

The question that remains to be clearly identified is the origin of insecticide pressures that select the resistances observed in mosquitoes from sub-Saharan Africa. In Burkina Faso, the emergence of the *ace-1R* mutation in *An. gambiae s.s.* populations is also associated with the insecticide treatment history with OP and CX of cotton. Since the mid 1990s and until recently a pest management strategy including four windows of treatment per cropping cycle using pyrethroids, OP/CX (such as chlorpyrifos, profenofos and trizophos) and organochlorines had been adopted in order to manage the pyrethroid-resistance of *Helicoverpa armigera* and *Bemisia tabaci* that emerged throughout the cotton belt. Some bioassays performed in 2003 on *An. gambiae* populations from four sites located in the cotton belt of western Burkina Faso revealed early resistance against CX and OP insecticides pre-empting the discovery of the genetic resistance mechanism revealed in further studies. In a previous study the agricultural use of insecticides was already implicated in the development of resistance to pyrethroids in *An. gambiae s.l.* populations. Then the geographical distribution of resistance decreased in *An. gambiae s.l.* populations from the Sudan savannah to Sahelian areas and the highest levels of resistance were found in sites of cotton cultivation. The areas under cotton cultivation have expanded dramatically in the last ten years (210,000 ha in 1996 to more than 520,000 ha in 2005). A corresponding increase in the level of insecticide use has also been reported reaching more than 3x106 litres of pesticide per cropping campaign. Furthermore, a clear knowledge concerning the practices of populations regarding the uses of insecticides in Africa is required.

Trends in Insecticide Resistance in Natural Populations

decision process based on social and cultural contexts.

these vector control tools.

threaten these strategies.

the insecticides used for control.

*Evolutionary Biology* 8.

*Res* 91: 265-272.

*Control Assoc* 2: 123-140.

*infectious Diseases* 4: 605-613.

**12. Acknowlegements** 

**13. References** 

of Malaria Vectors in Burkina Faso, West Africa: 10 Years' Surveys 497

in the implementation of control interventions that will preserve a long term efficacy of

Unfortunately reports of insecticide resistance in vector populations increase year by year and could jeopardize malaria vector control based on the use of insecticides. The use of insecticides for bednets impregnation or for IRS represents the primary means for malaria prevention worldwide. However the efficacy of such tools has been evaluated in areas where vectors are susceptible to insecticides. Moreover, mosquito resistance is not only due to the insecticides used for mosquito control, but to the many pesticide pollutions present in their environment which are generated by a large variety of human activities necessitating insect control for agriculture (large cultures, fruits and vegetables), animal and other household protections. These pollutions may dramatically affect resistance genes dynamics and

The overall pesticide pressures that select resistance in mosquitoes need to be clarified, both in terms of insecticides usage and quantity. It is also crucial to improve our knowledge on the practices of people regarding the use of insecticides and the reasons underlying their

Malaria vector control programs require up-to-date information on the distribution and composition of mosquito vector populations and the susceptibility of these populations to

Authors are grateful to Corus 6015 and National Malaria Control Programme of Burkina Faso which supported financially this study on resistance monitoring. We thank Nicole Pasteur for critical reading of the manuscript. We thank also *Malaria Journal* to have agreed

[1] Awolola TS, Oyewole IO, Amajoh CN, Idowu ET, Ajayi MB, Oduala A, *et al*., 2005.

[2] Beier J, Keating J, Githure J, Macdonald M, Impoinvil D, Novak R, 2008. Integrated

[3] Berticat C, Bonnet J, Duchon S, Agnew P, Weill M, Corbel V, 2008. Costs and benefits of

[4] Brogdon WG, McAllister JC, 1998. Insecticide resistance and vector control. *Emerging* 

[5] Brooke BD, Kloke G, Hunt RH, Koekemoer LL, Temu EA, Taylor ME, Small G,

[6] Brown AWA, 1986. Insecticide resistance in mosquitoes: a pragmatic review. J *Am Mosq* 

Distribution of the molecular forms of *Anopheles gambiae* and pyrethroids knock

multiple resistance to insecticides for *Culex quinquefasciatus* mosquitoes. *BMC* 

Hemingway J, Coetzee M, 2001. Bioassay and biochemical analyses of insecticide resistance in southern African *Anopheles funestus* (Diptera: Culicidae). *Bull Entomol* 

the use of figure published in *Malaria Journal* 2009, 8:70 doi:10.1186/1475-2875-8-70.

down resistance gene in Nigeria. *Acta Tropica* 95: 204-09

vector management for malaria control. *Malaria Journal* 7: S4.

## **10. Insecticide resistance and malaria vector control**

Although several studies in Ivory Coast and Burkina Faso had shown that ITNs may still achieve good control of PYR-target resistant *An. gambiae s.s.* populations (Darriet *et al*., 2000; Henry *et al*., 2005; Dabiré *et al*., 2006), recent results from experimental hut trials conducted in Southern Benin with lambdacyhalothrin (PYR) suggested that such ITNs may fail to control these field populations (Ngessan *et al*., 2007). Failure of indoor residual house spraying (IRS) with deltamethrin (PYR) had also occurred in South Africa where the malaria vector *An. funestus* had developed PYR-metabolic resistance. A recent study in Bioko Island (Equatorial Guinea) reported a failure of indoor residual spraying with deltamethrin on *An*. *gambiae* populations of the M molecular form carrying the Leu-Phe *kdr* mutation at a high frequency. These *kdr*-pyrethroid resistant populations were controlled after the introduction of a carbamate insecticide in IRS (Sharp *et al*., 2007). Thus the malaria outbreak in this country was only brought under control after reversion to DDT spraying (organochlorine insecticide).

A concern for the potential use of OP and CX as alternative for PYR is that target and metabolic resistances to these insecticides are already present in some *An. gambiae s.s.* populations in West Africa especially in Burkina Faso. Several studies have suggested that the use of agricultural pesticides, especially for cotton but also for vegetable crops, favored the emergence and facilitated the spread of insecticide resistance within mosquito populations. Other studies have given evidence for the selection of *kdr* alleles associated with the use of pyrethroids in ITNs and other domestic strategies of personal protection, especially in Kenya and Niger (Czeher *et al*., 2008). In countries supported by PMI (President Initiative against Malaria) such as Ghana, Senegal, Mali, Benin and Liberia in West Africa the large-scale pilot interventions implemented with bendiocarb 400mg/m2 in IRS could also contribute to select the OP/CX resistance. However, as no global health control program in Africa used OP and CX for mosquito control, it is necessary to clearly identify the origin of insecticide pressure that select these resistances.

## **11. Conclusions**

Reports of insecticide resistance in malaria vectors in West Africa especially in Burkina Faso indicate that insecticide resistance increases year after year and highlight the threat to the effectiveness of vector control strategies. In fact *An. gambiae s.l*. populations in Burkina Faso, and more broadly in West Africa, have evolved resistance to many of the insecticides classes used for vector control. Resistance may be conferred by target-site insensitivity such as *kdr* and *ace-1R*, other metabolic mechanisms or a combination of all as *kdr* and *ace-1R* resistance mechanisms occur concomitantly in the same populations of *An. gambiae* s.s. in the South-Western region of the country.

In conclusion the geographical distribution of insecticide resistance in *An. gambiae s.l.* populations was found in sites of cotton cultivation and vegetable in urban settlement that has expanded dramatically in the last ten years. But the role of agriculture in the selection of resistance in natural mosquito's populations needs to be clarified, both in terms of insecticides usage and quantity in order to devise strategies that may help to reduce the extension of resistance. Until the discovery of new insecticides or using new formulations of existing insecticides and also the use of genetically modified mosquitoes (GMM) and sterilised males techniques (SIT), it is crucial to integrate the regional vector resistance status in the implementation of control interventions that will preserve a long term efficacy of these vector control tools.

Unfortunately reports of insecticide resistance in vector populations increase year by year and could jeopardize malaria vector control based on the use of insecticides. The use of insecticides for bednets impregnation or for IRS represents the primary means for malaria prevention worldwide. However the efficacy of such tools has been evaluated in areas where vectors are susceptible to insecticides. Moreover, mosquito resistance is not only due to the insecticides used for mosquito control, but to the many pesticide pollutions present in their environment which are generated by a large variety of human activities necessitating insect control for agriculture (large cultures, fruits and vegetables), animal and other household protections. These pollutions may dramatically affect resistance genes dynamics and threaten these strategies.

The overall pesticide pressures that select resistance in mosquitoes need to be clarified, both in terms of insecticides usage and quantity. It is also crucial to improve our knowledge on the practices of people regarding the use of insecticides and the reasons underlying their decision process based on social and cultural contexts.

Malaria vector control programs require up-to-date information on the distribution and composition of mosquito vector populations and the susceptibility of these populations to the insecticides used for control.

## **12. Acknowlegements**

Authors are grateful to Corus 6015 and National Malaria Control Programme of Burkina Faso which supported financially this study on resistance monitoring. We thank Nicole Pasteur for critical reading of the manuscript. We thank also *Malaria Journal* to have agreed the use of figure published in *Malaria Journal* 2009, 8:70 doi:10.1186/1475-2875-8-70.

## **13. References**

496 Insecticides – Pest Engineering

Although several studies in Ivory Coast and Burkina Faso had shown that ITNs may still achieve good control of PYR-target resistant *An. gambiae s.s.* populations (Darriet *et al*., 2000; Henry *et al*., 2005; Dabiré *et al*., 2006), recent results from experimental hut trials conducted in Southern Benin with lambdacyhalothrin (PYR) suggested that such ITNs may fail to control these field populations (Ngessan *et al*., 2007). Failure of indoor residual house spraying (IRS) with deltamethrin (PYR) had also occurred in South Africa where the malaria vector *An. funestus* had developed PYR-metabolic resistance. A recent study in Bioko Island (Equatorial Guinea) reported a failure of indoor residual spraying with deltamethrin on *An*. *gambiae* populations of the M molecular form carrying the Leu-Phe *kdr* mutation at a high frequency. These *kdr*-pyrethroid resistant populations were controlled after the introduction of a carbamate insecticide in IRS (Sharp *et al*., 2007). Thus the malaria outbreak in this country was only brought under control after reversion to DDT spraying (organochlorine

A concern for the potential use of OP and CX as alternative for PYR is that target and metabolic resistances to these insecticides are already present in some *An. gambiae s.s.* populations in West Africa especially in Burkina Faso. Several studies have suggested that the use of agricultural pesticides, especially for cotton but also for vegetable crops, favored the emergence and facilitated the spread of insecticide resistance within mosquito populations. Other studies have given evidence for the selection of *kdr* alleles associated with the use of pyrethroids in ITNs and other domestic strategies of personal protection, especially in Kenya and Niger (Czeher *et al*., 2008). In countries supported by PMI (President Initiative against Malaria) such as Ghana, Senegal, Mali, Benin and Liberia in West Africa the large-scale pilot interventions implemented with bendiocarb 400mg/m2 in IRS could also contribute to select the OP/CX resistance. However, as no global health control program in Africa used OP and CX for mosquito control, it is necessary to clearly identify

Reports of insecticide resistance in malaria vectors in West Africa especially in Burkina Faso indicate that insecticide resistance increases year after year and highlight the threat to the effectiveness of vector control strategies. In fact *An. gambiae s.l*. populations in Burkina Faso, and more broadly in West Africa, have evolved resistance to many of the insecticides classes used for vector control. Resistance may be conferred by target-site insensitivity such as *kdr* and *ace-1R*, other metabolic mechanisms or a combination of all as *kdr* and *ace-1R* resistance mechanisms occur concomitantly in the same populations of *An. gambiae* s.s. in the South-

In conclusion the geographical distribution of insecticide resistance in *An. gambiae s.l.* populations was found in sites of cotton cultivation and vegetable in urban settlement that has expanded dramatically in the last ten years. But the role of agriculture in the selection of resistance in natural mosquito's populations needs to be clarified, both in terms of insecticides usage and quantity in order to devise strategies that may help to reduce the extension of resistance. Until the discovery of new insecticides or using new formulations of existing insecticides and also the use of genetically modified mosquitoes (GMM) and sterilised males techniques (SIT), it is crucial to integrate the regional vector resistance status

**10. Insecticide resistance and malaria vector control** 

the origin of insecticide pressure that select these resistances.

insecticide).

**11. Conclusions** 

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**23** 

*Portugal* 

**The Role of** *Anopheles gambiae*  **P450 Cytochrome in Insecticide** 

*Higiene e Medicina Tropical, Universidade Nova de Lisboa* 

*Centro de Malária e Outras Doenças Tropicais, UEI Parasitologia Médica, Instituto de* 

*Anopheles gambiae* is the major vector of malaria transmission in sub-Saharan Africa where the disease is responsible for the highest morbidity and mortality worldwide. Malaria, nowadays, is still a major burden causing the death of nearly one million people each year, mostly children under the age of five, and affecting those living in the poorest countries

Currently, the major obstacles to malaria eradication are the absence of a protective vaccine, the spread of parasite resistance to anti-malarial drugs and the mosquito resistance to insecticides. Controlling mosquito vectors is fundamental to reduce mosquito-borne diseases. In fact, it has been one of the most used and effective method to prevent malaria, namely trough insecticides spraying and impregnated bed nets. These methods are highly dependent on a single class of insecticides, the pyrethroids, which are the most frequently used compounds for indoor residual spraying, and the only insecticide class used for insecticide treated nets (WHO, 2010). The extensive use of a single class of insecticides further increases the risk of mosquitoes developing resistance, which could rapidly lead to a major public health problem mainly in sub-Saharan countries where insecticidal vector control is being used widely (WHO, 2010). Strategies to control malaria are still not enough to totally eliminate malaria transmission, having yet to overcome several difficulties as the development of parasite drug resistance and mosquito-vector insecticide resistance (Yassine & Osta, 2010). Unfortunately the emergence of mosquito populations capable of withstanding insecticide exposure is threatening the efficiency of these control measures.

Resistance has been defined as 'the inherited ability of a strain of some organisms to survive doses of a toxicant that would kill the majority of individuals in a normal population of the same species' (Scott, 1999). The evolution of insecticide-resistant mosquito strains is an increasing problem and one of the major obstacles for the control of medical and agricultural arthropod pests. Therefore, a better understanding of its genetic and biological basis is critical. Insecticide resistance can also lead to outbreaks of human diseases when

**1. Introduction** 

(World Health Organization [WHO], 2010).

**2. Insecticide resistance** 

**Resistance and Infection** 

Rute Félix and Henrique Silveira


## **The Role of** *Anopheles gambiae*  **P450 Cytochrome in Insecticide Resistance and Infection**

Rute Félix and Henrique Silveira

*Centro de Malária e Outras Doenças Tropicais, UEI Parasitologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa Portugal* 

## **1. Introduction**

502 Insecticides – Pest Engineering

[63] Wondji CS, Dabire KR, Tukur Z, Irving H, Djouaka D, Morgan JC, 2011. Identification

malaria vector *Anopheles funestus* in Africa. *Insect Bioch Mol Biol* 41: 484-491 [64] World Health Organisation Test procedures for insecticides resistance monitoring in

WHO/CDS/MAL, 1998, 12p

and distribution of a GABA receptor mutation conferring dieldrin resistance in the

malaria vectors, bio-efficacy and persistence of insecticides treated surfaces.

*Anopheles gambiae* is the major vector of malaria transmission in sub-Saharan Africa where the disease is responsible for the highest morbidity and mortality worldwide. Malaria, nowadays, is still a major burden causing the death of nearly one million people each year, mostly children under the age of five, and affecting those living in the poorest countries (World Health Organization [WHO], 2010).

Currently, the major obstacles to malaria eradication are the absence of a protective vaccine, the spread of parasite resistance to anti-malarial drugs and the mosquito resistance to insecticides. Controlling mosquito vectors is fundamental to reduce mosquito-borne diseases. In fact, it has been one of the most used and effective method to prevent malaria, namely trough insecticides spraying and impregnated bed nets. These methods are highly dependent on a single class of insecticides, the pyrethroids, which are the most frequently used compounds for indoor residual spraying, and the only insecticide class used for insecticide treated nets (WHO, 2010). The extensive use of a single class of insecticides further increases the risk of mosquitoes developing resistance, which could rapidly lead to a major public health problem mainly in sub-Saharan countries where insecticidal vector control is being used widely (WHO, 2010). Strategies to control malaria are still not enough to totally eliminate malaria transmission, having yet to overcome several difficulties as the development of parasite drug resistance and mosquito-vector insecticide resistance (Yassine & Osta, 2010). Unfortunately the emergence of mosquito populations capable of withstanding insecticide exposure is threatening the efficiency of these control measures.

