CHO supernatants and whole leaf extracts are prepared for purification.

+ BChE activity is determined spectrophotometrically (Grunwald at al., 1997), using butyrylthiocholine (BTC) (0.5 mM each) as substrate. One unit of enzyme activity is the amount required to hydrolyze 1 umol substrate/min. One mg MaBChE has 900 units of activity and one mg HuBChE has 700units.

Table 1. Expression levels of different forms of rBChE using CHO-and plant-based expression systems.

In addition to the tetrameric forms, a truncated monomeric form of rBChE (MW=~81KDa) that is incapable of oligomerization has also been produced by the insertion of a stop codon at G534 resulting in a monomeric form lacking 41 C-terminal residues (Blong et al., 1997). The smaller monomeric molecules may more rapidly gain access to the blood from muscle or lungs (depending on the route of delivery) with transiently higher bioavailablity in the plasma, which would be advantageous in emergency situations that require real time responses and rapid treatment or booster administrations.

Development of a Prophylactic Butyrylcholinesterase

**Et/E x 100%**

**Et/E x 100%**

**0% 20% 40% 60% 80% 100%**

**0% 20% 40% 60% 80% 100%** **6.4mM 3.2mM 1.6mM 0.8mM 0.4mM**

**6.4mM 3.2mM 1.6mM 0.8mM 0.4mM**

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 83

Since a 1 hour incubation of 0.016 uM plant-derived MaBChE (1.2U/ml) with 0.02uM paraoxon resulted in 80-90% inhibition of the enyme (Fig. 1A), the same conditions (incubation of paraoxon with rMaBChE at a final enzyme concentration of 0.04-0.05uM), was used to prepare inhibited rMaBChE. Reactivation of inhibited rMaBChE was then initiated by adding different concentrations of 2-PAM (0.4mM-6.4mM) for various times (Fig.2). The kinetics of reactivation of paraoxon-inhibited CHO- and plant-derived rMaBChE were

> **Et/E x 100%**

> **Et/E x 100%**

Fig. 2. Reactivation kinetics of paraoxon inhibited plant- and CHO-derived MaBChE by 2- PAM. A, C: Plant-derived MaBChE; B, D: CHO-derived MaBChE; A and B: Direct plot of the time course vs % reactivation; C and D: Semi-logarithmic plot of time course of reactivation. For inhibition controls, inhibited BChE was incubated with reaction buffer without 2-PAM.

**0% 20% 40% 60% 80% 100%**

**0% 20% 40% 60% 80% 100%** **6.4mM 3.2mM 1.6mM 0.8mM 0.4mM**

**6.4mM 3.2mM 1.6mM 0.8mM 0.4mM**

**0 60 120 180 240 300 360 420 480 Time (min)**

**0 60 120 180 240 300 360 420 480 Time (min)**

**3.2 Reactivation of paraoxon-inhibited plant-derived rMaBChE by 2-PAM** 

found to follow the simple first-order (mono-exponential) model.

**0 60 120 180 240 300 360 420 480 Time (min)**

**0 60 120 180 240 300 360 420 480 Time (min)**

A B

C D

Triplicate BChE assays were performed at the times indicated.

#### **3. In vitro biological properties of rMaBChE**

To test the chemical properties of CHO- and tobacco-derived rMaBChE, inhibition and reactivation assays using diisopropyl fluorophosphate (DFP) and paraoxon (diethyl 4 nitrophenyl phosphate) have been performed with and without the oxime 2-PAM (pyridine-2-aldoxime methochloride)(Luo et al., 2008). DFP is an OP compound that has been used as an experimental insecticide agent in neuroscience because of its ability to inhibit cholinesterase and cause delayed peripheral neuropathy. Paraoxon is an insecticide and will be described in detail in a later section. Following purification of the CHO supernatant and the plant leaf extract using procainamide sepharose, rMaBChE molecules conjugated with polyetheleneglycol (PEG) using succinimidyl-propionate-activated methoxy-PEG-20K (SPA-PEG-20K; Nektar Inc., Birmingham, AL) or Sunbright ME-200HS 20K PEG (NOF, Tokyo, Japan) (Chilukuri et al., 2008a; Cohen et al., 2001) to test the effects of PEGylation on enzyme plasma stability. Initially, the biochemical properties of both the unmodified and PEGylated forms of both monomeric and tetrameric rMaBChE were examined using DFP inhibition; bimolecular rate constants (ki ( 107) M-1 min-1) for inhibition of all the recombinants forms ranging from 2.58 - 2.23 ( 107) M-1 min-1 which were indistinguishable from the well characterized native HuBChE (2.29 +/- 0.1) and native MaBChE (2.22 +/- 0.1) (data not shown).

#### **3.1 Inhibition and reactivation of plant derived CHO-derived and plant-derived rBChE by paraoxon**

The kinetics of inhibition of both plant-derived and CHO-derived rMaBChE by paraoxon were further examined as shown in Fig. 1A. At low paraoxon concentrations (0.01 and 0.02*uM*), the reciprocal value of *Et/Et,0* was highly correlated with the reaction time; the reaction rate constant of plant-derived rMaBChE at 0.01*uM* paraoxon being slightly faster than that of CHO-derived MaBChE (0.035 *M-1min-1* vs 0.022 *M-1min-1 respectively).* These values follow the simple 2nd-order (reciprocal) model.

Fig. 1. Inhibition kinetics of plant- and CHO-derived\* rMaBChE by different concentrations of paraoxon (0.01uM - 0.10uM) A: Percent inhibition of BChE by paraoxon. (Percent BChE activity was obtained by dividing the BChE activity with paraoxon at each time point with control BChE activity at the same time point. B: Reciprocal plot of BChE inhibition by paraoxon.

#### **3.2 Reactivation of paraoxon-inhibited plant-derived rMaBChE by 2-PAM**

82 Insecticides – Basic and Other Applications

To test the chemical properties of CHO- and tobacco-derived rMaBChE, inhibition and reactivation assays using diisopropyl fluorophosphate (DFP) and paraoxon (diethyl 4 nitrophenyl phosphate) have been performed with and without the oxime 2-PAM (pyridine-2-aldoxime methochloride)(Luo et al., 2008). DFP is an OP compound that has been used as an experimental insecticide agent in neuroscience because of its ability to inhibit cholinesterase and cause delayed peripheral neuropathy. Paraoxon is an insecticide and will be described in detail in a later section. Following purification of the CHO supernatant and the plant leaf extract using procainamide sepharose, rMaBChE molecules conjugated with polyetheleneglycol (PEG) using succinimidyl-propionate-activated methoxy-PEG-20K (SPA-PEG-20K; Nektar Inc., Birmingham, AL) or Sunbright ME-200HS 20K PEG (NOF, Tokyo, Japan) (Chilukuri et al., 2008a; Cohen et al., 2001) to test the effects of PEGylation on enzyme plasma stability. Initially, the biochemical properties of both the unmodified and PEGylated forms of both monomeric and tetrameric rMaBChE were examined using DFP inhibition; bimolecular rate constants (ki ( 107) M-1 min-1) for inhibition of all the recombinants forms ranging from 2.58 - 2.23 ( 107) M-1 min-1 which were indistinguishable from the well characterized native HuBChE (2.29 +/- 0.1) and native MaBChE (2.22 +/- 0.1) (data not