## **2. Insecticide resistance**

Resistance has been defined as 'the inherited ability of a strain of some organisms to survive doses of a toxicant that would kill the majority of individuals in a normal population of the same species' (Scott, 1999). The evolution of insecticide-resistant mosquito strains is an increasing problem and one of the major obstacles for the control of medical and agricultural arthropod pests. Therefore, a better understanding of its genetic and biological basis is critical. Insecticide resistance can also lead to outbreaks of human diseases when

The Role of *Anopheles gambiae* 

*GSTE2-2* was able to metabolise DDT (Ortelli et al., 2003).

Kasai & Scott, 2000; Sabourault et al., 2001).

**3. Insect P450 cytochromes** 

proteins.

P450 Cytochrome in Insecticide Resistance and Infection 505

Karunaratne, 1998). There are many reports of over expression of carboxylesterases in insecticide resistant mosquitoes including *A. gambiae*, where enhanced production of carboxylesterases was observed in permethrin-resistant mosquitoes (Vulule et al., 1999). Glutathione S-transferases are a major class of detoxification enzymes that possess a wide range of substrates specificities (Enayati et al., 2005). Elevated GST activity has been implicated in resistance to several classes of insecticides (Ranson et al., 2001). Higher enzyme activity is usually due to an increase in the amount of one or more enzymes, either as a result of gene amplification or more commonly through increases in transcriptional rate, rather than qualitative changes in individual enzymes (Hemingway et al., 2004). The primary function of GSTs is the detoxification of both endogenous and xenobiotic compounds either directly or by catalysing the secondary metabolism of a vast array of compounds oxidised by P450 cytochromes (Wilce & Parker, 1994). GST enzymes metabolise insecticides by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione to produce water soluble metabolites that are more readily excreted (Wilce & Parker, 1994). They also contribute to the removal of toxic oxygen free radical species produced through the action of pesticides (Enayati et al., 2005). In *A. gambiae*  elevated GST levels were shown to be associated with DDT resistance (Ranson et al., 2001). Furthermore genetic mapping of the major *loci* conferring DDT resistance in *A. gambiae*  implicate both *cis*- and *trans*-acting factors in the overexpression of GSTs (Ranson et al., 2000b). GSTs in *A. gambiae* were over expressed in a DDT-resistant strain, but only one

P450 cytochromes are a complex family of enzymes that are involved in the metabolism of xenobiotics and have a role in the endogenous metabolism. P450 cytochromes mediated resistance is probably the most frequent type of insecticide resistance. They are involved in the metabolism of virtually all insecticides, leading to activation of the molecule in the case of organophosphorus insecticides, or more generally to detoxification (Scott & Wen, 2001). In most cases where a link between insecticide resistance and elevated P450 activity has been shown, the P450 cytochrome belongs to the *CYP6* family (Nikou et al., 2003; Djouaka et al., 2008; Müller et al., 2007; McLaughlin et al., 2008). Although being difficult the identification of the specific P450 cytochrome associated with resistance, several P450 cytochromes were already isolated from insecticide resistant strains (Dunkov, et al., 1997;

P450 Cytochromes are hemoproteins which act as terminal oxidases in monooxygenase systems. P450 cytochromes, whose name originated on its characteristic absorbance peak at 450 nm that appears when these enzymes are reduced and saturated with carbon-monoxide, constitute one of the oldest and largest super families of enzymes being found in almost all living organisms. In the literature, P450 enzymes are known by several names: cytochromes P450 monooxigenases, mixed functions oxidases, microsomal oxidases and heme thiolate

Insect P450s play a critical role in the metabolism of a wide variety of endogenous and exogenous compounds such as steroids, fatty acids and a wide range of xenobiotics and have also been implicated in vital processes like growth, development, feeding, reproduction, insecticide resistance and tolerance to plant toxins (Feyereisen, 1999; Scott et al., 1998; Scott, 1999). P450 cytochromes are also intimately involved in the synthesis and

vectors cannot be controlled. Hence, the elucidation of resistance mechanisms is extremely important for the development of tools to monitor resistance in populations, thereby contributing to mosquito control programs. Although the mechanisms by which insecticides become less effective are similar across all vector taxa, each resistance problem is potentially unique and may involve a complex pattern of resistance *foci* (Brogdon & McAllister, 1998).

The main forms of resistance mechanisms can be divided in two groups: target site resistance, which occurs when the insecticide no longer binds to its target, and metabolic resistance, which occurs when enhanced levels of modified activities of detoxification enzymes prevent the insecticide from reaching its site of action. Alone or in combination these mechanisms confer resistance, sometimes at high levels, to all classes of insecticides.

#### **2.1 Target site resistance**

Target site resistance is based on alterations of amino acids in the site of action where the insecticide is supposed to bind, causing the insecticide to be less effective or ineffective at all. Knock down resistance (*Kdr*) occurs due to a single or multiple substitutions in the sodium channel (Martinez-Torres et al., 1998; Ranson et al., 2000a); and alteration in acetylcholinesterase results in decreased sensitivity to insecticides (Mutero et al., 1994). Insecticide resistance has been reported from many insects including *A. gambiae* that showed the presence of insensitive acetylcholinesterase in two different populations that were resistant to carbosulfan, a carbamate insecticide (N'Guessan et al., 2003). Mutations at a single codon in the *Rdl* (resistance to dieldrin) gene have been documented in all dieldrinresistant insects, and confer both insensitivity to the insecticide and a decrease rate of desensitisation (ffrench-Constant et al., 1998). However, in *A. gambiae* this type of resistance mechanism has not been described so far. Those are examples of target site resistance that is not the object of the present review.

#### **2.2 Metabolic resistance**

Metabolic resistance usually involves over-expression of enzymes capable of detoxifying insecticides or modifications in the amino acid sequences that cause alterations in the levels and activity of detoxifying proteins. There are three major enzyme families involved in this type of resistance, glutathione-S-transferases (GST), carboxylesterases and P450 cytochromes. Carboxylesterases are mainly involved in organophosphate and carbamate and to a lesser extent in pyrethroid resistance, while P450 cytochromes are mainly involved in the metabolism of pyrethoids and to a lesser extent, detoxification of organophosphates and carbamates (Hemingway & Ranson, 2000). Glutathione S-transferases are involved in the detoxification of a wide range of xenobiotics, including the organochloride insecticide DDT (Enayati et al., 2005). In *A. gambiae* metabolic resistance to insecticides can be conferred by elevation in the activity of these three classes of detoxifying enzymes.

The over-expression of carboxylesterases as an evolutionary response to organophosphorus and carbamate insecticide selection pressure has been reported in several insects, including mosquitoes (Newcomb et al., 1997; Vulule et al., 1999; Zhu et al., 1999). Organophosphorus and carbamate inhibit B esterases by rapid esterification of the serine residue in the active site, usually followed by a slow hydrolysis of the new ester bond. Therefore, these insecticides can be considered as inhibitors of esterases, because they are poor substrates which have a high affinity for these enzymes (Hemingway & Karunaratne, 1998). Carboxylesterases in large amounts causes resistance as the insecticides are rapidly sequestered, even before reaching the target-site acetylcholinesterase (Hemingway &

vectors cannot be controlled. Hence, the elucidation of resistance mechanisms is extremely important for the development of tools to monitor resistance in populations, thereby contributing to mosquito control programs. Although the mechanisms by which insecticides become less effective are similar across all vector taxa, each resistance problem is potentially unique and may involve a complex pattern of resistance *foci* (Brogdon & McAllister, 1998). The main forms of resistance mechanisms can be divided in two groups: target site resistance, which occurs when the insecticide no longer binds to its target, and metabolic resistance, which occurs when enhanced levels of modified activities of detoxification enzymes prevent the insecticide from reaching its site of action. Alone or in combination these mechanisms confer resistance, sometimes at high levels, to all classes of insecticides.

Target site resistance is based on alterations of amino acids in the site of action where the insecticide is supposed to bind, causing the insecticide to be less effective or ineffective at all. Knock down resistance (*Kdr*) occurs due to a single or multiple substitutions in the sodium channel (Martinez-Torres et al., 1998; Ranson et al., 2000a); and alteration in acetylcholinesterase results in decreased sensitivity to insecticides (Mutero et al., 1994). Insecticide resistance has been reported from many insects including *A. gambiae* that showed the presence of insensitive acetylcholinesterase in two different populations that were resistant to carbosulfan, a carbamate insecticide (N'Guessan et al., 2003). Mutations at a single codon in the *Rdl* (resistance to dieldrin) gene have been documented in all dieldrinresistant insects, and confer both insensitivity to the insecticide and a decrease rate of desensitisation (ffrench-Constant et al., 1998). However, in *A. gambiae* this type of resistance mechanism has not been described so far. Those are examples of target site resistance that is

Metabolic resistance usually involves over-expression of enzymes capable of detoxifying insecticides or modifications in the amino acid sequences that cause alterations in the levels and activity of detoxifying proteins. There are three major enzyme families involved in this type of resistance, glutathione-S-transferases (GST), carboxylesterases and P450 cytochromes. Carboxylesterases are mainly involved in organophosphate and carbamate and to a lesser extent in pyrethroid resistance, while P450 cytochromes are mainly involved in the metabolism of pyrethoids and to a lesser extent, detoxification of organophosphates and carbamates (Hemingway & Ranson, 2000). Glutathione S-transferases are involved in the detoxification of a wide range of xenobiotics, including the organochloride insecticide DDT (Enayati et al., 2005). In *A. gambiae* metabolic resistance to insecticides can be conferred

The over-expression of carboxylesterases as an evolutionary response to organophosphorus and carbamate insecticide selection pressure has been reported in several insects, including mosquitoes (Newcomb et al., 1997; Vulule et al., 1999; Zhu et al., 1999). Organophosphorus and carbamate inhibit B esterases by rapid esterification of the serine residue in the active site, usually followed by a slow hydrolysis of the new ester bond. Therefore, these insecticides can be considered as inhibitors of esterases, because they are poor substrates which have a high affinity for these enzymes (Hemingway & Karunaratne, 1998). Carboxylesterases in large amounts causes resistance as the insecticides are rapidly sequestered, even before reaching the target-site acetylcholinesterase (Hemingway &

by elevation in the activity of these three classes of detoxifying enzymes.

**2.1 Target site resistance** 

not the object of the present review.

**2.2 Metabolic resistance** 

Karunaratne, 1998). There are many reports of over expression of carboxylesterases in insecticide resistant mosquitoes including *A. gambiae*, where enhanced production of carboxylesterases was observed in permethrin-resistant mosquitoes (Vulule et al., 1999).

Glutathione S-transferases are a major class of detoxification enzymes that possess a wide range of substrates specificities (Enayati et al., 2005). Elevated GST activity has been implicated in resistance to several classes of insecticides (Ranson et al., 2001). Higher enzyme activity is usually due to an increase in the amount of one or more enzymes, either as a result of gene amplification or more commonly through increases in transcriptional rate, rather than qualitative changes in individual enzymes (Hemingway et al., 2004). The primary function of GSTs is the detoxification of both endogenous and xenobiotic compounds either directly or by catalysing the secondary metabolism of a vast array of compounds oxidised by P450 cytochromes (Wilce & Parker, 1994). GST enzymes metabolise insecticides by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione to produce water soluble metabolites that are more readily excreted (Wilce & Parker, 1994). They also contribute to the removal of toxic oxygen free radical species produced through the action of pesticides (Enayati et al., 2005). In *A. gambiae*  elevated GST levels were shown to be associated with DDT resistance (Ranson et al., 2001). Furthermore genetic mapping of the major *loci* conferring DDT resistance in *A. gambiae*  implicate both *cis*- and *trans*-acting factors in the overexpression of GSTs (Ranson et al., 2000b). GSTs in *A. gambiae* were over expressed in a DDT-resistant strain, but only one *GSTE2-2* was able to metabolise DDT (Ortelli et al., 2003).

P450 cytochromes are a complex family of enzymes that are involved in the metabolism of xenobiotics and have a role in the endogenous metabolism. P450 cytochromes mediated resistance is probably the most frequent type of insecticide resistance. They are involved in the metabolism of virtually all insecticides, leading to activation of the molecule in the case of organophosphorus insecticides, or more generally to detoxification (Scott & Wen, 2001). In most cases where a link between insecticide resistance and elevated P450 activity has been shown, the P450 cytochrome belongs to the *CYP6* family (Nikou et al., 2003; Djouaka et al., 2008; Müller et al., 2007; McLaughlin et al., 2008). Although being difficult the identification of the specific P450 cytochrome associated with resistance, several P450 cytochromes were already isolated from insecticide resistant strains (Dunkov, et al., 1997; Kasai & Scott, 2000; Sabourault et al., 2001).

## **3. Insect P450 cytochromes**

P450 Cytochromes are hemoproteins which act as terminal oxidases in monooxygenase systems. P450 cytochromes, whose name originated on its characteristic absorbance peak at 450 nm that appears when these enzymes are reduced and saturated with carbon-monoxide, constitute one of the oldest and largest super families of enzymes being found in almost all living organisms. In the literature, P450 enzymes are known by several names: cytochromes P450 monooxigenases, mixed functions oxidases, microsomal oxidases and heme thiolate proteins.

Insect P450s play a critical role in the metabolism of a wide variety of endogenous and exogenous compounds such as steroids, fatty acids and a wide range of xenobiotics and have also been implicated in vital processes like growth, development, feeding, reproduction, insecticide resistance and tolerance to plant toxins (Feyereisen, 1999; Scott et al., 1998; Scott, 1999). P450 cytochromes are also intimately involved in the synthesis and

The Role of *Anopheles gambiae* 

Figure 1.

**3.4 Characterization / function** 

initiated (Guenguerich, 1991).

P450 Cytochrome in Insecticide Resistance and Infection 507

Cytochromes P450 enzymes catalyse thousands of different reactions, which are based on the activation of molecular oxygen, with insertion of one of its atoms into the substrate, and reduction of the other to form water (Guengerich, 1991). P450s use electrons from NAD(P)H to catalyse the activation of molecular oxygen, leading to the regiospecific and stereospecific oxidative attack of structurally diverse chemicals (Werck-Reichhart & Feyereisen, 2000). The interaction that occurs between P450 cytochromes and the NADPH–cytochrome P450 reductase is better expressed as a cyclic reaction (Guenguerich, 1991) as it is depicted in

The cycle is initiated by the binding of the substrate to the ferric form of the enzyme to form an enzyme-substrate complex, followed by a reduction of the ferric complex by an electron transferred from NADPH via NADPH-cytochrome P450 reductase. Next, the binding of molecular oxygen to the reduced complex forms an enzyme-oxygen-substrate complex followed by the transference of a second electron from NADPH via NADPH-cytochrome P450 reductase or from cytochrome *b5*. A second proton is added, which results in the breaking of the oxygen-oxygen bond, releasing one atom of oxygen as water. The oxygen atom remaining is transferred to the substrate, originating an oxidized product, which is released, and a ferric form of the enzyme is once more generated. Then the cycle is re-

Fig. 1. Catalytic mechanism of P450 enzymes, where S is the substrate.

degradation of insect hormones and pheromones, including 20-hydroxyecdysone and juvenile hormone (Feyereisen, 1999).

#### **3.1 Nomenclature**

To distinguish one of these cytochromes among all the P450s, a standardized nomenclature system was implemented (Nebert et al., 1991; Nelson et al., 1996). Each P450 is named with CYP, followed by an Arabical number for the gene family, a letter for the sub-family and another Arabical number for the gene. Cytochromes P450s with share more then 40% of the amino acids are usually grouped into the same family and members with >55% of the amino acids identical are normally grouped in the same sub-family. However, there are exceptions to these rules (Nelson et al., 1996). As it is based on amino acid similarities, no information regarding the function of each P450 should be assumed from its name.

#### **3.2 Structure**

P450s can be divided into classes depending on how electrons from NAD(P)H are delivered to the catalytic site. Class I P450s are found in eukaryotes and are associated with mitochondrial membranes. This class of enzymes requires both a FAD-containing reductase and an iron sulphur redoxin, and catalyzes several steps in the biosynthesis of steroid. Class II enzymes are the most common in eukaryotes and are found in the endoplasmic reticulum. These enzymes only require an FAD/FMN-containing P450 reductase for transfer of electrons. Their functions are extremely diverse and, in eukaryotes, include aspects of the biosynthesis and catabolism of signalling molecules and steroid hormones (Feyereisen, 1999). Class III enzymes are self-sufficient and require no electron donor. They are involved in the synthesis of signalling molecules. Finally, class IV enzymes receive electrons directly from NAD(P)H. Class I and II P450s from all organisms participate in the detoxification or sometimes the activation of xenobiotics and class III and IV enzymes are considered remains of the ancestral forms of P450s involved in detoxification of damaging activated oxygen species (Werck-Reichhart & Feyereisen, 2000).