**3.1 Inhibition and reactivation of plant derived CHO-derived and plant-derived rBChE** 

The kinetics of inhibition of both plant-derived and CHO-derived rMaBChE by paraoxon were further examined as shown in Fig. 1A. At low paraoxon concentrations (0.01 and 0.02*uM*), the reciprocal value of *Et/Et,0* was highly correlated with the reaction time; the reaction rate constant of plant-derived rMaBChE at 0.01*uM* paraoxon being slightly faster than that of CHO-derived MaBChE (0.035 *M-1min-1* vs 0.022 *M-1min-1 respectively).* These

**0**

 **A B** 

Fig. 1. Inhibition kinetics of plant- and CHO-derived\* rMaBChE by different concentrations of paraoxon (0.01uM - 0.10uM) A: Percent inhibition of BChE by paraoxon. (Percent BChE activity was obtained by dividing the BChE activity with paraoxon at each time point with control BChE activity at the same time point. B: Reciprocal plot of BChE inhibition by

**0 10 20 30 40 50 60 70 80 90 Time (min)**

**0.01uM \* 0.01uM 0.02uM 0.04uM**

**1**

**2**

**3**

**(Et/Et,0)-1**

**4**

**5**

**0.01uM \* 0.01uM 0.02uM 0.04uM 0.06uM 0.08uM 0.10uM \* 0.10uM**

**3. In vitro biological properties of rMaBChE** 

values follow the simple 2nd-order (reciprocal) model.

**0 10 20 30 40 50 60 70 80 90 Time (min)**

shown).

**by paraoxon**

**0%**

paraoxon.

**20% 40%**

**60%**

**(Et/Et,0) x 100%**

**80%**

**100%**

Since a 1 hour incubation of 0.016 uM plant-derived MaBChE (1.2U/ml) with 0.02uM paraoxon resulted in 80-90% inhibition of the enyme (Fig. 1A), the same conditions (incubation of paraoxon with rMaBChE at a final enzyme concentration of 0.04-0.05uM), was used to prepare inhibited rMaBChE. Reactivation of inhibited rMaBChE was then initiated by adding different concentrations of 2-PAM (0.4mM-6.4mM) for various times (Fig.2). The kinetics of reactivation of paraoxon-inhibited CHO- and plant-derived rMaBChE were found to follow the simple first-order (mono-exponential) model.

Fig. 2. Reactivation kinetics of paraoxon inhibited plant- and CHO-derived MaBChE by 2- PAM. A, C: Plant-derived MaBChE; B, D: CHO-derived MaBChE; A and B: Direct plot of the time course vs % reactivation; C and D: Semi-logarithmic plot of time course of reactivation. For inhibition controls, inhibited BChE was incubated with reaction buffer without 2-PAM. Triplicate BChE assays were performed at the times indicated.

Development of a Prophylactic Butyrylcholinesterase

in monkeys and mice (Rosenberg et al., 2010).

tetrameric form despite lack of oligomerization.

and monkeys (Cohen et al., 2004).

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 85

Generally pharmacokinetics of recombinant molecules differs considerably depending on the structure, glycoslyation, size, route of administration, immunogenicity, and animal model utilized. Interestingly, despite protein sequence identity, rBChE proteins, similar to many other recombinant biologics, have been shown to be rapidly cleared following injection (Saxena et al., 1998; Cohen et al., 2006) in contrast to the good plasma stability of native BChE. Thus, rBChE molecules require post-translational modification to provide protection as therapeutic scavengers. A common means of increasing the radius of the target molecule permitting slower renal clearance and prolonging plasma retention is by PEG conjugation. This has been successfully used with proteins, peptides, oliogonucleotides and antibody fragments to improve pharmacokinetic and immunological profiles (Kang et al., 2009). Accordingly, both monomeric and tetrameric forms of rMaBChE have been conjugated with 20KD PEG (without interference of in vitro biological properties) and the pharmacokinetic profiles of the unmodified and PEG-conjugated rMaBChE forms compared

Figure 3 shows the PK profiles in 24 monkeys following iv injection of 1.2 -3 mg/kg of unmodified or PEG-rMaBChE and illustrates several aspects of BChE clearance: (i) PEGrMaBChE exhibits good stability in the lower range of the native form; the hierarchy of clearance being native BChE ~ PEG-rMaBChE >>> unmodified monomeric rMaBChE > unmodified tetrameric rMaBChE. (ii) Surprisingly, five of the monkeys demonstrated unexpected dramatic decreases in BChE levels (shown in bold between days 150 and 230 days post injection). In each case, these decreases always occurred immediately after the weekend treatment of the grass surrounding the animal facility and presumably resulted from exposure of the housed monkeys to insecticide; highlighting the unintentional consequences of routine insecticide use on plasma BChE activity and (iii) despite very poor retention of the unmodified monomeric rBChE, administration of the PEGylated monomeric rMaBChE showed overlapping pharmacokinetic parameters with the larger PEG-rMaBChE

Importantly, the extended circulatory retention afforded by PEG conjugation of rMaBChE in monkeys (injected iv) was not observed in mice (injected ip) where unmodified and modified monomeric and tetrameric rMaBChE all exhibited the same high MRT and T1/2 (Rosenberg et al., 2010). This indicates that, depending on the parameter measured, the mouse model does not accurately predict the outcome in monkeys with MRT and T1/2 values appearing to be less predictive indicators of circulatory stability in macaques than parameters such as AUC and Cmax. Similar differential pharmacokinetic behaviour was observed following the administration of recombinant rhesus (Rh) and HuAChE in mice

These studies highlight the potential problems inherent in choosing an animal model to test human biologics. Notwithstanding the differences in pharmacokinetic behaviour of the same protein in different species and the high potential for immunogenicity in rodents due to the evolutionary distance between rodents and humans, other influences may also play a role in the circulatory stability of proteins following even the first injections into heterologous species. Table 2 shows the pharmacokinetic parameters (MRT, Cmax, Tmax, T1/2 and AUC ) following injection of different forms of BChE into several different animal species determined from the time course curve of blood BChE concentrations and using a Windows-based program for non-compartmental analysis. Several conclusions can be made.

The results indicate that both paraoxon-inhibited plant- and CHO-derived rMaBChE showed very similar patterns of reactivation by different concentrations of 2-PAM (Fig. 2A, 2B) with nearly 100 % reactivation of each rMaBChE form being achieved by 24 hours at >1.60 mM 2-PAM; the *kapp* values of CHO-rMaBChE ranging from 0.0014 to 0.004 min-1 and plant-rMaBChE from 0.0013 to 0.0051 min-1 (Fig. 2C, 2D). The reactivation *kapp*s at each 2- PAM concentration was linear when plotted against 2-PAM concentration (mM) expressed logrithmically.

#### **4. In vivo testing of rBChE**

In the area of insecticide exposure/contamination, there is a high likelihood that agricultural workers or military personnel will be exposed multiple times during their lives and thus multiple prophylactic treatments must be considered a possibly. This is often problematic since administration of heterologous HuBChE into macaques or other species eg mice has been shown to generate anti-BChE antibody responses and rapidly eliminate enzyme on repeated injections (Matzke et al., 1999; Chiluluri et al., 2008b; Sun et al., 2009). Thus, in vivo retention times of exogenously administered recombinant proteins can only be accurately assessed using homologous systems (rMaBChE macaques and rHuBChE humans) in which antibodies or other immune responses are not induced. In this context, homologous BChE enzyme has been shown to have a long half-life (8-12 days) with no adverse effects and no immunogenicity following either (i) transfusions of human plasma into humans (ii) daily administrations of partially purified native HuBChE into humans for several weeks (Jenkins et al., 1967; Cascio et al., 1988) or (iii) injection of purified native MaBChhE or PEG-rMaBChE into macaques (MRT= 200-300 h)(Rosenberg et al., 2002, 2010). These data are in contrast to exogenously administered heterologous HuBChE which displayed a rapid clearance in macaques (MRT = 33.7 h) (Raveh et al., 1989). While the choice of the animal model for PK, immunogenicity and efficacy testing is always important, the animal species utilized for the evaluation of an efficacious human cholinesterase bioscavenger is critical, since potential treatments against OP toxicity cannot be tested in humans and will require extensive testing in animal models and the Animal Rule (CFR 601.90 for biologics) for regulatory approval.

#### **4.1 Pharmacokinetics of clearance in rodent and macaque models**

Pharmacokinetic profiles following administration of biologics in many rodent and primate species are used to indicate the periods after administration that such biologics are likely to exhibit optimal benefit or protection. An efficacious therapeutic for preventing OP poisoning is a molecule that: (i) can bind and scavenge the OP before it reaches the targeted AChE in neuromuscular junctions and (ii) has the ability to remain at therapeutic levels in the blood for prolonged periods to counteract a known or impending OP exposure. The in vivo parameters generally used to assess PK performance after administration are mean retention time (MRT), maximal concentration (Cmax), time to reach maximal concentration (Tmax), elimination half life (T1/2) and area under the plasma concentration curve extrapolated to infinity (AUC).

The results indicate that both paraoxon-inhibited plant- and CHO-derived rMaBChE showed very similar patterns of reactivation by different concentrations of 2-PAM (Fig. 2A, 2B) with nearly 100 % reactivation of each rMaBChE form being achieved by 24 hours at >1.60 mM 2-PAM; the *kapp* values of CHO-rMaBChE ranging from 0.0014 to 0.004 min-1 and plant-rMaBChE from 0.0013 to 0.0051 min-1 (Fig. 2C, 2D). The reactivation *kapp*s at each 2- PAM concentration was linear when plotted against 2-PAM concentration (mM) expressed

In the area of insecticide exposure/contamination, there is a high likelihood that agricultural workers or military personnel will be exposed multiple times during their lives and thus multiple prophylactic treatments must be considered a possibly. This is often problematic since administration of heterologous HuBChE into macaques or other species eg mice has been shown to generate anti-BChE antibody responses and rapidly eliminate enzyme on repeated injections (Matzke et al., 1999; Chiluluri et al., 2008b; Sun et al., 2009). Thus, in vivo retention times of exogenously administered recombinant proteins can only be accurately assessed using homologous systems (rMaBChE macaques and rHuBChE humans) in which antibodies or other immune responses are not induced. In this context, homologous BChE enzyme has been shown to have a long half-life (8-12 days) with no adverse effects and no immunogenicity following either (i) transfusions of human plasma into humans (ii) daily administrations of partially purified native HuBChE into humans for several weeks (Jenkins et al., 1967; Cascio et al., 1988) or (iii) injection of purified native MaBChhE or PEG-rMaBChE into macaques (MRT= 200-300 h)(Rosenberg et al., 2002, 2010). These data are in contrast to exogenously administered heterologous HuBChE which displayed a rapid clearance in macaques (MRT = 33.7 h) (Raveh et al., 1989). While the choice of the animal model for PK, immunogenicity and efficacy testing is always important, the animal species utilized for the evaluation of an efficacious human cholinesterase bioscavenger is critical, since potential treatments against OP toxicity cannot be tested in humans and will require extensive testing in animal models and the Animal Rule (CFR 601.90 for biologics) for regulatory

**4.1 Pharmacokinetics of clearance in rodent and macaque models** 

Pharmacokinetic profiles following administration of biologics in many rodent and primate species are used to indicate the periods after administration that such biologics are likely to exhibit optimal benefit or protection. An efficacious therapeutic for preventing OP poisoning is a molecule that: (i) can bind and scavenge the OP before it reaches the targeted AChE in neuromuscular junctions and (ii) has the ability to remain at therapeutic levels in the blood for prolonged periods to counteract a known or impending OP exposure. The in vivo parameters generally used to assess PK performance after administration are mean retention time (MRT), maximal concentration (Cmax), time to reach maximal concentration (Tmax), elimination half life (T1/2) and area under the plasma concentration curve

logrithmically.

approval.

extrapolated to infinity (AUC).

**4. In vivo testing of rBChE** 

Generally pharmacokinetics of recombinant molecules differs considerably depending on the structure, glycoslyation, size, route of administration, immunogenicity, and animal model utilized. Interestingly, despite protein sequence identity, rBChE proteins, similar to many other recombinant biologics, have been shown to be rapidly cleared following injection (Saxena et al., 1998; Cohen et al., 2006) in contrast to the good plasma stability of native BChE. Thus, rBChE molecules require post-translational modification to provide protection as therapeutic scavengers. A common means of increasing the radius of the target molecule permitting slower renal clearance and prolonging plasma retention is by PEG conjugation. This has been successfully used with proteins, peptides, oliogonucleotides and antibody fragments to improve pharmacokinetic and immunological profiles (Kang et al., 2009). Accordingly, both monomeric and tetrameric forms of rMaBChE have been conjugated with 20KD PEG (without interference of in vitro biological properties) and the pharmacokinetic profiles of the unmodified and PEG-conjugated rMaBChE forms compared in monkeys and mice (Rosenberg et al., 2010).

Figure 3 shows the PK profiles in 24 monkeys following iv injection of 1.2 -3 mg/kg of unmodified or PEG-rMaBChE and illustrates several aspects of BChE clearance: (i) PEGrMaBChE exhibits good stability in the lower range of the native form; the hierarchy of clearance being native BChE ~ PEG-rMaBChE >>> unmodified monomeric rMaBChE > unmodified tetrameric rMaBChE. (ii) Surprisingly, five of the monkeys demonstrated unexpected dramatic decreases in BChE levels (shown in bold between days 150 and 230 days post injection). In each case, these decreases always occurred immediately after the weekend treatment of the grass surrounding the animal facility and presumably resulted from exposure of the housed monkeys to insecticide; highlighting the unintentional consequences of routine insecticide use on plasma BChE activity and (iii) despite very poor retention of the unmodified monomeric rBChE, administration of the PEGylated monomeric rMaBChE showed overlapping pharmacokinetic parameters with the larger PEG-rMaBChE tetrameric form despite lack of oligomerization.

Importantly, the extended circulatory retention afforded by PEG conjugation of rMaBChE in monkeys (injected iv) was not observed in mice (injected ip) where unmodified and modified monomeric and tetrameric rMaBChE all exhibited the same high MRT and T1/2 (Rosenberg et al., 2010). This indicates that, depending on the parameter measured, the mouse model does not accurately predict the outcome in monkeys with MRT and T1/2 values appearing to be less predictive indicators of circulatory stability in macaques than parameters such as AUC and Cmax. Similar differential pharmacokinetic behaviour was observed following the administration of recombinant rhesus (Rh) and HuAChE in mice and monkeys (Cohen et al., 2004).