Most P450s are approximately 500 amino acids long. The core of these proteins is formed by a four-helix bundle, two sets of *β* sheets, two helices and a coil called the "meander". A characteristic consensus sequence known as the P450 "signature" FXXGXXXCXG, located on the C-terminus of the heme binding region, contains a conserved cysteine that serves as a fifth ligand to the heme iron. There are two other conserved motifs specific of the P450 proteins. One is the DGXXT domain, which corresponds to the proton transfer groove on the distal site of the heme. Another is the EXXR domain, which is probably needed to stabilize the core structure located on the proximal side of heme (Werck-Reichhart & Feyereisen, 2000).

#### **3.3 Microssomal / mitochondrial**

In insects both mitochondrial and microssomal P450 systems have been described. The majority of P450 in insects are microssomal, located in the endoplasmic reticulum, and require the flavoprotein NADPH cytochrome P450 reductase as the main electron donor; however cytochrome *b5* is sometimes needed, depending of the substrate and of the P450 cytochrome involved. Mitochondrial P450 are also present, but, differently from microssomal P450, require ferridoxin and a NADPH ferridoxin reductase as electron donor (Scott & Wen, 2001).

### **3.4 Characterization / function**

506 Insecticides – Pest Engineering

degradation of insect hormones and pheromones, including 20-hydroxyecdysone and

To distinguish one of these cytochromes among all the P450s, a standardized nomenclature system was implemented (Nebert et al., 1991; Nelson et al., 1996). Each P450 is named with CYP, followed by an Arabical number for the gene family, a letter for the sub-family and another Arabical number for the gene. Cytochromes P450s with share more then 40% of the amino acids are usually grouped into the same family and members with >55% of the amino acids identical are normally grouped in the same sub-family. However, there are exceptions to these rules (Nelson et al., 1996). As it is based on amino acid similarities, no information

P450s can be divided into classes depending on how electrons from NAD(P)H are delivered to the catalytic site. Class I P450s are found in eukaryotes and are associated with mitochondrial membranes. This class of enzymes requires both a FAD-containing reductase and an iron sulphur redoxin, and catalyzes several steps in the biosynthesis of steroid. Class II enzymes are the most common in eukaryotes and are found in the endoplasmic reticulum. These enzymes only require an FAD/FMN-containing P450 reductase for transfer of electrons. Their functions are extremely diverse and, in eukaryotes, include aspects of the biosynthesis and catabolism of signalling molecules and steroid hormones (Feyereisen, 1999). Class III enzymes are self-sufficient and require no electron donor. They are involved in the synthesis of signalling molecules. Finally, class IV enzymes receive electrons directly from NAD(P)H. Class I and II P450s from all organisms participate in the detoxification or sometimes the activation of xenobiotics and class III and IV enzymes are considered remains of the ancestral forms of P450s involved in detoxification of damaging activated oxygen

Most P450s are approximately 500 amino acids long. The core of these proteins is formed by a four-helix bundle, two sets of *β* sheets, two helices and a coil called the "meander". A characteristic consensus sequence known as the P450 "signature" FXXGXXXCXG, located on the C-terminus of the heme binding region, contains a conserved cysteine that serves as a fifth ligand to the heme iron. There are two other conserved motifs specific of the P450 proteins. One is the DGXXT domain, which corresponds to the proton transfer groove on the distal site of the heme. Another is the EXXR domain, which is probably needed to stabilize the core structure located on the proximal side of heme (Werck-Reichhart &

In insects both mitochondrial and microssomal P450 systems have been described. The majority of P450 in insects are microssomal, located in the endoplasmic reticulum, and require the flavoprotein NADPH cytochrome P450 reductase as the main electron donor; however cytochrome *b5* is sometimes needed, depending of the substrate and of the P450 cytochrome involved. Mitochondrial P450 are also present, but, differently from microssomal P450, require ferridoxin and a NADPH ferridoxin reductase as electron donor

regarding the function of each P450 should be assumed from its name.

juvenile hormone (Feyereisen, 1999).

species (Werck-Reichhart & Feyereisen, 2000).

**3.1 Nomenclature** 

**3.2 Structure** 

Feyereisen, 2000).

(Scott & Wen, 2001).

**3.3 Microssomal / mitochondrial** 

Cytochromes P450 enzymes catalyse thousands of different reactions, which are based on the activation of molecular oxygen, with insertion of one of its atoms into the substrate, and reduction of the other to form water (Guengerich, 1991). P450s use electrons from NAD(P)H to catalyse the activation of molecular oxygen, leading to the regiospecific and stereospecific oxidative attack of structurally diverse chemicals (Werck-Reichhart & Feyereisen, 2000).

The interaction that occurs between P450 cytochromes and the NADPH–cytochrome P450 reductase is better expressed as a cyclic reaction (Guenguerich, 1991) as it is depicted in Figure 1.

The cycle is initiated by the binding of the substrate to the ferric form of the enzyme to form an enzyme-substrate complex, followed by a reduction of the ferric complex by an electron transferred from NADPH via NADPH-cytochrome P450 reductase. Next, the binding of molecular oxygen to the reduced complex forms an enzyme-oxygen-substrate complex followed by the transference of a second electron from NADPH via NADPH-cytochrome P450 reductase or from cytochrome *b5*. A second proton is added, which results in the breaking of the oxygen-oxygen bond, releasing one atom of oxygen as water. The oxygen atom remaining is transferred to the substrate, originating an oxidized product, which is released, and a ferric form of the enzyme is once more generated. Then the cycle is reinitiated (Guenguerich, 1991).

Fig. 1. Catalytic mechanism of P450 enzymes, where S is the substrate.

The Role of *Anopheles gambiae* 

of *A. gambiae* (Awolola et al., 2009).

of these insecticides (Mclaughlin et al., 2008).

*gambiae* population (Müller et al., 2008).

in *A. gambiae*.

al., 2005).

2006).

P450 Cytochrome in Insecticide Resistance and Infection 509

several P450 cytochromes that were highly expressed in the *A. gambiae* permethrin or DDTresistant strains (David et al., 2005). Of notice is the P450 cytochrome *CYP325A3,* which belongs to a class that was not associated with insecticide resistance before and which was highly over-expressed in an *A. gambiae* permethrin resistant strain. Additionally, *CYP325A3*  was later reported as constitutively over-expressed in a Nigerian pyrethroid resistant strain

In 2007, studies regarding a recently colonised strain of *A. gambiae* from Ghana identified genes whose expression levels were associated with pyrethroid resistance. Among these were three P450 cytochromes (*CYP6M2, CYP6Z2* and *CYP6Z3*) (Muller et al., 2007). These results, together with their location within a cluster of P450 cytochromes in the right arm of chromosome 3 (3R), which is in close association with a pyrethroid resistance QTL (Ranson et al., 2004), strongly support their involvement in insecticide resistance. A subsequent study showed that *CYP6Z2* displays broad substrate specificity, which may be associated with xenobiotics metabolism and detoxification (Mclaughlin et al., 2008). Despite, *CYP6Z2* being able to bind to permethrin and cypermethrin, *CYP6Z2* does not metabolise neither one

In 2008, Djouaka et al. also identified several P450 cytochromes over-expressed in one or more pyrethroid resistant populations of *A. gambiae.* Among these were *CYP6P3* and once again *CYP6M2*. Both genes showed high levels of over-expression in all the resistant populations, but the first was the gene that showed greatest differences. In the same year, *CYP6P3* was also identified as being up-regulated in another highly permethrin resistant *A.* 

Recent studies on *A. gambiae* recombinant proteins *CYP6M2* (Stevenson et al., 2011) and *CYP6P3* (Müller et al., 2008) demonstrated that these enzymes could metabolise pyrethroids. Thus, the up regulation of these P450 cytochromes in pyrethroid resistant populations, strongly supports a key role for these genes to confer pyrethroid resistance

Highly expressed P450s have been also reported in DDT resistant strains of *A. gambiae*  (David et al., 2005). *CYP6Z1* and *CYP12F1* were strongly over-expressed together with other genes, suggesting that multiple genes could contribute to the DDT resistance phenotype. The slightly over-expression of the electron donor cytochrome P450 reductase in the DDT resistant strain further supported a P450-based resistance mechanism in *A. gambiae* (David et

As the above P450 cytochromes, *CYP314A1* was also found to be over-expressed in a DDT resistant strain of *A. gambiae* from Kenia (Vontas et al., 2005), suggesting a possible involvement in the insecticide resistance phenotype. Both *CYP6Z1* and *CYP6Z2* were overexpressed in DDT resistant strains of *A. gambiae* (David et al. 2005). Although being very similar, these two cytochromes have predicted substrate cavities dramatically different and *CYP6Z1* was predicted to be the only one capable of metabolizing DDT. Chiu et al. (2008) through biochemical characterisations supported these predictions and identified *CYP6Z1* as the only P450 cytochrome capable of metabolising DDT, demonstrating its potential as a

Another evidence of the involvement of P450s in insecticide resistance is the fact that silencing the main electron donor of P450 cytochromes, the cytochrome P450 reductase, by RNAi, greatly increased the susceptibility of *A. gambiae* to permethrin, emphasising the important chemoprotective role of P450 cytochromes in this process (Lycett et al.,

target to reduce *A. gambiae* resistance to DDT (Chiu et al., 2008).

## **3.5 Diversity and specificity**

The huge diversity of P450 cytochromes is probably due to an extensive process of gene duplication and cases of gene amplification, conversion, genome duplication, gene loss and lateral transfer (Werck-Reichhart & Feyereisen, 2000). Due to their extremely diverse functions, they can be found with different patterns of expression in all types of tissues and in almost all types of organisms. Although being expressed in a wide range of tissues, insect P450s have their highest activity associated with midgut, fat body and malpighian tubules (Feyereisen, 1999; Scott, 1999).

Additionally, P450s metabolise a large number of substrates, probably due to the existence of numerous P450 isoforms and to the broad specificity of some isoforms (Scott & Wen, 2001). Nevertheless the substrate specificity and type of reaction catalysed by each P450 cytochrome is still not well understood.

Their diversity enables individual P450 cytochromes to display different expression patterns related to life stages, tissues, inducers/inhibitors and substrates. There are P450s that are expressed in all life stages (*CYP12* genes) while others are only expressed in adults (*CYP6Z1*) or in larval stages (*CYP6Z3*) (Nikou et al., 2003). Although being found expressed in almost all types of tissues, there are P450s which are tissue specific, while others are expressed everywhere (Feyereisen, 1999; Scott et al., 1998; Scott & Wen, 2001). Expression of P450 cytochromes may also be sex specific, as some P450s showed higher levels of expression in males compared with females (Muller et al., 2007; Nikou et al., 2003).

A large variation in substrate specificity can also be found among different P450s, some being capable of metabolising several substrates while others have only one known substrate (Scott, 1999; Scott et al., 1998). There can be also some overlapping substrate specificity among P450 cytochromes, so that one compound could be metabolised by several enzymes. The production of one or several metabolites from a single substrate also differs depending on the P450s. P450s show a vast variation in response to inducers and inhibitors, each P450 can be induced/inhibited by one or several compounds. Some P450s can also remain unaltered while others are induced or repressed (Scott et al., 1998).

## **4.** *Anopheles gambiae* **P450 cytochromes and insecticide resistance**

The *A. gambiae* genome has 111 annotated P450 cytochromes (Ranson et al., 2002). The great interest in these cytochromes derives from their role in the oxidative metabolism of insecticides, but only in few cases a definitive link between an increased expression of a specific P450 cytochrome and increased insecticide metabolism has been established.

Increasing reports of specific *A. gambiae* P450 cytochromes being involved in insecticide resistance have been published in the past. The involvement of P450s in pyrethroid resistance started to be demonstrated in *A. gambiae* from Kenyan villages, in synergistic studies using specific P450 cytochrome inhibitors and also given the detection of increased heme levels in resistant mosquitoes (Vulule et al., 1999).

In 2003, Nikou et al., verified that a P450 cytochrome (*CYP6Z1*) was over-expressed in a pyrethroid-resistant strain of *A. gambiae,* and the development of her work pointed to an implication of the involvement of this P450 in conferring pyrethroid resistance to this mosquito (Nikou et al., 2003).

Later, a microarray *chip* was constructed containing fragments from 230 genes associated with detoxification (David et al., 2005) to further study the metabolic based insecticide resistance in *A. gambiae*. From this work resulted the identification of, among other genes,

The huge diversity of P450 cytochromes is probably due to an extensive process of gene duplication and cases of gene amplification, conversion, genome duplication, gene loss and lateral transfer (Werck-Reichhart & Feyereisen, 2000). Due to their extremely diverse functions, they can be found with different patterns of expression in all types of tissues and in almost all types of organisms. Although being expressed in a wide range of tissues, insect P450s have their highest activity associated with midgut, fat body and malpighian tubules

Additionally, P450s metabolise a large number of substrates, probably due to the existence of numerous P450 isoforms and to the broad specificity of some isoforms (Scott & Wen, 2001). Nevertheless the substrate specificity and type of reaction catalysed by each P450

Their diversity enables individual P450 cytochromes to display different expression patterns related to life stages, tissues, inducers/inhibitors and substrates. There are P450s that are expressed in all life stages (*CYP12* genes) while others are only expressed in adults (*CYP6Z1*) or in larval stages (*CYP6Z3*) (Nikou et al., 2003). Although being found expressed in almost all types of tissues, there are P450s which are tissue specific, while others are expressed everywhere (Feyereisen, 1999; Scott et al., 1998; Scott & Wen, 2001). Expression of P450 cytochromes may also be sex specific, as some P450s showed higher levels of

A large variation in substrate specificity can also be found among different P450s, some being capable of metabolising several substrates while others have only one known substrate (Scott, 1999; Scott et al., 1998). There can be also some overlapping substrate specificity among P450 cytochromes, so that one compound could be metabolised by several enzymes. The production of one or several metabolites from a single substrate also differs depending on the P450s. P450s show a vast variation in response to inducers and inhibitors, each P450 can be induced/inhibited by one or several compounds. Some P450s can also

The *A. gambiae* genome has 111 annotated P450 cytochromes (Ranson et al., 2002). The great interest in these cytochromes derives from their role in the oxidative metabolism of insecticides, but only in few cases a definitive link between an increased expression of a

Increasing reports of specific *A. gambiae* P450 cytochromes being involved in insecticide resistance have been published in the past. The involvement of P450s in pyrethroid resistance started to be demonstrated in *A. gambiae* from Kenyan villages, in synergistic studies using specific P450 cytochrome inhibitors and also given the detection of increased

In 2003, Nikou et al., verified that a P450 cytochrome (*CYP6Z1*) was over-expressed in a pyrethroid-resistant strain of *A. gambiae,* and the development of her work pointed to an implication of the involvement of this P450 in conferring pyrethroid resistance to this

Later, a microarray *chip* was constructed containing fragments from 230 genes associated with detoxification (David et al., 2005) to further study the metabolic based insecticide resistance in *A. gambiae*. From this work resulted the identification of, among other genes,

expression in males compared with females (Muller et al., 2007; Nikou et al., 2003).

remain unaltered while others are induced or repressed (Scott et al., 1998).

heme levels in resistant mosquitoes (Vulule et al., 1999).

mosquito (Nikou et al., 2003).

**4.** *Anopheles gambiae* **P450 cytochromes and insecticide resistance** 

specific P450 cytochrome and increased insecticide metabolism has been established.

**3.5 Diversity and specificity** 

(Feyereisen, 1999; Scott, 1999).

cytochrome is still not well understood.

several P450 cytochromes that were highly expressed in the *A. gambiae* permethrin or DDTresistant strains (David et al., 2005). Of notice is the P450 cytochrome *CYP325A3,* which belongs to a class that was not associated with insecticide resistance before and which was highly over-expressed in an *A. gambiae* permethrin resistant strain. Additionally, *CYP325A3*  was later reported as constitutively over-expressed in a Nigerian pyrethroid resistant strain of *A. gambiae* (Awolola et al., 2009).

In 2007, studies regarding a recently colonised strain of *A. gambiae* from Ghana identified genes whose expression levels were associated with pyrethroid resistance. Among these were three P450 cytochromes (*CYP6M2, CYP6Z2* and *CYP6Z3*) (Muller et al., 2007). These results, together with their location within a cluster of P450 cytochromes in the right arm of chromosome 3 (3R), which is in close association with a pyrethroid resistance QTL (Ranson et al., 2004), strongly support their involvement in insecticide resistance. A subsequent study showed that *CYP6Z2* displays broad substrate specificity, which may be associated with xenobiotics metabolism and detoxification (Mclaughlin et al., 2008). Despite, *CYP6Z2* being able to bind to permethrin and cypermethrin, *CYP6Z2* does not metabolise neither one of these insecticides (Mclaughlin et al., 2008).