These studies highlight the potential problems inherent in choosing an animal model to test human biologics. Notwithstanding the differences in pharmacokinetic behaviour of the same protein in different species and the high potential for immunogenicity in rodents due to the evolutionary distance between rodents and humans, other influences may also play a role in the circulatory stability of proteins following even the first injections into heterologous species. Table 2 shows the pharmacokinetic parameters (MRT, Cmax, Tmax, T1/2 and AUC ) following injection of different forms of BChE into several different animal species determined from the time course curve of blood BChE concentrations and using a Windows-based program for non-compartmental analysis. Several conclusions can be made.

Development of a Prophylactic Butyrylcholinesterase

**BChE Dose**

BChE Dose

nat: native, Mon: monomeric, Tet: tetramer.

heterologous systems.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 87

**Human and Mouse BChE**

(hr)

(hr)

1.9 mg/kg Monkey iv 200 2097 26 157

1.9 mg/kg Monkey iv 134 1724 24 97

8.75 mg/kg (20,000 U) Monkey im 3822 33 10.3 73.5

**AUC** (U/ml.h)

AUC (U/ml.h)

**Cmax** (U/ml)

Cmax (U/ml) **Tmax** (hr)

Tmax (hr)

**T1/2**  (hr)

T1/2 (hr)

[Units, mg, mg/kg] **Animal Route MRT**

natHuBChE 11.5 mg (8,000 U) Monkey im 582 16.2 9.5

natHuBChE (Lenz,2005) 5.25 mg/kg (12,000 U) Monkey im 2576 21 9.27 79.3

natHuBChE (Sun, 2005) 34 mg/kg (30,000 U) Monkey iv 73 16,538 222 0 37 natHuBChE (Sun, 2009) 100 U Mouse im 48 1,300 19 21 natMaBChE \* 100 U Mouse im 73 2,500 25 24

Monkey BChE

[Units, mg, mg/kg] Animal Route MRT

natMaBChE\* 2.9 mg/kg Monkey iv 224 4431 38 143 (Rosenberg, 2010) 2.9 mg/kg Monkey iv 307 4299 40 126

PEG-rMaBChE\* 2.9 mg/kg Monkey iv 168 2141 33 112 (Rosenberg, 2010) 2.9 mg/kg Monkey iv 223 3312 39 85

MRT: mean retention time, Cmax: maximal concentration, Tmax: time to reach maximal concentration, T1/2: elimination half life, AUC: area under the plasma concentration curve extrapolated to infinity.

The BChE molecule is a soluble protein, protected from proteolysis by a heavy sugar coating from nine N-linked glycans (Li et al., 2008). N-glycosylation is one of the major posttranslational modifications of proteins and can be critical to their bioavailability. Importantly, while the first steps in the *N*-glycosylation pathway, leading to the formation of oligomannosidic structures, are conserved in plants and animals, the final steps in the formation of complex N-glycans may differ with the expression system used. Thus, in contrast to native HuBChE molecules which have highly sialylated bi- and triantennary type glycans (Saxena et al., 1998; Kolarich et al. 2008) containing the N-acetyl neuraminic acid (NANA, NeuAc) form of sialic acid (Varki, 2001), rHuBChE molecules may exhibit undersialyated or immunogenic non-human glycan structures that accelerate in vivo clearance

Table 2. Pharmacokinetic parameters of different forms of BChE in homologous**\*** and

1.8 mg/kg Monkey iv 142 4010 37

natMaBChE (unpub)\* 1.8 mg/kg Monkey iv 142 2950 27

PEG-rMaBChE (unpub)\* 3.0 mg/kg Monkey iv 4359 51 PEG-rHuBChE (unpub)\* 3.0 mg/kg Monkey iv 1101 40

**4.2 The role of glycosylation and oligomerization on pharmacokinetics** 

natHuBChE (Raveh,1997) 11.5 mg (8,000 U) Monkey iv 33 710

natMaBChE \* 3 -5 mg/kg (7,000 U) Monkey iv 191 (Rosenbberg, 2002) 1.3 - 1.65 mg/kg (3,000 U) Monkey iv 50

Fig. 3. Pharmacokinetics of clearance following iv injection of 1.2 - 3.0 mg/kg rMaBChE into 24 monkeys. Each line represents a single monkey. Different forms of rMaBChE were used except for 4 macaques receiving native BChE.

For example, while the Cmax following first injections appear to be similar in any animal model at comparable doses, the AUC, MRT and T1/2 are often significantly higher in homologous systems (e.g. PEG-rMaBChE into macaques and native mouse (Mo) BChE into mice) than heterologous injections (native HuBChE into monkeys or mice or PEG-rHuBChE into monkeys). This indicates that heterologous proteins, even when PEGylated and given at a time when anti-BChE titers are absent or low, appear to be eliminated faster than homologous proteins suggesting that pharmacokinetic parameters are less than optimal in all heterologous systems.

It should also be noted, that while PEG conjugation markedly improves the pharmacokinetic profile of therapeutic rMaBChE and other biologics, effects relating to immunogenicity have been mixed. Thus, reduced immunogenicity has been observed following PEGylation of enzymes, cytokines and hormones, while administration of PEGylated interferon-1a to monkeys actually resulted in increased immunogenicity (Pepinsky et al., 2001). In the case of rHuBChE produced in HEK-293 cells, PEGylation failed to eliminate immunogenicity in mice as demonstrated by the rapid clearance of a repeat 100U injection of (heterologous) PEG-rHuBChE, coincident with induction of high levels of serum anti-BChE antibody ( Sun et al., 2009). Likewise, when tested in a sandwich ELISA, the presence of 4–7 PEG molecules per rMaBChE monomer did not prevent the binding of BChE epitopes to either an anti-BChE MAb or a polyclonal rabbit anti-BChE antibody when antigen concentrations were increased to as little as 4–8 U/ml (Rosenberg et al., 2010) which, as mentioned above, is in the range of BChE in normal plasma. These studies raise the question whether chemical modification by PEG will be able to mask any "foreign" rBChE epitopes, such as non-human glycans, sufficient to prevent humoral immune responses and also highlights the importance of using homologous animal models to perform in vivo PK, immunogenicity and efficacy testing.

**PEG-tet 1 PEG-tet 2 PEG-tet 3 PEG-tet 4 PEG-tet 11 PEG-tet 12 PEG-tet 13 PEG-tet 14 native 5 native 6 native 7 native 8 non-PEG tet 9 non-PEG tet 10 non-PEG tet 15 non-PEG tet 16 PEG-mon 17 PEG-mon 18 PEG-mon 23 PEG-mon 24 non-PEG-mon 19 non-PEG-mon 20 non-PEG-mon 21 non-PEG-mon 22**

**0 50 100 150 200 250 300 Time (hr)**

Fig. 3. Pharmacokinetics of clearance following iv injection of 1.2 - 3.0 mg/kg rMaBChE into 24 monkeys. Each line represents a single monkey. Different forms of rMaBChE were used

For example, while the Cmax following first injections appear to be similar in any animal model at comparable doses, the AUC, MRT and T1/2 are often significantly higher in homologous systems (e.g. PEG-rMaBChE into macaques and native mouse (Mo) BChE into mice) than heterologous injections (native HuBChE into monkeys or mice or PEG-rHuBChE into monkeys). This indicates that heterologous proteins, even when PEGylated and given at a time when anti-BChE titers are absent or low, appear to be eliminated faster than homologous proteins suggesting that pharmacokinetic parameters are less than optimal in

It should also be noted, that while PEG conjugation markedly improves the pharmacokinetic profile of therapeutic rMaBChE and other biologics, effects relating to immunogenicity have been mixed. Thus, reduced immunogenicity has been observed following PEGylation of enzymes, cytokines and hormones, while administration of PEGylated interferon-1a to monkeys actually resulted in increased immunogenicity (Pepinsky et al., 2001). In the case of rHuBChE produced in HEK-293 cells, PEGylation failed to eliminate immunogenicity in mice as demonstrated by the rapid clearance of a repeat 100U injection of (heterologous) PEG-rHuBChE, coincident with induction of high levels of serum anti-BChE antibody ( Sun et al., 2009). Likewise, when tested in a sandwich ELISA, the presence of 4–7 PEG molecules per rMaBChE monomer did not prevent the binding of BChE epitopes to either an anti-BChE MAb or a polyclonal rabbit anti-BChE antibody when antigen concentrations were increased to as little as 4–8 U/ml (Rosenberg et al., 2010) which, as mentioned above, is in the range of BChE in normal plasma. These studies raise the question whether chemical modification by PEG will be able to mask any "foreign" rBChE epitopes, such as non-human glycans, sufficient to prevent humoral immune responses and also highlights the importance of using homologous animal models to perform in vivo PK,

except for 4 macaques receiving native BChE.

all heterologous systems.

immunogenicity and efficacy testing.

**BChE activity (U/ml)**


MRT: mean retention time, Cmax: maximal concentration, Tmax: time to reach maximal concentration, T1/2: elimination half life, AUC: area under the plasma concentration curve extrapolated to infinity. nat: native, Mon: monomeric, Tet: tetramer.

Table 2. Pharmacokinetic parameters of different forms of BChE in homologous**\*** and heterologous systems.

#### **4.2 The role of glycosylation and oligomerization on pharmacokinetics**

The BChE molecule is a soluble protein, protected from proteolysis by a heavy sugar coating from nine N-linked glycans (Li et al., 2008). N-glycosylation is one of the major posttranslational modifications of proteins and can be critical to their bioavailability. Importantly, while the first steps in the *N*-glycosylation pathway, leading to the formation of oligomannosidic structures, are conserved in plants and animals, the final steps in the formation of complex N-glycans may differ with the expression system used. Thus, in contrast to native HuBChE molecules which have highly sialylated bi- and triantennary type glycans (Saxena et al., 1998; Kolarich et al. 2008) containing the N-acetyl neuraminic acid (NANA, NeuAc) form of sialic acid (Varki, 2001), rHuBChE molecules may exhibit undersialyated or immunogenic non-human glycan structures that accelerate in vivo clearance

Development of a Prophylactic Butyrylcholinesterase

**4.3 Effects of the route of administration on pharmacokinetics** 

rodents (Lenz et al., 2005; Mumford et al, 2010; Saxena, et al., 2011).

apoplast (complex) (Stoger et al., 2005)

monkeys following im and sc injections.

**4.3.1 Intramuscular delivery of PEG-rMaBChE** 

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 89

terminating in sialic acid (Paccalet et al., 2007; Castilho et al., 2010). In addition, different glycoforms of plant derived proteins can be generated by protein targeting to different compartments (i) cytosol (aglycosylated) (ii) ER (high mannose) or (iii) secreted into the

As mentioned, delivery of PEG-rBChE as a pre-exposure modality is disadvantaged by its large size and a 1:1 stoichiometry between the enzyme and OP requiring high doses due to the high LD50 of many insecticides (ug-mg/kg levels). The route of systemic delivery of high doses of native BChE (MW~350KDa) and tetrameric PEG-rMaBChE (MW>800KDa) will determine the pharmacokinetics (PK) of clearance and is critical to efficacy and safety. Currently very little monkey data exists on the delivery of a stoichiometrically equivalent dose of PEG-rBChE calculated to protect against a known LD50 of a toxic OP insecticide. Although immediate release requiring intravenous (iv) injection may be necessary in certain high threat situations, these are usually impractical in the field. Needleless cutaneous delivery via the dermis and epidermis (chemical mediators, electroporation) appear quite promising, but are unlikely to deliver high doses. Thus, self-administered transdermal injections through the skin either by subcutaneous (sc) or intramuscular (im) routes have been the approaches most commonly used; virtually all human vaccines currently on the market being administered via these routes. Traditionally, autoinjectors, devices for im delivery of a self administered single dose of a drug are used in the military to protect personnel from chemical warfare agents and are currently used to deliver morphine for pain and atropine, diazepam and 2-PAM-Cl for first-aid against nerve agents. For this reason, most animal protection studies with OP bioscavengers have routinely been delivered im to

Despite all the pharmacokinetics data generated using im and sc routes of delivery of many drugs and biologics, little is known about the factors that govern the rate and extent of protein absorption from the injection site and the role of the lymphatic system in the transport of large molecules to the systemic circulation. With smaller molecules, the time to maximal concentration is usually shorter following im injections compared to sc injections where absorption is slow and prolonged and accounts for the lag in entering the blood. However with larger therapeutic molecules (MW>16KDa), the lymphatics are thought by some groups to be the primary route of absorption from sc (and im) injection sites. Large molecules are thought to exit the interstitium via cleft like openings into the lymph and enter the systemic circulation via the thoracic duct (Supersaxo et al., 1990; Porter et al., 2001; McLennan et L., 2006). To assess the effects of different routes of delivery, pharmacokinetic behaviour using different doses of PEG-rMaBChE tetrameric molecules was compared in

Four monkeys each received an im injection of either 2.5 or 3 mg/kg of PEG-rMaBChE. As shown in Fig. 4, unlike the delivery of the smaller native HuBChE which appear to behave uniformly following im injection (Lenz et al., 2005), the much larger PEG-conjugated form exhibits very variable results when delivered into the muscle with Tmax values in the 4 macaques having values of 8, 24, 48 and 48 hr respectively; the 8-hour peak looking more like an iv injection than an im injection. It is not clear whether this more rapid exit from the

due to rapid uptake by asialoglycoprotein and mannose receptors in the liver or by antibody-mediated mechanisms (Park et al., 2005). For example, CHO cells produce recombinant proteins which contain human-like glycans that may be undersialyted, compared to those produced in livestock systems which append the non-human galactose- -1,3-galactose and the N-glycolyl neuraminic (NGNA, NeuGc) form of sialic acid (Chung et al., 2008; Diaz et al., 2009) and those produced in plants which are non-sialylated and append the non-human -1,2 xylose and -1,3 fucose containing glycans (Altmann, 2007).