In 2008, Djouaka et al. also identified several P450 cytochromes over-expressed in one or more pyrethroid resistant populations of *A. gambiae.* Among these were *CYP6P3* and once again *CYP6M2*. Both genes showed high levels of over-expression in all the resistant populations, but the first was the gene that showed greatest differences. In the same year, *CYP6P3* was also identified as being up-regulated in another highly permethrin resistant *A. gambiae* population (Müller et al., 2008).

Recent studies on *A. gambiae* recombinant proteins *CYP6M2* (Stevenson et al., 2011) and *CYP6P3* (Müller et al., 2008) demonstrated that these enzymes could metabolise pyrethroids. Thus, the up regulation of these P450 cytochromes in pyrethroid resistant populations, strongly supports a key role for these genes to confer pyrethroid resistance in *A. gambiae*.

Highly expressed P450s have been also reported in DDT resistant strains of *A. gambiae*  (David et al., 2005). *CYP6Z1* and *CYP12F1* were strongly over-expressed together with other genes, suggesting that multiple genes could contribute to the DDT resistance phenotype. The slightly over-expression of the electron donor cytochrome P450 reductase in the DDT resistant strain further supported a P450-based resistance mechanism in *A. gambiae* (David et al., 2005).

As the above P450 cytochromes, *CYP314A1* was also found to be over-expressed in a DDT resistant strain of *A. gambiae* from Kenia (Vontas et al., 2005), suggesting a possible involvement in the insecticide resistance phenotype. Both *CYP6Z1* and *CYP6Z2* were overexpressed in DDT resistant strains of *A. gambiae* (David et al. 2005). Although being very similar, these two cytochromes have predicted substrate cavities dramatically different and *CYP6Z1* was predicted to be the only one capable of metabolizing DDT. Chiu et al. (2008) through biochemical characterisations supported these predictions and identified *CYP6Z1* as the only P450 cytochrome capable of metabolising DDT, demonstrating its potential as a target to reduce *A. gambiae* resistance to DDT (Chiu et al., 2008).

Another evidence of the involvement of P450s in insecticide resistance is the fact that silencing the main electron donor of P450 cytochromes, the cytochrome P450 reductase, by RNAi, greatly increased the susceptibility of *A. gambiae* to permethrin, emphasising the important chemoprotective role of P450 cytochromes in this process (Lycett et al., 2006).

The Role of *Anopheles gambiae* 

P450 Cytochrome in Insecticide Resistance and Infection 511

including P450 cytochromes that were differently expressed in the *P. berghei* infected blood meal (*CYP9L1, CYP304B1* and *CYP305A1*). A second study focused on the role of *A. gambiae*  detoxification enzymes, from the three major families involved in detoxification, GSTs, carboxylesterases and P450 cytochromes, in the response to *Plasmodium* infection (Félix et al., 2010). In this study the impact of *P. berghei* infection was analyzed at two time points: one day following the blood meal, during which parasites invade the midgut epithelium, and eleven days after the blood meal when sporozoites were starting to be released to the hemolymph; in two different tissues, midgut and fat body. At day one after the *Plasmodium*  infected blood meal they found 17 P450 cytochromes down-regulated and 5 P450 cytochromes up-regulated, including *CYP9L1, CYP304B1, CYP325H1, CYP6M2* and *CYP6Z2* in the midgut, and 5 P450 up-regulated and 1 down-regulated in the fat body, including *CYP12F2, CYP6M2, CYP6M3* and *CYP4G17*. At eleven days after an infected blood meal they found 2 P450 cytochromes up-regulated and 3 down-regulated in the midgut and 1 P450 cytochrome up-regulated and 1 down-regulated in the fat body. The high number of P450 cytochromes differently expressed by the presence of *P. berghei* parasites in different phases of infection and in different tissues suggests that P450 cytochromes are deeply involved in the mosquito response to *Plasmodium* infection, having an important role in different development stages of the parasite and covering different tissues of the mosquito. More specifically, these P450 cytochromes might have a direct role in *Plasmodium* response during the parasite invasion of the midgut epithelium as this is the moment and tissue where more P450 were differentially expressed. The over expression of these P450 cytochromes could be part of a mosquito response mechanism to parasite invasion occurring in the midgut. One possibility is that P450s are involved in the cytoskeleton rearrangement (Vlachou et al., 2005; Vlachou & Kafatos, 2005), or alternatively P450s could be involved in the production of nitric oxide and other reactive oxygen radicals that are induced by *Plasmodium* invasion of the midgut epithelium (Han et al., 2000; Luckhart et al., 1998). The blood meal *per si* generates metabolic changes that are also expected to increase the oxidative stress in the mosquito midgut, which is augmented by the presence of *Plasmodium* parasites (Molina-Cruz et al., 2008). Moreover, other parasite killing mechanisms also induce oxidative stress inside the host which, although helping to eliminate the parasite, are also toxic to the host cell. The high level of oxidative stress inside the host cell could trigger cellular and molecular regulation of these P450 cytochromes, at

this time point, being responsible for host detoxification and parasite elimination.

Mosquito hemocytes mediate important cellular immune responses including phagocythosis, encapsulation and secrete immune factors such as antimicrobial peptides and mediate melanization. Recently, studies were made to characterize the role of *A. gambiae*  hemocytes in mosquito immunity, consisting in a genome-wide transcriptomic analysis of adult female hemocytes following infection by bacteria and *Plasmodium* parasites (Baton et al., 2009). This work showed that *CYP325H1* and *CYP6M1* were differently expressed in the presence of *Micrococcus luteus*, a Gram-positive bacteria (Baton et al., 2009), reinforcing the role of P450 cytochromes in response to microbial challenge. This work also showed that *CYP325H1* was differently expressed 24 hours after the infected blood meal, during *P. berghei* ookinete invasion of the midgut epithelium. Moreover, *CYP6AG1* and *CYP6M3* were also differentially expressed 19 days after the infected blood meal, during *P. berghei* sporozoite migration through the hemolymph (Baton et al., 2009), suggesting that P450 cytochromes have a role in the response to malaria infection by hemocytes. Another study aiming to analyze the transcriptional profile of circulating *A. gambiae* hemocytes during *P.* 

Nevertheless, although P450s have been clearly associated with insecticide resistance, the identification of specific P450 cytochromes responsible for insecticide resistance is still extremely difficult.

## **5.** *Anopheles gambiae* **P450 cytochromes and malaria infection**

P450 cytochromes have also been implicated in other vital processes as in *A. gambiae* response to bacterial challenge and to parasite invasion, but the real importance and function of these cytochromes in this process is still not well understood.

A genome expression analysis of *A. gambiae* was made to identify which genes responded to injury, bacterial challenge and malaria infection (Dimopoulos et al., 2002). This study identified three P450 cytochromes, one associated with injury, microbial challenge and oxidative stress; the second associated with the response to septic injury which is similar to a bacterial infection *in vivo*; and the third associated with the response to malaria infection and the presence of lipopolysaccharide (Dimopoulos et al., 2002).

The involvement of P450 cytochromes in response to microbial challenge was established when two P450 cytochromes (*CYP4C27* and *CYP306A1*) were differently expressed in the presence of Gram negative (*Salmonella thyphimurium*) or Gram positive (*Staphylococcus aureus*) bacteria (Aguilar et al., 2005). This involvement was even more evident when a study, trying to implicate the mosquito midgut microbiota in the defense against malaria parasites, showed that there were ten P450s differently expressed in response to *Escherichia coli* and *S. aureus* in the *A. gambiae* midgut twelve hours after an uninfected blood meal (Dong et al., 2009). Between the P450 cytochromes differently expressed there were *CYP4H17, CYP6M3, CYP6AG1, CYP9J5*, two of them were mitochondrial cytochromes, *CYP49A1* and *CYP12F4* (Dong et al., 2009).

Regarding the relation between P450 cytochromes and the response to malaria infection, it was partly unveiled for the first time in a study about the midgut epithelial responses to *Plasmodium* invasion (Vlachou et al., 2005). The study revealed that P450 cytochromes were differentially expressed during different phases of the midgut invasion (before invasion, during invasion and after invasion) as well as when they compared *Plasmodium* wild-type infection with *Plasmodium* that were unable to invade the epithelium. (Vlachou et al., 2005). P450s that stood out in this study were *CYP305A1, CYP304B1, CYP6Z1* and *CYP6M4* (Vlachou et al., 2005). The role of P450 cytochromes in the *A. gambiae* response to malaria infection has been reinforced in the last years. Comparing the *A. gambiae* response to two different *Plasmodium* parasites -*P. berghei* and *Plasmodium falciparum -* showed that the mosquito induced slightly different immune responses to each parasite, and that the mosquito was capable of sensing infected blood constituents and mount an immune response, even in the absence of invading ookinetes (Dong et al., 2006). Although there were different responses between the three experimental groups, in all of them there were P450s differentially expressed in the midgut (*CYP6AG1, CYP6M4, CYP6M1, CYP9J5* and *CYP12F3*) and in the fat body (*CYP6AG1* and *CYP4G17*), reinforcing, their involvement in response to malaria infection.

Further evidence of the link between P450 cytochromes and the mosquito's response to malaria infection came from different studies. First, the effect on gene regulation of the presence of chloroquine in an uninfected blood meal and in a *Plasmodium* infected blood meal was investigated (Abrantes et al., 2008). This work showed that chloroquine affects the abundance of transcripts which encode proteins involved in a variety of processes,

Nevertheless, although P450s have been clearly associated with insecticide resistance, the identification of specific P450 cytochromes responsible for insecticide resistance is still

P450 cytochromes have also been implicated in other vital processes as in *A. gambiae* response to bacterial challenge and to parasite invasion, but the real importance and

A genome expression analysis of *A. gambiae* was made to identify which genes responded to injury, bacterial challenge and malaria infection (Dimopoulos et al., 2002). This study identified three P450 cytochromes, one associated with injury, microbial challenge and oxidative stress; the second associated with the response to septic injury which is similar to a bacterial infection *in vivo*; and the third associated with the response to malaria infection

The involvement of P450 cytochromes in response to microbial challenge was established when two P450 cytochromes (*CYP4C27* and *CYP306A1*) were differently expressed in the presence of Gram negative (*Salmonella thyphimurium*) or Gram positive (*Staphylococcus aureus*) bacteria (Aguilar et al., 2005). This involvement was even more evident when a study, trying to implicate the mosquito midgut microbiota in the defense against malaria parasites, showed that there were ten P450s differently expressed in response to *Escherichia coli* and *S. aureus* in the *A. gambiae* midgut twelve hours after an uninfected blood meal (Dong et al., 2009). Between the P450 cytochromes differently expressed there were *CYP4H17, CYP6M3, CYP6AG1, CYP9J5*, two of them were mitochondrial cytochromes,

Regarding the relation between P450 cytochromes and the response to malaria infection, it was partly unveiled for the first time in a study about the midgut epithelial responses to *Plasmodium* invasion (Vlachou et al., 2005). The study revealed that P450 cytochromes were differentially expressed during different phases of the midgut invasion (before invasion, during invasion and after invasion) as well as when they compared *Plasmodium* wild-type infection with *Plasmodium* that were unable to invade the epithelium. (Vlachou et al., 2005). P450s that stood out in this study were *CYP305A1, CYP304B1, CYP6Z1* and *CYP6M4* (Vlachou et al., 2005). The role of P450 cytochromes in the *A. gambiae* response to malaria infection has been reinforced in the last years. Comparing the *A. gambiae* response to two different *Plasmodium* parasites -*P. berghei* and *Plasmodium falciparum -* showed that the mosquito induced slightly different immune responses to each parasite, and that the mosquito was capable of sensing infected blood constituents and mount an immune response, even in the absence of invading ookinetes (Dong et al., 2006). Although there were different responses between the three experimental groups, in all of them there were P450s differentially expressed in the midgut (*CYP6AG1, CYP6M4, CYP6M1, CYP9J5* and *CYP12F3*) and in the fat body (*CYP6AG1* and *CYP4G17*), reinforcing, their involvement in response to

Further evidence of the link between P450 cytochromes and the mosquito's response to malaria infection came from different studies. First, the effect on gene regulation of the presence of chloroquine in an uninfected blood meal and in a *Plasmodium* infected blood meal was investigated (Abrantes et al., 2008). This work showed that chloroquine affects the abundance of transcripts which encode proteins involved in a variety of processes,

**5.** *Anopheles gambiae* **P450 cytochromes and malaria infection** 

function of these cytochromes in this process is still not well understood.

and the presence of lipopolysaccharide (Dimopoulos et al., 2002).

*CYP49A1* and *CYP12F4* (Dong et al., 2009).

malaria infection.

extremely difficult.

including P450 cytochromes that were differently expressed in the *P. berghei* infected blood meal (*CYP9L1, CYP304B1* and *CYP305A1*). A second study focused on the role of *A. gambiae*  detoxification enzymes, from the three major families involved in detoxification, GSTs, carboxylesterases and P450 cytochromes, in the response to *Plasmodium* infection (Félix et al., 2010). In this study the impact of *P. berghei* infection was analyzed at two time points: one day following the blood meal, during which parasites invade the midgut epithelium, and eleven days after the blood meal when sporozoites were starting to be released to the hemolymph; in two different tissues, midgut and fat body. At day one after the *Plasmodium*  infected blood meal they found 17 P450 cytochromes down-regulated and 5 P450 cytochromes up-regulated, including *CYP9L1, CYP304B1, CYP325H1, CYP6M2* and *CYP6Z2* in the midgut, and 5 P450 up-regulated and 1 down-regulated in the fat body, including *CYP12F2, CYP6M2, CYP6M3* and *CYP4G17*. At eleven days after an infected blood meal they found 2 P450 cytochromes up-regulated and 3 down-regulated in the midgut and 1 P450 cytochrome up-regulated and 1 down-regulated in the fat body. The high number of P450 cytochromes differently expressed by the presence of *P. berghei* parasites in different phases of infection and in different tissues suggests that P450 cytochromes are deeply involved in the mosquito response to *Plasmodium* infection, having an important role in different development stages of the parasite and covering different tissues of the mosquito. More specifically, these P450 cytochromes might have a direct role in *Plasmodium* response during the parasite invasion of the midgut epithelium as this is the moment and tissue where more P450 were differentially expressed. The over expression of these P450 cytochromes could be part of a mosquito response mechanism to parasite invasion occurring in the midgut. One possibility is that P450s are involved in the cytoskeleton rearrangement (Vlachou et al., 2005; Vlachou & Kafatos, 2005), or alternatively P450s could be involved in the production of nitric oxide and other reactive oxygen radicals that are induced by *Plasmodium* invasion of the midgut epithelium (Han et al., 2000; Luckhart et al., 1998). The blood meal *per si* generates metabolic changes that are also expected to increase the oxidative stress in the mosquito midgut, which is augmented by the presence of *Plasmodium* parasites (Molina-Cruz et al., 2008). Moreover, other parasite killing mechanisms also induce oxidative stress inside the host which, although helping to eliminate the parasite, are also toxic to the host cell. The high level of oxidative stress inside the host cell could trigger cellular and molecular regulation of these P450 cytochromes, at this time point, being responsible for host detoxification and parasite elimination.

Mosquito hemocytes mediate important cellular immune responses including phagocythosis, encapsulation and secrete immune factors such as antimicrobial peptides and mediate melanization. Recently, studies were made to characterize the role of *A. gambiae*  hemocytes in mosquito immunity, consisting in a genome-wide transcriptomic analysis of adult female hemocytes following infection by bacteria and *Plasmodium* parasites (Baton et al., 2009). This work showed that *CYP325H1* and *CYP6M1* were differently expressed in the presence of *Micrococcus luteus*, a Gram-positive bacteria (Baton et al., 2009), reinforcing the role of P450 cytochromes in response to microbial challenge. This work also showed that *CYP325H1* was differently expressed 24 hours after the infected blood meal, during *P. berghei* ookinete invasion of the midgut epithelium. Moreover, *CYP6AG1* and *CYP6M3* were also differentially expressed 19 days after the infected blood meal, during *P. berghei* sporozoite migration through the hemolymph (Baton et al., 2009), suggesting that P450 cytochromes have a role in the response to malaria infection by hemocytes. Another study aiming to analyze the transcriptional profile of circulating *A. gambiae* hemocytes during *P.* 

The Role of *Anopheles gambiae* 

**7. Acknowledgments** 

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**8. References** 

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processes in this interaction might help mitigate the problem of insecticide resistance, and

We would like to thank to Fundação da Ciência e Tecnologia (FCT) for for the financial support provided by a PhD fellowship grant (SFRH/BD/28024/2006) and research funds

Abrantes, P.; Dimopoulos, G.; Grosso, A.R.; do Rosário, V.E. & Silveira, H. (2008).