The relationship between sialic acid levels and oligomerization of recombinant molecules with their circulatory longevity has been extensively studied. For example, administration to mice of recombinant bovine and rhesus acetylcholinesterase (rBoAChE, rRhAChE) as well as plant-derived rHuBChE have supported the idea that pharmacokinetic behaviour is governed by hierarchical rules (Kronman et al., 2000); efficient enzyme tetramerization and high sialic acid occupancy both being required for optimal plasma retention. However, other data from monkey and mice studies do not closely obey these classical rules for circulatory retention. For example: (i) the requirement for tetramerization of rAChE molecules was less important when performed in macaques rather than mice (Cohen et al., 2004) (ii) CHO-derived monomeric PEG-rMaBChE resulted in high MRT when injected into in monkeys (Fig.3, Rosenberg et al., 2010) and (iii) the MRT and T1/2 of unmodified and PEG-modified monomeric rMaBChE were both unexpectedly high following injection into mice; PEG-conjugation offering no significant advantages.

While the short lived circulatory retention of asialylated BChE attests to the importance of sialylation in retention/clearance, the degree to which silaic acid occupancy is required does not always seem straight forward. Thus, although the rapid clearance of monomeric (13% non-silayted) and tetrameric (25% nonsialyted) rMaBChE in monkeys, compared to the native or PEGylated forms, has been thought to result from undersialylation, glycan analysis by MALDI-TOF of the highly stable native HuBChE and MaBChE proteins indicates that these also contain a significant percentage of nonsialyted or undersilayted proteins. For example, native HuBChE contains 23% monosialyted glycans (99.9% NANA) and a significant percentage of non-sialyted glycans (Kolarich et al., 2008) while native MaBChE is comprised of 21.3% non-sialayted glycans and 21.8% monosialylated glycans ( 99.9% NGNA) (Rosenberg, unp. data). This means that heterologous animal models invariably involve the administration of native or CHO-derived human proteins containing NANA into animals containing the NGNA form of sialic acid (monkeys, rodents). These findings showing either high percentages of undersialylated glycans in the stable native proteins and those showing lower pharmacokinetic parameters following heterologous injections, raise the interesting question as to whether the type of sialic acid type as well as the degree of sialic acid occupancy may determine the rate of clearance of recombinant glycoproteins.

It is also important to note that recent engineering of different expression systems is now permitting the production of glycoproteins with human-like glycans. For example, while the inability to perform appropriate *N*-glycosylation has been a major limitation of plants as expression systems, these are being overcome by new approaches involving the generation of knockout or knockdown plants that: (i) completely lack xylosyl transferase (XylT) and fucosyl transferase (FucT) activity (Strasser et al., 2004) and accumulate high amounts of human-like N-glycan structures that contain no 1,2-xylose or core a1,3-fucose (ii) lack complex N-glycans resulting from the inactivity of N-acetlyglucosaminyltransferase 1 (GnT1) (Strasser et al, 2005; Wenderoth & von Schaewen, 2000) and (iii) contain glycans terminating in sialic acid (Paccalet et al., 2007; Castilho et al., 2010). In addition, different glycoforms of plant derived proteins can be generated by protein targeting to different compartments (i) cytosol (aglycosylated) (ii) ER (high mannose) or (iii) secreted into the apoplast (complex) (Stoger et al., 2005)

#### **4.3 Effects of the route of administration on pharmacokinetics**

88 Insecticides – Basic and Other Applications

due to rapid uptake by asialoglycoprotein and mannose receptors in the liver or by antibody-mediated mechanisms (Park et al., 2005). For example, CHO cells produce recombinant proteins which contain human-like glycans that may be undersialyted, compared to those produced in livestock systems which append the non-human galactose- -1,3-galactose and the N-glycolyl neuraminic (NGNA, NeuGc) form of sialic acid (Chung et al., 2008; Diaz et al., 2009) and those produced in plants which are non-sialylated and append the non-human -1,2 xylose and -1,3 fucose containing glycans (Altmann, 2007). The relationship between sialic acid levels and oligomerization of recombinant molecules with their circulatory longevity has been extensively studied. For example, administration to mice of recombinant bovine and rhesus acetylcholinesterase (rBoAChE, rRhAChE) as well as plant-derived rHuBChE have supported the idea that pharmacokinetic behaviour is governed by hierarchical rules (Kronman et al., 2000); efficient enzyme tetramerization and high sialic acid occupancy both being required for optimal plasma retention. However, other data from monkey and mice studies do not closely obey these classical rules for circulatory retention. For example: (i) the requirement for tetramerization of rAChE molecules was less important when performed in macaques rather than mice (Cohen et al., 2004) (ii) CHO-derived monomeric PEG-rMaBChE resulted in high MRT when injected into in monkeys (Fig.3, Rosenberg et al., 2010) and (iii) the MRT and T1/2 of unmodified and PEG-modified monomeric rMaBChE were both unexpectedly high following injection into

While the short lived circulatory retention of asialylated BChE attests to the importance of sialylation in retention/clearance, the degree to which silaic acid occupancy is required does not always seem straight forward. Thus, although the rapid clearance of monomeric (13% non-silayted) and tetrameric (25% nonsialyted) rMaBChE in monkeys, compared to the native or PEGylated forms, has been thought to result from undersialylation, glycan analysis by MALDI-TOF of the highly stable native HuBChE and MaBChE proteins indicates that these also contain a significant percentage of nonsialyted or undersilayted proteins. For example, native HuBChE contains 23% monosialyted glycans (99.9% NANA) and a significant percentage of non-sialyted glycans (Kolarich et al., 2008) while native MaBChE is comprised of 21.3% non-sialayted glycans and 21.8% monosialylated glycans ( 99.9% NGNA) (Rosenberg, unp. data). This means that heterologous animal models invariably involve the administration of native or CHO-derived human proteins containing NANA into animals containing the NGNA form of sialic acid (monkeys, rodents). These findings showing either high percentages of undersialylated glycans in the stable native proteins and those showing lower pharmacokinetic parameters following heterologous injections, raise the interesting question as to whether the type of sialic acid type as well as the degree of sialic acid occupancy may determine the rate of clearance of recombinant glycoproteins. It is also important to note that recent engineering of different expression systems is now permitting the production of glycoproteins with human-like glycans. For example, while the inability to perform appropriate *N*-glycosylation has been a major limitation of plants as expression systems, these are being overcome by new approaches involving the generation of knockout or knockdown plants that: (i) completely lack xylosyl transferase (XylT) and fucosyl transferase (FucT) activity (Strasser et al., 2004) and accumulate high amounts of human-like N-glycan structures that contain no 1,2-xylose or core a1,3-fucose (ii) lack complex N-glycans resulting from the inactivity of N-acetlyglucosaminyltransferase 1 (GnT1) (Strasser et al, 2005; Wenderoth & von Schaewen, 2000) and (iii) contain glycans

mice; PEG-conjugation offering no significant advantages.