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therefore contribute to the control of malaria and other human diseases.

from projects POCTI/SAU-IMI/59489/2004 and PTDC/SAUMII/102596/2008.

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*berghei* infection showed that *CYP6Z1*, *CYP6M2*, *CYP6M3* and *CYP12F2* were differently expressed at 24-28 hours after an infective blood meal (Pinto et al., 2009), valuing the importance of P450 cytochromes on the hemocyte response to malaria parasite invasion.

## **6. Conclusion**

The role of P450 cytochromes during *Plasmodium* invasion is still poorly understood, but it may play out to be of utmost importance to combat malaria transmission. Here, we intend to bring an update review on the connection between P450 cytochromes and the *A. gambiae*  response to malaria infection, identifying several P450 cytochromes that probably are, directly or indirectly, involved in the response to *Plasmodium* invasion. We have also reviewed the implication of P450 cytochromes in *A. gambiae* insecticide resistance. However, uncovering the objective role of these cytochromes in insecticide resistance, that is naming specific cytochromes and describing in detail the processes in which those specific P450s are involved is still extremely difficult.

The consistent detection of differential expression of P450 cytochromes, in studies about either insecticide resistance or the response to malaria infection, suggests that the role of these P450s could be similar in these two processes. Nevertheless, the real importance and function of P450 cytochromes in these processes is still not well understood neither the possibility of interplay between infection and insecticide resistance. One of the P450 cytochromes with expression altered in response to insecticides and *Plasmodium* infection was *CYP6M2* that, was highly over-expressed in a pyrethroid-resistant strain of *A. gambiae*  mosquitoes (Muller et al., 2007) and also highly over-expressed in response to *Plasmodium*  infection in both the midgut and the fat body 1 day after an infected blood meal (Félix et al., 2010). These results suggest that the role of *CYP6M2* might be the same in response to insecticides and infection, or that these two processes might share the activation mechanism of *CYP6M2* expression. *CYP6M2* could also function as an endogenous mediator, acting as the first response to different challenges, which would explain being increased by parasite infection and insecticide exposure. Similar to *CYP6M2* is *CYP6Z1*, yet another P450 cytochrome that was over-expressed in insecticides-resistant strains of *A. gambiae* (David et al., 2005; Nikou et al., 2003) and was also over-expressed in response to *Plasmodium* infection (Vlachou et al., 2005). The increase in the expression of this P450 could function as an immediate response to an exogenous challenge or *A. gambiae* could have the same mechanism of response, including over-expression of specific P450 cytochromes, to parasite infection and insecticide exposure. *CYP6Z2* was highly over-expressed in a pyrethroidresistant strain (Müller et al., 2007), but opposite to *CYP6M2*, was down-regulated in the midgut of *A. gambiae* at day 1 and day 11 after an infected blood meal (Félix et al., 2010). These results suggest a different role for *CYP6Z2* in response to the insecticide and to parasite infection, however, we have to take into account that, although being able to bind to permethrin and cypermethrin, *CYP6Z2* does not metabolise these compounds (Mclaughlin et al., 2008). So the over-expression of *CYP6Z2* in a pyrethroid-resistant strain might be associated with different processes other than insecticide resistance.

A more complete knowledge about the factors involved in P450 cytochromes response to malaria infection and insecticide resistance is extremely needed for the implementation of efficient malaria and vector control programmes, including strategies able to adapt to different types of resistance. Although the interaction of insecticides with P450 enzymes has been studied, many of its aspects still remains poorly understood. Grasping the underlying processes in this interaction might help mitigate the problem of insecticide resistance, and therefore contribute to the control of malaria and other human diseases.

## **7. Acknowledgments**

We would like to thank to Fundação da Ciência e Tecnologia (FCT) for for the financial support provided by a PhD fellowship grant (SFRH/BD/28024/2006) and research funds from projects POCTI/SAU-IMI/59489/2004 and PTDC/SAUMII/102596/2008.

## **8. References**

512 Insecticides – Pest Engineering

*berghei* infection showed that *CYP6Z1*, *CYP6M2*, *CYP6M3* and *CYP12F2* were differently expressed at 24-28 hours after an infective blood meal (Pinto et al., 2009), valuing the importance of P450 cytochromes on the hemocyte response to malaria parasite invasion.

The role of P450 cytochromes during *Plasmodium* invasion is still poorly understood, but it may play out to be of utmost importance to combat malaria transmission. Here, we intend to bring an update review on the connection between P450 cytochromes and the *A. gambiae*  response to malaria infection, identifying several P450 cytochromes that probably are, directly or indirectly, involved in the response to *Plasmodium* invasion. We have also reviewed the implication of P450 cytochromes in *A. gambiae* insecticide resistance. However, uncovering the objective role of these cytochromes in insecticide resistance, that is naming specific cytochromes and describing in detail the processes in which those specific P450s are

The consistent detection of differential expression of P450 cytochromes, in studies about either insecticide resistance or the response to malaria infection, suggests that the role of these P450s could be similar in these two processes. Nevertheless, the real importance and function of P450 cytochromes in these processes is still not well understood neither the possibility of interplay between infection and insecticide resistance. One of the P450 cytochromes with expression altered in response to insecticides and *Plasmodium* infection was *CYP6M2* that, was highly over-expressed in a pyrethroid-resistant strain of *A. gambiae*  mosquitoes (Muller et al., 2007) and also highly over-expressed in response to *Plasmodium*  infection in both the midgut and the fat body 1 day after an infected blood meal (Félix et al., 2010). These results suggest that the role of *CYP6M2* might be the same in response to insecticides and infection, or that these two processes might share the activation mechanism of *CYP6M2* expression. *CYP6M2* could also function as an endogenous mediator, acting as the first response to different challenges, which would explain being increased by parasite infection and insecticide exposure. Similar to *CYP6M2* is *CYP6Z1*, yet another P450 cytochrome that was over-expressed in insecticides-resistant strains of *A. gambiae* (David et al., 2005; Nikou et al., 2003) and was also over-expressed in response to *Plasmodium* infection (Vlachou et al., 2005). The increase in the expression of this P450 could function as an immediate response to an exogenous challenge or *A. gambiae* could have the same mechanism of response, including over-expression of specific P450 cytochromes, to parasite infection and insecticide exposure. *CYP6Z2* was highly over-expressed in a pyrethroidresistant strain (Müller et al., 2007), but opposite to *CYP6M2*, was down-regulated in the midgut of *A. gambiae* at day 1 and day 11 after an infected blood meal (Félix et al., 2010). These results suggest a different role for *CYP6Z2* in response to the insecticide and to parasite infection, however, we have to take into account that, although being able to bind to permethrin and cypermethrin, *CYP6Z2* does not metabolise these compounds (Mclaughlin et al., 2008). So the over-expression of *CYP6Z2* in a pyrethroid-resistant strain might be

associated with different processes other than insecticide resistance.

A more complete knowledge about the factors involved in P450 cytochromes response to malaria infection and insecticide resistance is extremely needed for the implementation of efficient malaria and vector control programmes, including strategies able to adapt to different types of resistance. Although the interaction of insecticides with P450 enzymes has been studied, many of its aspects still remains poorly understood. Grasping the underlying

**6. Conclusion** 

involved is still extremely difficult.


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in the malaria vector, *Anopheles gambiae*. *Insect Molecular Biology*, 13 (4), 379-386, ISSN 0962-1075


**24** 

*Argentina* 

**Genetic Toxicological Profile of Carbofuran and** 

It's well known that the pesticide usages in agriculture have led increase in food production worldwide. Although the benefits of conventional agricultural practices have been immense, they utilize levels of pesticides and fertilizers that can result in a negative impact on the environment (WHO, 1988). Only for the 2006-2007, the total world pesticide amount employed was approximately 5.2 billion pounds (www.epa.gov). Their application is still the most effective and accepted method for the plant and animal protection from a large number of pests, being the environment consequently and inevitably exposed to these chemicals. Herbicides accounted for the largest portion of total use, followed by other pesticides, like insecticides and fungicides (www.epa.gov). The goal in pesticide investigation and development is identifying the specificity of action of a pesticide toward the organisms it is supposed to kill (Cantelli-Forti et al., 1993). Only the target organisms should be affected by the application of the product. However, because pesticides are designed and selected for their biological activity, toxicity on non-target organisms frequently remains a significant potential risk (Cantelli-Forti et al., 1993). The benefits in using pesticides must be weighed against their deleterious effects on human health, biological interactions with non-target organisms, pesticide resistance and/or accumulation of these chemicals in the environment (WHO, 1988). Pesticides are high volume, widely used environmental chemicals and there is continuous debate concerning their probable role in both acute and chronic human health effects (Cantelli-Forti et al., 1993; Hodgson & Levi, 1996). Among the potential risk effects of agricultural chemicals, carcinogenesis is of special concern. The genetic toxicities of pesticides have been determined by numerous factors like their biological accumulation or degradation in the environment, their metabolism in humans, and their action in cellular components such as DNA, RNA and proteins (Shirasu, 1975). It seems essential the determination of the genotoxic risks of these pesticides before they are used in agriculture. Therefore, the carcinogenic and mutagenic potential of a large amount of pesticides has been the object of an extensive and wide investigation (WHO, 1990). These results have great predictive value for the carcinogenicity of several pesticides (IARC, 1987). The International Agency for Research on Cancer (IARC) has reviewed the potential carcinogenicity of a wide range of insecticides, fungicides, herbicides and other similar compounds. Fifty-six pesticides have been classified with carcinogenic potential in different laboratory animals (IARC, 2003). Among them, and as a brief example, chemicals compounds as phenoxy acid herbicides, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), lindane,

**1. Introduction** 

**Pirimicarb Carbamic Insecticides** 

*Faculty of Natural Sciences and Museum, National University of La Plata* 

Sonia Soloneski and Marcelo L. Larramendy

Zhu, Y.C.; Dowdy, A.K. & Baker, J.E. (1999). Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid *Anisopteromalus calandrae* (Hymenoptera: Pteromalidae). *Insect Molecular Biology*, 29(5), 417-421, ISSN 0962-1075

## **Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides**

Sonia Soloneski and Marcelo L. Larramendy *Faculty of Natural Sciences and Museum, National University of La Plata Argentina* 

## **1. Introduction**

518 Insecticides – Pest Engineering

Zhu, Y.C.; Dowdy, A.K. & Baker, J.E. (1999). Differential mRNA expression levels and gene

*Biology*, 29(5), 417-421, ISSN 0962-1075

sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid *Anisopteromalus calandrae* (Hymenoptera: Pteromalidae). *Insect Molecular* 

> It's well known that the pesticide usages in agriculture have led increase in food production worldwide. Although the benefits of conventional agricultural practices have been immense, they utilize levels of pesticides and fertilizers that can result in a negative impact on the environment (WHO, 1988). Only for the 2006-2007, the total world pesticide amount employed was approximately 5.2 billion pounds (www.epa.gov). Their application is still the most effective and accepted method for the plant and animal protection from a large number of pests, being the environment consequently and inevitably exposed to these chemicals. Herbicides accounted for the largest portion of total use, followed by other pesticides, like insecticides and fungicides (www.epa.gov). The goal in pesticide investigation and development is identifying the specificity of action of a pesticide toward the organisms it is supposed to kill (Cantelli-Forti et al., 1993). Only the target organisms should be affected by the application of the product. However, because pesticides are designed and selected for their biological activity, toxicity on non-target organisms frequently remains a significant potential risk (Cantelli-Forti et al., 1993). The benefits in using pesticides must be weighed against their deleterious effects on human health, biological interactions with non-target organisms, pesticide resistance and/or accumulation of these chemicals in the environment (WHO, 1988). Pesticides are high volume, widely used environmental chemicals and there is continuous debate concerning their probable role in both acute and chronic human health effects (Cantelli-Forti et al., 1993; Hodgson & Levi, 1996). Among the potential risk effects of agricultural chemicals, carcinogenesis is of special concern. The genetic toxicities of pesticides have been determined by numerous factors like their biological accumulation or degradation in the environment, their metabolism in humans, and their action in cellular components such as DNA, RNA and proteins (Shirasu, 1975). It seems essential the determination of the genotoxic risks of these pesticides before they are used in agriculture. Therefore, the carcinogenic and mutagenic potential of a large amount of pesticides has been the object of an extensive and wide investigation (WHO, 1990). These results have great predictive value for the carcinogenicity of several pesticides (IARC, 1987). The International Agency for Research on Cancer (IARC) has reviewed the potential carcinogenicity of a wide range of insecticides, fungicides, herbicides and other similar compounds. Fifty-six pesticides have been classified with carcinogenic potential in different laboratory animals (IARC, 2003). Among them, and as a brief example, chemicals compounds as phenoxy acid herbicides, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), lindane,

Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 521

applied worldwide (EPA, 2006). The most sensitive and appropriate effect associated with the use of carbofuran is its toxicity following acute exposure (HSDB, 2011). On the basis of its acute toxicity, it has been classified as a highly hazardous member (class Ib) by WHO (2009) and highly toxic compound (category I) by EPA (2006) based on its potency by the oral and inhalation exposure routes. In spite of the recommendation and regulation proposed by the United States Environmental Protection Agency (EPA) concerning the use of this carbamate within the United States of America, its application has been recently cancelled all over the Northern country by the same organization since 2009 (www.epa.gov). However, the contamination of environment with this compound can by far occur, particularly taking into consideration those countries where it is still in use and the probability of long-term low dose exposure becomes increased. Due to its extensive employment in agriculture and household, contamination of food, water and air has become serious and undesirable health problem for humans, animals and wildlife. Large quantities

of this carbamate are particularly applied to different environments worldwide.

Fig. 1. Chemical structure of carborufan. Source: INCHEM (www.inchem.org).

toxic to mammals (Gupta, 1994; WHO-FAO, 2004, 2009; WHO, 2009).

reported so far is presented in Table 1.

Metabolism of carbofuran has been extensively studied in plants and animals (Dorough & Casida, 1964; Metcalf et al., 1968). In mammals, it reversibly inhibits acethylcholinesterase by carbamylation as well as others non-specific serine-containing enzymes, such as carboxylesterases and butyrylcholinesterases (Gupta, 1994). This results in accumulation of acetylcholine at nerve synapses and myoneural junctions leading to cholinergic signs and causing toxic effects (Karczmar, 1998). Epidemiological studies suggested that exposure to carbofuran may be associated with increased risk of gastrointestinal, neurological, cardiac dysfunction, and retinal degeneration (Cole et al., 1998; Kamel et al., 2000; Peter & Cherian, 2000). Carbofuran represents an acute poison when absorbed into the gastrointestinal tract by inhalation of dust and spray mist and minimally poison thought the intact skin contact (Gupta, 1994). In summary, carbofuran is reported to be teratogenic, embryotoxic and highly

Genotoxicity and cytotoxicity studies have been conducted with this *N*-methyl carbamate member using several end-points on different cellular systems. A summary of the results

The compound produced both conflicting and inconclusive results in mutagenicity tests varying according to either the end-point assessed (WHO, 1988, 2000-2002, 2009; WHO-FAO, 2004, 2009). When mutagenic activity was assessed in bacterial systems either positive or negative results have been reported. Carbofuran has been found to be non-mutagenic in *Salmonella typhimurium* since negative or weak positive response were observed in the number of mitotic recombinants regardless of the presence or absence of a rat liver

methoxychlor, toxaphene, and some organophosphates have been reported with a carcinogenic potential in human studies (IARC, 2003).

Numerous well known pesticides have been tested in a wide variety of mutagenicity as well as DNA, chromosomal, and cellular damage endpoints (IARC, 2003). Several investigations have been reported positive associations between exposure and pesticide risk (Shirasu, 1975; Bolognesi et al., 1993, 2009, 2011; Pavanello & Clonfero, 2000; Bolognesi, 2003; Clark & Snedeker, 2005; Castillo-Cadena et al., 2006).

## **2. Carbamic insecticides**

The carbamates are chemicals mainly used in agriculture as insecticides, fungicides, herbicides, nematocides, and/or sprout inhibitors (IARC, 1976). These chemicals are part of the large group of synthetic pesticides that have been developed, produced, and used on a large scale within the last 50 years. Additionally, they are used as biocides for industrial or other applications as household products including gardens and homes (IARC, 1976).

During the last decades, considerable amounts of pesticides belonging to the class of carbamates have been released into the environment. Humans may be exposed to carbamates through food and drinking water around residences, schools, and commercial buildings, among others (IARC, 1976). Consequently, carbamates are potentially harmful to the health of different kinds of organisms (EPA, 2004). Among all classes of pesticides, carbamates are most commonly used compounds because organophosphates and organochlorines are extremely toxic and possess delayed neurotoxic effects (Hour et al., 1998). They share with organophosphates the ability to inhibit cholinesterase enzymes and therefore share similar symptomatology throughout acute and chronic exposures. Likewise, exposure can occur by several routes in the same individual due to multiple uses, and there is likely to be additive toxicity with simultaneous exposure to organophosphates (IARC, 1976).

The *N*-methyl carbamates are a group of closely related pesticides employed in homes, gardens and agriculture that may affect the functioning of the nervous system (EPA, 2007). Toxicological characteristics of the *N*-methyl carbamates involve maximal cholinesterase enzyme inhibition followed by a rapid recovery, typically from minutes to hours (EPA, 2007). Several compounds namely aldicarb, carbaryl, carbofuran, formetanate HCl, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, and thiodicarb are included as members of the *N*-methyl carbamate class (EPA, 2007).

## **3. Carbofuran. Genotoxicity and cytotoxicity profiles**

Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate; CASRN: 1563-66-2) is one of the most widely granular employed *N*-methyl carbamate esters with both contact and systemic activity. Carbofuran is a derivative of carbamic acid being its chemical structure formula shown in Fig. 1.

Carbofuran is a relatively unstable compound that breaks down in the environment within weeks or months (www.inchem.org). It is registered on a variety of agricultural uses to control soil-dwelling and foliar-feeding insects, mites and nematodes on a variety of field, fruit, forage, grain, seed, and fiber crops (EPA, 2006). Carbofuran is a systemic, broad spectrum insecticide and nematocide registered *N*-methyl carbamate for control of soil and foliar pests. It has been reported for 2006 that nearly one million pounds of carbofuran was

methoxychlor, toxaphene, and some organophosphates have been reported with a

Numerous well known pesticides have been tested in a wide variety of mutagenicity as well as DNA, chromosomal, and cellular damage endpoints (IARC, 2003). Several investigations have been reported positive associations between exposure and pesticide risk (Shirasu, 1975; Bolognesi et al., 1993, 2009, 2011; Pavanello & Clonfero, 2000; Bolognesi, 2003; Clark &

The carbamates are chemicals mainly used in agriculture as insecticides, fungicides, herbicides, nematocides, and/or sprout inhibitors (IARC, 1976). These chemicals are part of the large group of synthetic pesticides that have been developed, produced, and used on a large scale within the last 50 years. Additionally, they are used as biocides for industrial or other applications as household products including gardens and homes (IARC, 1976). During the last decades, considerable amounts of pesticides belonging to the class of carbamates have been released into the environment. Humans may be exposed to carbamates through food and drinking water around residences, schools, and commercial buildings, among others (IARC, 1976). Consequently, carbamates are potentially harmful to the health of different kinds of organisms (EPA, 2004). Among all classes of pesticides, carbamates are most commonly used compounds because organophosphates and organochlorines are extremely toxic and possess delayed neurotoxic effects (Hour et al., 1998). They share with organophosphates the ability to inhibit cholinesterase enzymes and therefore share similar symptomatology throughout acute and chronic exposures. Likewise, exposure can occur by several routes in the same individual due to multiple uses, and there is likely to be additive toxicity with simultaneous exposure to organophosphates (IARC,

The *N*-methyl carbamates are a group of closely related pesticides employed in homes, gardens and agriculture that may affect the functioning of the nervous system (EPA, 2007). Toxicological characteristics of the *N*-methyl carbamates involve maximal cholinesterase enzyme inhibition followed by a rapid recovery, typically from minutes to hours (EPA, 2007). Several compounds namely aldicarb, carbaryl, carbofuran, formetanate HCl, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, and thiodicarb are included as

Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate; CASRN: 1563-66-2) is one of the most widely granular employed *N*-methyl carbamate esters with both contact and systemic activity. Carbofuran is a derivative of carbamic acid being its chemical

Carbofuran is a relatively unstable compound that breaks down in the environment within weeks or months (www.inchem.org). It is registered on a variety of agricultural uses to control soil-dwelling and foliar-feeding insects, mites and nematodes on a variety of field, fruit, forage, grain, seed, and fiber crops (EPA, 2006). Carbofuran is a systemic, broad spectrum insecticide and nematocide registered *N*-methyl carbamate for control of soil and foliar pests. It has been reported for 2006 that nearly one million pounds of carbofuran was

carcinogenic potential in human studies (IARC, 2003).

members of the *N*-methyl carbamate class (EPA, 2007).

structure formula shown in Fig. 1.

**3. Carbofuran. Genotoxicity and cytotoxicity profiles** 

Snedeker, 2005; Castillo-Cadena et al., 2006).

**2. Carbamic insecticides** 

1976).

applied worldwide (EPA, 2006). The most sensitive and appropriate effect associated with the use of carbofuran is its toxicity following acute exposure (HSDB, 2011). On the basis of its acute toxicity, it has been classified as a highly hazardous member (class Ib) by WHO (2009) and highly toxic compound (category I) by EPA (2006) based on its potency by the oral and inhalation exposure routes. In spite of the recommendation and regulation proposed by the United States Environmental Protection Agency (EPA) concerning the use of this carbamate within the United States of America, its application has been recently cancelled all over the Northern country by the same organization since 2009 (www.epa.gov). However, the contamination of environment with this compound can by far occur, particularly taking into consideration those countries where it is still in use and the probability of long-term low dose exposure becomes increased. Due to its extensive employment in agriculture and household, contamination of food, water and air has become serious and undesirable health problem for humans, animals and wildlife. Large quantities of this carbamate are particularly applied to different environments worldwide.

Fig. 1. Chemical structure of carborufan. Source: INCHEM (www.inchem.org).

Metabolism of carbofuran has been extensively studied in plants and animals (Dorough & Casida, 1964; Metcalf et al., 1968). In mammals, it reversibly inhibits acethylcholinesterase by carbamylation as well as others non-specific serine-containing enzymes, such as carboxylesterases and butyrylcholinesterases (Gupta, 1994). This results in accumulation of acetylcholine at nerve synapses and myoneural junctions leading to cholinergic signs and causing toxic effects (Karczmar, 1998). Epidemiological studies suggested that exposure to carbofuran may be associated with increased risk of gastrointestinal, neurological, cardiac dysfunction, and retinal degeneration (Cole et al., 1998; Kamel et al., 2000; Peter & Cherian, 2000). Carbofuran represents an acute poison when absorbed into the gastrointestinal tract by inhalation of dust and spray mist and minimally poison thought the intact skin contact (Gupta, 1994). In summary, carbofuran is reported to be teratogenic, embryotoxic and highly toxic to mammals (Gupta, 1994; WHO-FAO, 2004, 2009; WHO, 2009).

Genotoxicity and cytotoxicity studies have been conducted with this *N*-methyl carbamate member using several end-points on different cellular systems. A summary of the results reported so far is presented in Table 1.

The compound produced both conflicting and inconclusive results in mutagenicity tests varying according to either the end-point assessed (WHO, 1988, 2000-2002, 2009; WHO-FAO, 2004, 2009). When mutagenic activity was assessed in bacterial systems either positive or negative results have been reported. Carbofuran has been found to be non-mutagenic in *Salmonella typhimurium* since negative or weak positive response were observed in the number of mitotic recombinants regardless of the presence or absence of a rat liver

Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 523

+/-

Blevins et al., 1977a; Waters et al., 1982; Haworth & Lawlor, 1983;

Yoon et al., 2001 Hour et al., 1998 Gentile et al., 1982; Moriya et al., 1983; Hour

et al., 1998

Simmon, 1979 Saxena et al., 1997

Nicolaou, 1996

SRI, 1979 SRI, 1979

1982

Simmon, 1979

Das et al., 2003

Thilagar, 1983b Thilagar, 1983c Gentile et al., 1982; Soloneski et al., 2008 Lin et al., 2007 Georgian et al., 1985

SRI, 1979

Gentile et al., 1982

Naravaneni & Jamil, 2005




1 – 50 mg/ml - Simmon, 1979

16 – 1 780 g/ml +/- Kirby, 1983a, b


+ -

+ +


+ +

100 – 10 000 g/plate

1 – 10 000 g/plate 0.1 – 100 g/plate

1 – 5 000 g/plate 1 – 5 000 g/plate

*Vibrio fischeri* (M169), S9 +/- 175 g/plate + Canna-Michaelidou &

0 – 5 mg/6-mm disk 0 – 5 mg/6-mm disk

V79 cells NA + Wojciechowski et al.,

0.1 – 1 000 g/ml

0.1 – 1 000 g/ml 0 – 100 g/ml

Human skin fibroblasts NA + Blevins et al., 1977b

12.5 – 312.5 µg/ml 12.5 – 2 500 g/ml 5 – 100 g/ml

0.04 – 0.32 g/ml

NA

0.5 – 4.0 M

End-point/Test System Concentrationa Results References

*In vitro* assays Ames test

*Salmonella typhimurium*, S9 +/-

*Salmonella* lactam assay, S9 +/-

Pol A reverse mutation *Escherichia coli* (WP2), S9 +/- *Escherichia coli* (K-12)

DNA damage and repair *Escherichia coli* (W3110-p3478) *Bacillus subtilis* (H17-M45)

Mitotic recombination *Saccharomyces cerevisiae* (D3),

Gene mutation assay

Cell mutation tk locus Mouse lymphoma L5178 Y

Human fibroblasts (WI-38), S9

HL NA

DNA fragmentation analysis

Human lung fibroblasts Primary rat hepatocytes

*A*lkaline comet assay

cells, S9 +/-

UDS

+/-

SCE assay

CHO cells, S9 +/- CHO cells, S9 +/- CHO-K1 cells

CHO-W8 cells

HL

Mutatox test

S9 +/-

metabolic activation system (Blevins et al., 1977a; Gentile et al., 1982; Waters et al., 1982; Haworth & Lawlor, 1983; Hour et al., 1998; Yoon et al., 2001). These results indicate that carbofuran cannot be considered mutagenic in bacterial systems. However, it was active in *Salmonella typhimurium* TA1538 and TA98 strains in the presence or absence of S9 metabolic system (Gentile et al., 1982; Moriya et al., 1983; Hour et al., 1998). Whereas the insecticide did not induce reverse mutations in *Escherichia coli* (Simmon, 1979), it has been claimed as a relatively weaker mutagen with the repair defective Ames *Escherichia coli* K-12 test (Saxena et al., 1997). Similarly, positive results have been found after exposure in *Vibrio fischeri*  regardless the absence or presence of S9 metabolic system (Canna-Michaelidou & Nicolaou, 1996). When DNA damage and repair assays were performed, carbofuran was also negative in both *Escherichia coli* and *Bacillus subtilis* bacterial systems (SRI, 1979). Similar negative results were also found after carbofuran exposure in *Saccharomyces cerevisiae* mitotic recombination assay (Simmon, 1979).

The mammalian *in vitro* gene mutation assay systems generated results consistent with the microbial gene mutation assays, although they were generally more responsive. When a mammalian cell system was employed for mutagenic screening, carbofuran was found to be positive in V79 cells (Wojciechowski et al., 1982). Similar results were reported for the cell mutation assay in mouse lymphoma L5178 cells (Kirby, 1983a, b). Unscheduled DNA synthesis was monitored in human fibroblasts and primary rat hepatocytes following treatment with the insecticide with and without S9 fraction. Both negative and positive results were obtained for the same endpoint in human primary fibroblasts regardless of the presence or absence of a rat liver metabolic activation system (Simmon, 1979; Gentile et al., 1982) but negative results were obtained in primary rat hepatocyte cultures (SRI, 1979). Single-strand breaks detected by alkaline comet assay were induced in *in vitro* human peripheral lymphocytes (Das et al., 2003; Naravaneni & Jamil, 2005). The induction of DNA fragmentation on human skin fibroblasts have been found to be enhanced after *in vitro*  carbofuran treatment (Blevins et al., 1977b).

As opposed to mutation assays that detect specific gene defects, the chromosomal assays evaluate the structure of the whole chromosome. Five studies of carbofuran have evaluated the induction of sister chromatid exchanges in mammalian cell cultures. In one of the first studies, carbofuran was negative in Chinese hamster ovary cells regardless of the presence or absence of S9 fraction (Thilagar, 1983b). However, other authors reported positive results for the same cell system (Gentile et al., 1982; Thilagar, 1983c; Lin et al., 2007; Soloneski et al., 2008) as well as human lymphocytes (Georgian et al., 1985). Similarly, the effects on chromosomal structure following exposure to carbofuran were investigated in Chinese hamster ovary and primary human lymphocytes cells. While carbofuran did not induce *in vitro* chromosome damage in Chinese hamster ovary cells with or without metabolic system activation (Thilagar, 1983a), positive results were reported to occur not only in the same cellular system (Lin et al., 2007) but also in human lymphocytes *in vitro* (Pilinskaia & Stepanova, 1984; Das et al., 2003). However, inconclusive response for this endpoint has been also reported to occur in the latter system after carbofuran exposure (Naravaneni & Jamil, 2005). Positive results have been also reported for the ability of carbofuran to induce micronuclei in both Chinese hamster ovary cells and human lymphocytes *in vitro* with and without S9 metabolic fraction (Soloneski et al., 2008; Mladinic et al., 2009).

Several assays have been developed to assess the ability of carbofuran to cause cytotoxic effects on different cellular systems. Negative response was observed in both *Escherichia coli* and *Bacillus subtilis* bacterial systems (Simmon, 1979). When the analysis of cell-cycle

metabolic activation system (Blevins et al., 1977a; Gentile et al., 1982; Waters et al., 1982; Haworth & Lawlor, 1983; Hour et al., 1998; Yoon et al., 2001). These results indicate that carbofuran cannot be considered mutagenic in bacterial systems. However, it was active in *Salmonella typhimurium* TA1538 and TA98 strains in the presence or absence of S9 metabolic system (Gentile et al., 1982; Moriya et al., 1983; Hour et al., 1998). Whereas the insecticide did not induce reverse mutations in *Escherichia coli* (Simmon, 1979), it has been claimed as a relatively weaker mutagen with the repair defective Ames *Escherichia coli* K-12 test (Saxena et al., 1997). Similarly, positive results have been found after exposure in *Vibrio fischeri*  regardless the absence or presence of S9 metabolic system (Canna-Michaelidou & Nicolaou, 1996). When DNA damage and repair assays were performed, carbofuran was also negative in both *Escherichia coli* and *Bacillus subtilis* bacterial systems (SRI, 1979). Similar negative results were also found after carbofuran exposure in *Saccharomyces cerevisiae* mitotic

The mammalian *in vitro* gene mutation assay systems generated results consistent with the microbial gene mutation assays, although they were generally more responsive. When a mammalian cell system was employed for mutagenic screening, carbofuran was found to be positive in V79 cells (Wojciechowski et al., 1982). Similar results were reported for the cell mutation assay in mouse lymphoma L5178 cells (Kirby, 1983a, b). Unscheduled DNA synthesis was monitored in human fibroblasts and primary rat hepatocytes following treatment with the insecticide with and without S9 fraction. Both negative and positive results were obtained for the same endpoint in human primary fibroblasts regardless of the presence or absence of a rat liver metabolic activation system (Simmon, 1979; Gentile et al., 1982) but negative results were obtained in primary rat hepatocyte cultures (SRI, 1979). Single-strand breaks detected by alkaline comet assay were induced in *in vitro* human peripheral lymphocytes (Das et al., 2003; Naravaneni & Jamil, 2005). The induction of DNA fragmentation on human skin fibroblasts have been found to be enhanced after *in vitro* 

As opposed to mutation assays that detect specific gene defects, the chromosomal assays evaluate the structure of the whole chromosome. Five studies of carbofuran have evaluated the induction of sister chromatid exchanges in mammalian cell cultures. In one of the first studies, carbofuran was negative in Chinese hamster ovary cells regardless of the presence or absence of S9 fraction (Thilagar, 1983b). However, other authors reported positive results for the same cell system (Gentile et al., 1982; Thilagar, 1983c; Lin et al., 2007; Soloneski et al., 2008) as well as human lymphocytes (Georgian et al., 1985). Similarly, the effects on chromosomal structure following exposure to carbofuran were investigated in Chinese hamster ovary and primary human lymphocytes cells. While carbofuran did not induce *in vitro* chromosome damage in Chinese hamster ovary cells with or without metabolic system activation (Thilagar, 1983a), positive results were reported to occur not only in the same cellular system (Lin et al., 2007) but also in human lymphocytes *in vitro* (Pilinskaia & Stepanova, 1984; Das et al., 2003). However, inconclusive response for this endpoint has been also reported to occur in the latter system after carbofuran exposure (Naravaneni & Jamil, 2005). Positive results have been also reported for the ability of carbofuran to induce micronuclei in both Chinese hamster ovary cells and human lymphocytes *in vitro* with and

Several assays have been developed to assess the ability of carbofuran to cause cytotoxic effects on different cellular systems. Negative response was observed in both *Escherichia coli* and *Bacillus subtilis* bacterial systems (Simmon, 1979). When the analysis of cell-cycle

without S9 metabolic fraction (Soloneski et al., 2008; Mladinic et al., 2009).

recombination assay (Simmon, 1979).

carbofuran treatment (Blevins et al., 1977b).


Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 525

+ +

+ + - +




Saxena et al., 2010 Saxena et al., 2010 Woodruff et al., 1983 Chauhan et al., 2000

Putman, 1983b, a Zeljezic et al., 2009

Zhou et al., 2005 Chauhan et al., 2000

Pilinskaia & Stepanova,

Aly, 1998

1984

NA NA

NA

NA

20 – 80 ppm 20 – 80 ppm

mg/Kg bw

3.8 – 1.9 (for 4 days)

0.1 – 1.0 mg/Kg bw

0.6 – 10 mg/Kg bw

0.1 – 0.4 mg/Kg bw 5.7 – 1.9 (for 4 days)

*Allium cepa* 20 – 80 ppm + Saxena et al., 2010

carbofuran; \*, from agricultural workers occupationally exposed to carbofuran.

UDS, unscheduled DNA synthesis; SCE, sister chromatid exchange; HL, human lymphocytes; CCP, cell-

Table 1. Evaluation of carbofuran-induced genotoxicity and cytotoxicity on different target systems. a, expressed as reported by authors; b, exposed to pesticide mixture containing

progression on mammalian cells was studied, carbofuran gave negative results in Chinese hamster ovary cells and lung fibroblasts (Yoon et al., 2001; Lin et al., 2007). On the other hand, Soloneski and co-workers (2008) reported a delay in the cell-cycle progression of Chinese hamster ovary cells after the insecticide treatment. Carbofuran was tested *in vitro* in the porcine brain tubulin assembly assay for detecting whether the chemical can be considered as a microtubule poison and an aneuploidy agent. A dose-dependent reduction in the degree of polymerization of tubulins was reported in porcine cells after *in vitro* treatment (Stehrer-Schmid & Wolf, 1995). Controversial results were reported for the cell viability assay in mammalian cells, e.g., Chinese hamster lung and ovary cells after the exposure (Yoon et al., 2001; Soloneski et al., 2008). Finally, whereas carbofuran-induced apoptosis has been reported in rat cortical cells (Kim et al., 2004), negative results have been also observed in mouse brain microvascular entothelial cells and Chinese lung fibroblasts (Yoon et al., 2001; Jung et al., 2003). Similar end-points for both genotoxicity and cytotoxicity were also applied in *in vivo* systems. Carbofuran has been reported as a non inducer agent of mutations in plants cells, at least in *Zea mays* (Gentile et al., 1982), in the *Drosophila melanogaster* sex-linked recessive lethal test (DeGraff, 1983; Gee, 1983), and in the mice dominant-lethal mutagenicity test (FMC, 1971). Negative results have been obtained for the induction of unscheduled DNA synthesis in primary rat hepatocytes (Valencia, 1981, 1983). Controversial observations have been reported for the induction of DNA single-strand breaks assayed by the alkaline comet assay. Positive results were reported in circulating erythrocytes from occupationally exposed workers (Castillo-Cadena et al., 2006) whereas no induction was observed in mouse peripheral lymphocytes exposed *in vivo* (Zhou et al., 2005). It should be noted that the former positive

SCE

Rat

HL**<sup>b</sup>**

Micronuclei

Alteration in CCP

*Allium cepa Allium sativum* 

Mouse peripheral lymphocytes

Chromosomal aberrations

*Drosophila melanogaster* Mouse bone marrow cells

Rat bone marrow cells

Mouse bone marrow cells

Mouse peripheral lymphocytes Mouse bone marrow cells

cycle proliferation; NA, data not available.



Thilagar, 1983a Lin et al., 2007

Das et al., 2003

Simmon, 1979 Simmon, 1979

Lin et al., 2007 Soloneski et al., 2008 Yoon et al., 2001

Yoon et al., 2001 Soloneski et al., 2008

Yoon et al., 2001 Jung et al., 2003

Kim et al., 2004

Zhou et al., 2005 Castillo-Cadena et al.,

2006

1995

Soloneski et al., 2008 Mladinic et al., 2009

1984

Naravaneni & Jamil, 2005 Pilinskaia & Stepanova,

+

+ +





+

 - +

50 – 2 500 g/ml 0.04 – 0.32 g/ml

100 – 300 g/ml

10 – 100 g/ml 0.008 g/ml

1 – 500 mg/ml 1 – 500 mg/ml

0.04 – 0.32g/ml 50 – 100g/ml

Porcine cells 100 – 2 000 mol/l + Stehrer-Schmid & Wolf,

30 M

30 M

30 M 3 – 30 M

500 M

50 – 100 g/ml

*Zea mays* NA - Gentile et al., 1982

Mice 0.025 – 0.5 mg/Kg/day - FMC, 1971

NA

*Drosophila melanogaster* 0 – 10 ppm - DeGraff, 1983; Gee, 1983

Rat hepatocytes 5 – 10 ppm - Valencia, 1981; 1983

0.1 – 0.4 mg/Kg bw

NA

NA

Chromosomal aberrations

CHO cells, S9 +/- CHO-W8 cells

Micronuclei assay CHO-K1 cells HL, S9 +/-

Growth inhibition *Escherichia coli Bacillus subtilis* 

Alteration in CCP CHO-W8 cells CHO-K1 cells CHL cells

Cell viability CHL cells CHO-K1 cells

Apoptosis CHL cells

endothelial cells Rat cortical cells

*In vivo* assays

UDS

HL**\***

Reverse mutation

Alkaline comet assay

Brain tubulin assembly assay

Mouse brain microvascular

Sex-linked recessive lethal test

Dominant –lethal mutagenicity

Mouse peripheral lymphocytes

HL HL

HLb


UDS, unscheduled DNA synthesis; SCE, sister chromatid exchange; HL, human lymphocytes; CCP, cellcycle proliferation; NA, data not available.

Table 1. Evaluation of carbofuran-induced genotoxicity and cytotoxicity on different target systems. a, expressed as reported by authors; b, exposed to pesticide mixture containing carbofuran; \*, from agricultural workers occupationally exposed to carbofuran.

progression on mammalian cells was studied, carbofuran gave negative results in Chinese hamster ovary cells and lung fibroblasts (Yoon et al., 2001; Lin et al., 2007). On the other hand, Soloneski and co-workers (2008) reported a delay in the cell-cycle progression of Chinese hamster ovary cells after the insecticide treatment. Carbofuran was tested *in vitro* in the porcine brain tubulin assembly assay for detecting whether the chemical can be considered as a microtubule poison and an aneuploidy agent. A dose-dependent reduction in the degree of polymerization of tubulins was reported in porcine cells after *in vitro* treatment (Stehrer-Schmid & Wolf, 1995). Controversial results were reported for the cell viability assay in mammalian cells, e.g., Chinese hamster lung and ovary cells after the exposure (Yoon et al., 2001; Soloneski et al., 2008). Finally, whereas carbofuran-induced apoptosis has been reported in rat cortical cells (Kim et al., 2004), negative results have been also observed in mouse brain microvascular entothelial cells and Chinese lung fibroblasts (Yoon et al., 2001; Jung et al., 2003). Similar end-points for both genotoxicity and cytotoxicity were also applied in *in vivo* systems.

Carbofuran has been reported as a non inducer agent of mutations in plants cells, at least in *Zea mays* (Gentile et al., 1982), in the *Drosophila melanogaster* sex-linked recessive lethal test (DeGraff, 1983; Gee, 1983), and in the mice dominant-lethal mutagenicity test (FMC, 1971). Negative results have been obtained for the induction of unscheduled DNA synthesis in primary rat hepatocytes (Valencia, 1981, 1983). Controversial observations have been reported for the induction of DNA single-strand breaks assayed by the alkaline comet assay. Positive results were reported in circulating erythrocytes from occupationally exposed workers (Castillo-Cadena et al., 2006) whereas no induction was observed in mouse peripheral lymphocytes exposed *in vivo* (Zhou et al., 2005). It should be noted that the former positive

Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 527



+ -


+

+ + -

Trueman, 1980 Callander, 1995

Clay, 1996 Clay, 1996

Soloneski & Larramendy, 2010 Wildgoose et al., 1987

Anderson et al., 1980

Vera Candioti et al., 2010b Vera Candioti et al., 2010a Jones & Howard, 1989

Pilinskaia, 1982

End-point/System Concentrationa Results References

5 000 g/plate

*Escherichia coli* 5 000 g/plate - Callander, 1995

*Aspergillus nidulans* NA - Käfer et al., 1982

100 mg/ml + S9

10 – 300 g/ml 500 µg/ml

HL 50 – 500 g/ml + Ündeger & Basaran, 2005

CHO-K1 cells 100 – 200 g/ml + Soloneski & Larramendy, 2010

CHO-K1 cells 100 – 300 g/ml + Soloneski & Larramendy, 2010

*Drosophila melanogaster* NA + Aguirrezabalaga et al., 1994

Mice 20 mg/Kg bw - McGregor, 1974

Rat liver cells 200 mg/Kg bw - Kennelly, 1990

bw NA

50/–100 mg/Kg

50 – 157 mg/L 80 – 250 mg/L 69.3 mg/Kg bw

Table 2. Evaluation of Pirimicarb-induced genotoxicity and cytotoxicity on different target systems. a, expressed as reported by authors; UDS, unscheduled DNA synthesis; SCE, sister chromatid exchange; HL, human lymphocytes; CCP, cell-cycle proliferation; NA, data not

*Salmonella typhimurium*, S9 +/- 2 500 g/plate

Mouse lymphoma L5178 Y cells 1 400 mg/ml – S9

*In vitro* assays Ames test

Pol A reverse mutation

Cell mutation tk locus

Chromosomal aberrations

Eye mosaic system w/w+

Dominant lethal mutation

Chromosomal aberrations Rat bone marrow cells

*Cnesterodon decemmaculatus* 

Micronuclei assay

*Rhinella arenarum*  Rat bone marrow cells

available.

Alkaline comet

SCE assay

CHO-K1 cells HL, S9 +/-

*In vivo* assays

UDS

HL\*

Alteration in CCP

Recessive lethal gene mutation

results could not be totally committed to carbofuran but to other pesticides, since the cohort of donors included in the study was exposed to a panel of other pesticides. Several reports were able to revealed that carbofuran increased the frequency of sister chromatid exchanges in mammalian cells from mouse and rats exposed *in vivo* (Gentile et al., 1982; Aly, 1998), and chromosomal aberrations in plants from *Allium* (Saxena et al., 2010), and mammals including occupationally exposed workers (Putman, 1983a, b; Pilinskaia & Stepanova, 1984; Chauhan et al., 2000; Zeljezic et al., 2009) but not in insects (Woodruff et al., 1983) as well as in rodent cells (Putman, 1983a, b; Pilinskaia & Stepanova, 1984). When the micronuclei induction end-point was employed in mouse, no induction was found either in bone marrow cells (Chauhan et al., 2000) or circulating lymphocytes (Zhou et al., 2005). Finally, alterations in the progression of the cell-cycle were reported to occur after carbofuran exposure in plants when the *Allium cepa* model was employed (Saxena et al., 2010).

## **4. Pirimicarb. Genotoxicity and cytotoxicity profiles**

Pirimicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate, CASRN: 23103-98-2) is a dimethylcarbamate insecticide member with both contact and systemic activity. Similar to carbofuran, pirimicarb is a derivative of carbamic acid being its chemical structure formula shown in Fig. 2.

Fig. 2. Chemical structure of pirimicarb. Source: INCHEM (www.inchem.org)

Based on its acute toxicity, pirimicarb has been classified as a moderately hazardous compound (class II) by WHO (http://www.who.int/ipcs/publications/pesticides hazard/en/) and slightly to moderately toxic (category II-III) by EPA (1974a). Among carbamate pesticides, pirimicarb is registered as a fast-acting selective aphicide mostly used in a broad range of crops, including cereals, sugar beet, potatoes, fruit, and vegetables, and is relatively non-toxic to beneficial predators, parasites, and bees (WHO-FAO, 2004, 2009). It acts by contact, translaminar, vapor, and systemic action. Its mode of action is inhibiting acetylcholinesterase activity (WHO-FAO, 2004, 2009).

Available information on the genotoxic and cytotoxic properties of pirimicarb is limited and inconsistent. Only few data are available in the literature (WHO-FAO, 2004, 2009). Genotoxicity and cytotoxicity studies have been conducted with this carbamate using several end-points on different cellular systems. A summary of the results reported so far is presented in Table 2.

Pirimicarb has been generally recognized as non-genotoxic in bacteria, yeast and fungi as well as in mammalian cells (EPA, 1974b). It has been reported to be non-mutagenic in *Salmonella typhimurium* when the Ames reversion mutagenicity test for the TA1535, TA1538, TA98, and TA100 strains after S9 metabolic activation has been used (Trueman, 1980; Callander, 1995). Furthermore, similar situation was observed in both *Escherichia coli* and *Aspergillus nidulans* when the reverse mutation assay or recessive lethal gene mutation test


results could not be totally committed to carbofuran but to other pesticides, since the cohort of donors included in the study was exposed to a panel of other pesticides. Several reports were able to revealed that carbofuran increased the frequency of sister chromatid exchanges in mammalian cells from mouse and rats exposed *in vivo* (Gentile et al., 1982; Aly, 1998), and chromosomal aberrations in plants from *Allium* (Saxena et al., 2010), and mammals including occupationally exposed workers (Putman, 1983a, b; Pilinskaia & Stepanova, 1984; Chauhan et al., 2000; Zeljezic et al., 2009) but not in insects (Woodruff et al., 1983) as well as in rodent cells (Putman, 1983a, b; Pilinskaia & Stepanova, 1984). When the micronuclei induction end-point was employed in mouse, no induction was found either in bone marrow cells (Chauhan et al., 2000) or circulating lymphocytes (Zhou et al., 2005). Finally, alterations in the progression of the cell-cycle were reported to occur after carbofuran exposure in plants when the *Allium cepa*

Pirimicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate, CASRN: 23103-98-2) is a dimethylcarbamate insecticide member with both contact and systemic activity. Similar to carbofuran, pirimicarb is a derivative of carbamic acid being its chemical

Based on its acute toxicity, pirimicarb has been classified as a moderately hazardous compound (class II) by WHO (http://www.who.int/ipcs/publications/pesticides hazard/en/) and slightly to moderately toxic (category II-III) by EPA (1974a). Among carbamate pesticides, pirimicarb is registered as a fast-acting selective aphicide mostly used in a broad range of crops, including cereals, sugar beet, potatoes, fruit, and vegetables, and is relatively non-toxic to beneficial predators, parasites, and bees (WHO-FAO, 2004, 2009). It acts by contact, translaminar, vapor, and systemic action. Its mode of action is inhibiting

Available information on the genotoxic and cytotoxic properties of pirimicarb is limited and inconsistent. Only few data are available in the literature (WHO-FAO, 2004, 2009). Genotoxicity and cytotoxicity studies have been conducted with this carbamate using several end-points on different cellular systems. A summary of the results reported so far is

Pirimicarb has been generally recognized as non-genotoxic in bacteria, yeast and fungi as well as in mammalian cells (EPA, 1974b). It has been reported to be non-mutagenic in *Salmonella typhimurium* when the Ames reversion mutagenicity test for the TA1535, TA1538, TA98, and TA100 strains after S9 metabolic activation has been used (Trueman, 1980; Callander, 1995). Furthermore, similar situation was observed in both *Escherichia coli* and *Aspergillus nidulans* when the reverse mutation assay or recessive lethal gene mutation test

Fig. 2. Chemical structure of pirimicarb. Source: INCHEM (www.inchem.org)

model was employed (Saxena et al., 2010).

structure formula shown in Fig. 2.

**4. Pirimicarb. Genotoxicity and cytotoxicity profiles** 

acetylcholinesterase activity (WHO-FAO, 2004, 2009).

presented in Table 2.