As mentioned, delivery of PEG-rBChE as a pre-exposure modality is disadvantaged by its large size and a 1:1 stoichiometry between the enzyme and OP requiring high doses due to the high LD50 of many insecticides (ug-mg/kg levels). The route of systemic delivery of high doses of native BChE (MW~350KDa) and tetrameric PEG-rMaBChE (MW>800KDa) will determine the pharmacokinetics (PK) of clearance and is critical to efficacy and safety. Currently very little monkey data exists on the delivery of a stoichiometrically equivalent dose of PEG-rBChE calculated to protect against a known LD50 of a toxic OP insecticide. Although immediate release requiring intravenous (iv) injection may be necessary in certain high threat situations, these are usually impractical in the field. Needleless cutaneous delivery via the dermis and epidermis (chemical mediators, electroporation) appear quite promising, but are unlikely to deliver high doses. Thus, self-administered transdermal injections through the skin either by subcutaneous (sc) or intramuscular (im) routes have been the approaches most commonly used; virtually all human vaccines currently on the market being administered via these routes. Traditionally, autoinjectors, devices for im delivery of a self administered single dose of a drug are used in the military to protect personnel from chemical warfare agents and are currently used to deliver morphine for pain and atropine, diazepam and 2-PAM-Cl for first-aid against nerve agents. For this reason, most animal protection studies with OP bioscavengers have routinely been delivered im to rodents (Lenz et al., 2005; Mumford et al, 2010; Saxena, et al., 2011).

Despite all the pharmacokinetics data generated using im and sc routes of delivery of many drugs and biologics, little is known about the factors that govern the rate and extent of protein absorption from the injection site and the role of the lymphatic system in the transport of large molecules to the systemic circulation. With smaller molecules, the time to maximal concentration is usually shorter following im injections compared to sc injections where absorption is slow and prolonged and accounts for the lag in entering the blood. However with larger therapeutic molecules (MW>16KDa), the lymphatics are thought by some groups to be the primary route of absorption from sc (and im) injection sites. Large molecules are thought to exit the interstitium via cleft like openings into the lymph and enter the systemic circulation via the thoracic duct (Supersaxo et al., 1990; Porter et al., 2001; McLennan et L., 2006). To assess the effects of different routes of delivery, pharmacokinetic behaviour using different doses of PEG-rMaBChE tetrameric molecules was compared in monkeys following im and sc injections.

#### **4.3.1 Intramuscular delivery of PEG-rMaBChE**

Four monkeys each received an im injection of either 2.5 or 3 mg/kg of PEG-rMaBChE. As shown in Fig. 4, unlike the delivery of the smaller native HuBChE which appear to behave uniformly following im injection (Lenz et al., 2005), the much larger PEG-conjugated form exhibits very variable results when delivered into the muscle with Tmax values in the 4 macaques having values of 8, 24, 48 and 48 hr respectively; the 8-hour peak looking more like an iv injection than an im injection. It is not clear whether this more rapid exit from the

Development of a Prophylactic Butyrylcholinesterase

leading to reduced receptor saturation.

**0**

the choice route of delivery.

performed in parallel.

**5**

**10**

**15**

**BChE activity (U/ml)**

**20**

**25**

**30**

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 91

dose. However, while Cmax was generally associated with dose, there was a good deal of overlap between the 3 mg/kg and 5mg/kg doses; the larger doses being retained at higher levels in the blood for many days. This once again raises the question as to whether a high dose of very large molecules can leave the site of the sc injection and enter the blood at levels required for protection. By contrast 3 mg/kg delivered iv reaches a peak of >50 U/ml. It is important to note that despite the apparent low bioavailability of sc administered proteins compared to those given intravenously (17-65%), sc delivery often produces equivalent efficacy to iv administration and is assumed to be due to prolonged absorption

> 2.5 mg/kg 2.5 mg/kg 3 mg/kg 3 mg/kg 5 mg/kg 5 mg/kg 5 mg/kg 5 mg/kg

**0 50 100 150 200 250 300 Time (hr)**

injected with the doses indicated in 2-4 ml sc between the shoulder blades.

Fig. 5. Pharmacokinetics of PEG-rMaBChE delivered by sc injection. Eight monkeys were

A direct comparison of the pharmacokinetic parameters following im versus sc injections of 4 monkeys at does of 2.5 mg/kg and 3 mg/kg is shown in Table 3 and indicates that the im and sc values are quite similar. Overall, the results indicate that for a very high MW protein such as PEG-rMaBChE or PEG-rHuBChE, neither im or sc administrarion are optimal to achieve good plasma retention with high PK parameters. For this reason, a different nonparenteral route of delivery via the lung, where the high MW becomes an advantage, is now

Parameters Subcutaneous injection Intramuscular injection

MRT (h) 62.23 90.12 110.2 73.4 49.37 60.99 58.6 108.0 T1/2 (h) 25.2 42.3 77.8 37.8 23.3 19.4 24.0 58.7 Cmax (U/ml) 19.6 18.3 12.3 11.0 23.1 20.3 16.5 9.8 AUC (U/ml·h) 1706 1856 1489 1128 1762 1675 1089 1367 Table 3. Comparison of the pharmacokinetics parameters four following sc and im injections

Four individual monkeys Four individual monkeys

muscle injection site into the blood reflects a more vascularised muscle or whether im delivery has more potential to damage blood vessels and promote faster draining. It is clear however that delivery of large doses of a therapeutic such as PEG-rHuBChE will require many im injections to achieve required peak values and will increase the likelihood of targeting a blood vessel. The stoichiometric dose of BChE required to protect humans against 2 LD50 of soman has been considered to be 3 mg/kgm (200 mg/70 kg); the antidotal efficacy of BChE being contingent upon both the rate of OP detoxification and its levels in blood (Raveh, 1997; Ashani & Pistinner, 2004). It would be unlikely that Cmax values (20 and 23 U/ml at 3 mg/kg and 17 and 10 U/kg at 2.5 mg/kg) following im administration would be sufficient for protection. In addition, the variable times of peak enzyme make it difficult to choose a time for prophylactic dosing.

#### **4.3.2 Subcutaneous delivery of PEG-rMaBChE**

Extensive pharmacokinetics have been performed on many well known biologics in monkeys and humans, either PEGylated or unmodified, using the sc routes of delivery (Boelaert et al., 1989; Ramakrishnan et al., 2003; Heatherington et al., 2001; Radwanski et al., 1987; Mager et al., 2005), although extrapolation from these studies may be problematic because all used considerably smaller molecules than native or PEG-rBChE. Generally, sc injections have been the delivery route of choice for compounds with limited oral bioavailability, as a means of modifying or extending the release profiles of these molecules, or as a means of delivering drugs that require large quantities (Yang, 2003) since larger volumes may be injected. In one case, a highly concentrated form of a therapeutic requiring large doses for its effects has be prepared as a crystalline and successfully delivered sc in a small volume (Yang et al. 2003).

Fig. 4. Pharmacokinetic profiles of PEG-rMaBChE delivered by im injection. Four monkeys were injected into the thigh muscles using a 1-ml syringe.