Table 2. Evaluation of Pirimicarb-induced genotoxicity and cytotoxicity on different target systems. a, expressed as reported by authors; UDS, unscheduled DNA synthesis; SCE, sister chromatid exchange; HL, human lymphocytes; CCP, cell-cycle proliferation; NA, data not available.

Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 529

Buenos Aires, Argentina) on mammalian Chinese hamster ovary cells as well as circulating erythrocytes of the fish *Cnesterodon decemmaculatus* and the amphibian *Rhinella arenarum* 

A summary of the results obtained is presented in Fig. 3. The figure clearly reveals that all compounds assayed were able to inflict damage at chromosomal and cellular level

We observed that carbofuran/Furadan® and pirimicarb/Aficida® caused SCEs on mammalian cells indicating that they have a clastogenic activity (Fig. 3A). It has been suggested that at the chromosomal level, the induction of SCEs is a reliable indicator for the screening of clastogens, since the bioassay is more sensitive than the analysis of clastogeninduced chromosomal aberrations (Palitti et al., 1982). The results also demonstrate the ability of pirimicarb/Aficida® to induce DNA damage quali- and quantitative analyzed by the frequency of chromosomal aberrations (Fig. 3B). Furthermore, a putative clastogenic/aneugenic activity exerted *in vitro* by carbofuran/Furadan® and *in viv*o by pirimicarb/Aficida® was also demonstrated by the ability of the pesticide-induced micronuclei (Fig. 3C). The analysis of the proliferative replication (Fig. 3D) and the mitotic indexes (Fig. 3E) demonstrated that both carbofuran/Furadan® and pirimicarb/Aficida® were able to delay the cell-cycle progression as well as to exert a marked reduction of the cellular mitotic activity on mammalian cells *in vitro*. Besides, carbofuran/Furadan® and pirimicarb/Aficida® were able to induced a clear cellular cytotoxicity. This deleterious effect was estimated by a loss of lysosomal activity (indicated by a decrease in the uptake of neutral red), as well as alteration in energy metabolism (measured by mitochondrial succinic dehydrogenase activity in the MTT assay), as clearly revealed in insecticides-treated Chinese

Overall, the results revealed, depending upon the endpoint employed, that the damage induced by the commercial formulations of both insecticides is, in general, greater than that produced by the pure pesticides (Fig. 3). Unfortunately, the identity of the components present in the excipient formulations was not made available by the manufacturers. These final remarks are in accord with previous observations not only reported by us but also by other research groups indicating the presence of xenobiotics within the composition of the commercial formulations with genotoxic and cytotoxic effects (David, 1982; Lin & Garry, 2000; Soloneski et al., 2001, 2002, 2003, 2008; González et al., 2007a, b, 2009; Elsik et al., 2008; Molinari et al., 2009; Soloneski & Larramendy, 2010). These observations highlight that, in agriculture, agrochemicals are generally not used as a single active ingredient but as part of a complex commercial formulations. Thus, both the workers as well as non-target organisms are exposed to the simultaneous action of the active ingredient and a variety of other chemical/s contained in the formulated product. Hence, risk assessment must also consider additional geno-cytotoxic effects caused by the

Finally, the results highlight that a whole knowledge of the toxic effect/s of the active ingredient of a pesticide is not enough in biomonitoring studies as well as that agrochemical/s toxic effect/s should be evaluated according to the commercial formulation available in market. Furthermore, the deleterious effect/s of the excipient/s present within the commercial formulation should be neither discarded nor underestimated. The importance of further studies on this type of pesticide in order to achieve a complete knowledge on its genetic toxicology seems to be, then, more than

tadpoles were also estimated.

hamster ovary cells (Fig. 3F).

excipient/s.

evident.

regardless of the cellular system used as target.

were respectively applied (Käfer et al., 1982; Callander, 1995). Negative and positive results were obtained for the induction of mutagenicity in mouse lymphoma L5178Y cells regardless of the presence or absence of a rat liver metabolic activation system (Clay, 1996). Furthermore, the induction of DNA single strand breaks, estimated by the alkaline comet assay, was evaluated revealing positive results in human lymphocytes exposed *in vitro* to pirimicarb (Ündeger & Basaran, 2005). Similar positive results were found when the sister chromatid exchange assay was performed in Chinese hamster ovary cells (Soloneski & Larramendy, 2010). Although a significant increase in chromosomal aberrations has been reported in Chinese hamster ovary cells after pirimicarb exposure (Soloneski & Larramendy, 2010), Wildgoose and coworkers (1987) observed negative results in human lymphocytes with or without S9 metabolic activation. Finally, the induction of alterations in the cell-cycle progression in Chinese hamster ovary cells was reported to occur after *in vitro* exposure to pirimicarb (Soloneski & Larramendy, 2010).

In *in vivo* genotoxic and cytotoxic studies, pirimicarb was able to induce different types of lesions. It has been reported the ability of the insecticide to give positive results by using the eye mosaic system *white/white*+ (*w/w*+) somatic mutation and recombination test (SMART) when *Drosophila melanogaster* was employed as experimental model (Aguirrezabalaga et al., 1994). However, McGregor and co-workers (1974) reported negative results in mice when the dominant lethal mutation assay was performed. Similar negative results were found by Kenelly (1990) using the unscheduled DNA synthesis in rat liver cells. At the chromosomal level, pirimicarb did not induce chromosomal alterations in bone marrow cells of Wistar male rats after oral administration (Anderson et al., 1980). Contrarily, Pilinskaia (1982) observed a significant increase of chromosomal aberrations in the peripheral blood lymphocytes from occupational workers after pirimicarb exposure. Finally, when the micronuclei induction end-point was employed, positive results were reported in erythrocytes of the fish *Cnesterodon decemmaculatus* and *Rhinella arenarum* tadpoles by Vera Candioti and collaborators (Vera Candioti et al., 2010a, b). Lastly, when a mammal model was employed for the micronuclei detection, Jones and Howard (1989) found negative results in rat bone marrow cells.

### **5. Comparison of the genotoxicity and cytotoxicity of carbofuran and pirimicarb and some Argentinean technical formulations**

One of the goals of our research group is to compare the genotoxic and cytotoxic effects exerted by the pesticide active ingredients (Pestanal®, Riedel-de Haën, Germany) and their technical formulations commonly used in Argentina on vertebrate cells both *in vitro* and *in vivo*.

The evaluation was performed using end-points for genotoxicity [Sister Chromatid Exchange, Chromosome Aberration, and Micronuclei frequencies] and cytotoxicity [Mitotic Index, Cell Viability, Proliferative Rate Index, Erythroblasts/Erythrocytes Ratio, 3(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Neutral Red assays] (Soloneski et al., 2008; Soloneski & Larramendy, 2010; Vera Candioti et al., 2010a, b).

We comparatively evaluated the genotoxic and cytotoxic *in vitro* effects on mammalian Chinese hamster ovary cells induced by the pure insecticide carbofuran and its commercial formulation Furadan® (47% carbofuran, FMC Argentina S.A., Buenos Aires, Argentina). Similarly, the genotoxic and cytotoxic *in vitro*-*in vivo* effects induced by the pure insecticide pirimicarb and its commercial formulation Aficida® (50% pirimicarb, Syngenta Agro S.A.,

were respectively applied (Käfer et al., 1982; Callander, 1995). Negative and positive results were obtained for the induction of mutagenicity in mouse lymphoma L5178Y cells regardless of the presence or absence of a rat liver metabolic activation system (Clay, 1996). Furthermore, the induction of DNA single strand breaks, estimated by the alkaline comet assay, was evaluated revealing positive results in human lymphocytes exposed *in vitro* to pirimicarb (Ündeger & Basaran, 2005). Similar positive results were found when the sister chromatid exchange assay was performed in Chinese hamster ovary cells (Soloneski & Larramendy, 2010). Although a significant increase in chromosomal aberrations has been reported in Chinese hamster ovary cells after pirimicarb exposure (Soloneski & Larramendy, 2010), Wildgoose and coworkers (1987) observed negative results in human lymphocytes with or without S9 metabolic activation. Finally, the induction of alterations in the cell-cycle progression in Chinese hamster ovary cells was reported to occur after *in vitro* exposure to

In *in vivo* genotoxic and cytotoxic studies, pirimicarb was able to induce different types of lesions. It has been reported the ability of the insecticide to give positive results by using the eye mosaic system *white/white*+ (*w/w*+) somatic mutation and recombination test (SMART) when *Drosophila melanogaster* was employed as experimental model (Aguirrezabalaga et al., 1994). However, McGregor and co-workers (1974) reported negative results in mice when the dominant lethal mutation assay was performed. Similar negative results were found by Kenelly (1990) using the unscheduled DNA synthesis in rat liver cells. At the chromosomal level, pirimicarb did not induce chromosomal alterations in bone marrow cells of Wistar male rats after oral administration (Anderson et al., 1980). Contrarily, Pilinskaia (1982) observed a significant increase of chromosomal aberrations in the peripheral blood lymphocytes from occupational workers after pirimicarb exposure. Finally, when the micronuclei induction end-point was employed, positive results were reported in erythrocytes of the fish *Cnesterodon decemmaculatus* and *Rhinella arenarum* tadpoles by Vera Candioti and collaborators (Vera Candioti et al., 2010a, b). Lastly, when a mammal model was employed for the micronuclei detection, Jones and Howard (1989) found negative

**5. Comparison of the genotoxicity and cytotoxicity of carbofuran and** 

One of the goals of our research group is to compare the genotoxic and cytotoxic effects exerted by the pesticide active ingredients (Pestanal®, Riedel-de Haën, Germany) and their technical formulations commonly used in Argentina on vertebrate cells both *in vitro* and *in* 

The evaluation was performed using end-points for genotoxicity [Sister Chromatid Exchange, Chromosome Aberration, and Micronuclei frequencies] and cytotoxicity [Mitotic Index, Cell Viability, Proliferative Rate Index, Erythroblasts/Erythrocytes Ratio, 3(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Neutral Red assays]

We comparatively evaluated the genotoxic and cytotoxic *in vitro* effects on mammalian Chinese hamster ovary cells induced by the pure insecticide carbofuran and its commercial formulation Furadan® (47% carbofuran, FMC Argentina S.A., Buenos Aires, Argentina). Similarly, the genotoxic and cytotoxic *in vitro*-*in vivo* effects induced by the pure insecticide pirimicarb and its commercial formulation Aficida® (50% pirimicarb, Syngenta Agro S.A.,

(Soloneski et al., 2008; Soloneski & Larramendy, 2010; Vera Candioti et al., 2010a, b).

**pirimicarb and some Argentinean technical formulations** 

pirimicarb (Soloneski & Larramendy, 2010).

results in rat bone marrow cells.

*vivo*.

Buenos Aires, Argentina) on mammalian Chinese hamster ovary cells as well as circulating erythrocytes of the fish *Cnesterodon decemmaculatus* and the amphibian *Rhinella arenarum*  tadpoles were also estimated.

A summary of the results obtained is presented in Fig. 3. The figure clearly reveals that all compounds assayed were able to inflict damage at chromosomal and cellular level regardless of the cellular system used as target.

We observed that carbofuran/Furadan® and pirimicarb/Aficida® caused SCEs on mammalian cells indicating that they have a clastogenic activity (Fig. 3A). It has been suggested that at the chromosomal level, the induction of SCEs is a reliable indicator for the screening of clastogens, since the bioassay is more sensitive than the analysis of clastogeninduced chromosomal aberrations (Palitti et al., 1982). The results also demonstrate the ability of pirimicarb/Aficida® to induce DNA damage quali- and quantitative analyzed by the frequency of chromosomal aberrations (Fig. 3B). Furthermore, a putative clastogenic/aneugenic activity exerted *in vitro* by carbofuran/Furadan® and *in viv*o by pirimicarb/Aficida® was also demonstrated by the ability of the pesticide-induced micronuclei (Fig. 3C). The analysis of the proliferative replication (Fig. 3D) and the mitotic indexes (Fig. 3E) demonstrated that both carbofuran/Furadan® and pirimicarb/Aficida® were able to delay the cell-cycle progression as well as to exert a marked reduction of the cellular mitotic activity on mammalian cells *in vitro*. Besides, carbofuran/Furadan® and pirimicarb/Aficida® were able to induced a clear cellular cytotoxicity. This deleterious effect was estimated by a loss of lysosomal activity (indicated by a decrease in the uptake of neutral red), as well as alteration in energy metabolism (measured by mitochondrial succinic dehydrogenase activity in the MTT assay), as clearly revealed in insecticides-treated Chinese hamster ovary cells (Fig. 3F).

Overall, the results revealed, depending upon the endpoint employed, that the damage induced by the commercial formulations of both insecticides is, in general, greater than that produced by the pure pesticides (Fig. 3). Unfortunately, the identity of the components present in the excipient formulations was not made available by the manufacturers. These final remarks are in accord with previous observations not only reported by us but also by other research groups indicating the presence of xenobiotics within the composition of the commercial formulations with genotoxic and cytotoxic effects (David, 1982; Lin & Garry, 2000; Soloneski et al., 2001, 2002, 2003, 2008; González et al., 2007a, b, 2009; Elsik et al., 2008; Molinari et al., 2009; Soloneski & Larramendy, 2010). These observations highlight that, in agriculture, agrochemicals are generally not used as a single active ingredient but as part of a complex commercial formulations. Thus, both the workers as well as non-target organisms are exposed to the simultaneous action of the active ingredient and a variety of other chemical/s contained in the formulated product. Hence, risk assessment must also consider additional geno-cytotoxic effects caused by the excipient/s.

Finally, the results highlight that a whole knowledge of the toxic effect/s of the active ingredient of a pesticide is not enough in biomonitoring studies as well as that agrochemical/s toxic effect/s should be evaluated according to the commercial formulation available in market. Furthermore, the deleterious effect/s of the excipient/s present within the commercial formulation should be neither discarded nor underestimated. The importance of further studies on this type of pesticide in order to achieve a complete knowledge on its genetic toxicology seems to be, then, more than evident.

Genetic Toxicological Profile of Carbofuran and Pirimicarb Carbamic Insecticides 531

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Fig. 3. Comparative genotoxicity and cytotoxicity effects induced by carbofuran and pirimicarb pure herbicides Pestanal® (grey) and their technical formulations Furadan® and Aficida® (black) commonly used in Argentina on mammalian Chinese hamster ovary cells (cylinders) and *in vivo* piscine and amphibian erythrocytes (bars). Results are expressed as fold-time values over control data. Evaluation was performed using end-points for genotoxicity [Sister Chromatid Exchanges (A), Chromosome Aberrations (B), Micronuclei (C)] and cytotoxicity [Proliferative Rate Index (D), Mitotic Index (E), 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Neutral Red (NR) (F)].

## **6. Acknowledgements**

This study was supported by grants from the National University of La Plata (Grant Numbers 11/N564, 11/N619) and the National Council for Scientific and Technological Research (CONICET, PIP N° 0106) from Argentina.

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530 Insecticides – Pest Engineering

Fig. 3. Comparative genotoxicity and cytotoxicity effects induced by carbofuran and pirimicarb pure herbicides Pestanal® (grey) and their technical formulations Furadan® and Aficida® (black) commonly used in Argentina on mammalian Chinese hamster ovary cells (cylinders) and *in vivo* piscine and amphibian erythrocytes (bars). Results are expressed as fold-time values over control data. Evaluation was performed using end-points for genotoxicity [Sister Chromatid Exchanges (A), Chromosome Aberrations (B), Micronuclei (C)] and cytotoxicity [Proliferative Rate Index (D), Mitotic Index (E), 3(4,5-Dimethylthiazol-

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## *Edited by Farzana Perveen*

This book is compiled of 24 Chapters divided into 4 Sections. Section A focuses on toxicity of organic and inorganic insecticides, organophosphorus insecticides, toxicity of fenitrothion and permethrin, and dichlorodiphenyltrichloroethane (DDT). Section B is dedicated to vector control using insecticides, biological control of mosquito larvae by Bacillus thuringiensis, metabolism of pyrethroids by mosquito cytochrome P40 susceptibility status of Aedes aegypti, etc. Section C describes bioactive natural products from sapindacea, management of potato pests, flower thrips, mango mealy bug, pear psylla, grapes pests, small fruit production, boll weevil and tsetse fly using insecticides. Section D provides information on insecticide resistance in natural population of malaria vector, role of Anopheles gambiae P450 cytochrome, genetic toxicological profile of carbofuran and pirimicarp carbamic insecticides, etc. The subject matter in this book should attract the reader's concern to support rational decisions regarding the use of pesticides.

Insecticides - Pest Engineering

Insecticides

Pest Engineering

*Edited by Farzana Perveen*

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