Figure 5 shows the pharmacokinetic profiles following sc delivery of the tetrameric PEGrMaBChE at 2.5, 3 and 5 mg/kg. Tmax values were all consistently ~48 hrs, regardless of the

muscle injection site into the blood reflects a more vascularised muscle or whether im delivery has more potential to damage blood vessels and promote faster draining. It is clear however that delivery of large doses of a therapeutic such as PEG-rHuBChE will require many im injections to achieve required peak values and will increase the likelihood of targeting a blood vessel. The stoichiometric dose of BChE required to protect humans against 2 LD50 of soman has been considered to be 3 mg/kgm (200 mg/70 kg); the antidotal efficacy of BChE being contingent upon both the rate of OP detoxification and its levels in blood (Raveh, 1997; Ashani & Pistinner, 2004). It would be unlikely that Cmax values (20 and 23 U/ml at 3 mg/kg and 17 and 10 U/kg at 2.5 mg/kg) following im administration would be sufficient for protection. In addition, the variable times of peak enzyme make it

Extensive pharmacokinetics have been performed on many well known biologics in monkeys and humans, either PEGylated or unmodified, using the sc routes of delivery (Boelaert et al., 1989; Ramakrishnan et al., 2003; Heatherington et al., 2001; Radwanski et al., 1987; Mager et al., 2005), although extrapolation from these studies may be problematic because all used considerably smaller molecules than native or PEG-rBChE. Generally, sc injections have been the delivery route of choice for compounds with limited oral bioavailability, as a means of modifying or extending the release profiles of these molecules, or as a means of delivering drugs that require large quantities (Yang, 2003) since larger volumes may be injected. In one case, a highly concentrated form of a therapeutic requiring large doses for its effects has be prepared as a crystalline and successfully delivered sc in a

> **0 50 100 150 200 250 300 Time (hr)**

Fig. 4. Pharmacokinetic profiles of PEG-rMaBChE delivered by im injection. Four monkeys

Figure 5 shows the pharmacokinetic profiles following sc delivery of the tetrameric PEGrMaBChE at 2.5, 3 and 5 mg/kg. Tmax values were all consistently ~48 hrs, regardless of the

2.5 mg/kg 2.5 mg/kg 3 mg/kg 3 mg/kg

difficult to choose a time for prophylactic dosing.

**4.3.2 Subcutaneous delivery of PEG-rMaBChE** 

**0**

were injected into the thigh muscles using a 1-ml syringe.

**5**

**10**

**15**

**BChE activity (U/ml)**

**20**

**25**

small volume (Yang et al. 2003).

dose. However, while Cmax was generally associated with dose, there was a good deal of overlap between the 3 mg/kg and 5mg/kg doses; the larger doses being retained at higher levels in the blood for many days. This once again raises the question as to whether a high dose of very large molecules can leave the site of the sc injection and enter the blood at levels required for protection. By contrast 3 mg/kg delivered iv reaches a peak of >50 U/ml. It is important to note that despite the apparent low bioavailability of sc administered proteins compared to those given intravenously (17-65%), sc delivery often produces equivalent efficacy to iv administration and is assumed to be due to prolonged absorption leading to reduced receptor saturation.

Fig. 5. Pharmacokinetics of PEG-rMaBChE delivered by sc injection. Eight monkeys were injected with the doses indicated in 2-4 ml sc between the shoulder blades.

A direct comparison of the pharmacokinetic parameters following im versus sc injections of 4 monkeys at does of 2.5 mg/kg and 3 mg/kg is shown in Table 3 and indicates that the im and sc values are quite similar. Overall, the results indicate that for a very high MW protein such as PEG-rMaBChE or PEG-rHuBChE, neither im or sc administrarion are optimal to achieve good plasma retention with high PK parameters. For this reason, a different nonparenteral route of delivery via the lung, where the high MW becomes an advantage, is now the choice route of delivery.


Table 3. Comparison of the pharmacokinetics parameters four following sc and im injections performed in parallel.

Development of a Prophylactic Butyrylcholinesterase

for post-exposure atropine and oximes.

propazine to inhibit BChE activity.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 93

poisoning and death than any other OP insecticide (Lotti & Moretto, 2005) and was recently phased out of use in the US. In humans, parathion is absorbed via skin, mucous membranes, and orally and is rapidly metabolized to paraoxon which can result in headaches, convulsions, poor vision, vomiting, abdominal pain, severe diarrhea, unconsciousness, tremor, dyspnea and finally lung-edema as well as respiratory arrest. Symptoms of severe poisoning are known to last for extended periods of time, sometimes months. Additionally, peripheral neuropathy including paralysis is noticed as late sequelae after recovery from acute intoxication (http://extoxnet.orst.edu/pips/parathio.htm). Parathion has been extensively used for committing suicide and potentially for the deliberate killing of people.

**4.4.2 Aerosolized PEG-rMaBChE protection against aerosolized paraoxon exposure**  As an alternative to delivering high doses of a large molecule into the systemic circulation by sc or im routes, studies are currently being performed using aerosol therapy for delivering rBChE directly to the lung in order to create an effective "pulmonary bioshield" that will detoxify incoming inhaled insecticide in situ and prevent or reduce respiratory toxicity. This takes advantage of the large size of the molecule which will be retained in the lung due to its inability to pass through the lung endothelium into the blood. In this context, inhalation serves as a major means of intoxication because of rapid accesses of the OP to the blood. An efficient pre-exposure pulmonary therapeutic in the form of aerosolized PEGrBChE could be delivered before a known use/release of insecticides and prevent the lung damage and delayed neuropathy often associated with exposure, while reducing the need

Maxwell et al. (2006) have recently shown that for OP compounds (including the insecticides paraoxon, DFP and dichlorvos) the primary mechanism of in vivo toxicity is the inhibition of AChE and the residual unexplained variation in OP toxicity represents <10% of the total variation in toxicity. Almost all of the variation in the LD50 of OP compounds in rats was explained by the variation in their in vitro rate constants for inhibition of AChE. Thus, to develop a paraoxon/monkey animal model for aerosolized insecticide exposure and to avoid unnecessary stressing and killing of monkeys in developing the model, the dose of aerosolized paraoxon required to achieve a ~50% inhibition of RBC AChE and serum BChE has been used initially as a readout for toxicity and a basis from which to analyse protection by CHO-derived rMaBChE. Thus, paraoxon which is not neutralized in the lung will enter the blood and can be measured by the inhibition of AChE and BChE activity in lysed whole blood using using a modified assay (Ellman et al, 1961) with 5,5' dithiobis(2-nitrobenzoic acid), the substrate acetyl-thiocholine (ATC) and 20uM etho-

Initially, the dose of aerosolized paraoxon required to produce ~50% inhibition of red blood cell (RBC) AChE and BChE in the circulation was first determined in mice prior to the macaque studies. The LD50 of paraoxon in rodents has been established using oral, percutaneous (pc) and subcutaneous (sc) routes (mice: 760 ug/kg orally; 270 - 800 ug/kg sc and for rats: 1800 ug/kg orally and 200 - 430 sc (reviewed in Levine, 2006; Villa et al., 2007). Milatovic et al. (1996) showed that a single acute injection of 0.09, 0.12, or 0.19 mg/kg paraoxon in rats, representing 40% LD50, 52% LD50 and 83% LD50 respectively, did not produce signs of cholinergic hyperactively. In the present study, the effective dose of aerosolized paraoxon resulting in 50% inhibition in mice was found to be 150-180 ug/kg which is less toxic than the parenteral route. In addition, aerosolized BChE given 24 hr prior

#### **4.4 Protection studies with PEG-rMaBChE**

Many studies have demonstrated efficacy of native HuBChE, both pre-and post–exposure, in rodents and monkeys to protect against OP nerve agents delivered by sc injection, iv injection or vapour. (Lenz et al., 2005; Sun et al., 2008; Saxena et al., 2011; Mumford et al, 2010). Protection has also been shown in mice and guinea pigs using PEG-rBChE produced in goat and plants (Huang et al. 2007, Geyer et al., 2010). However, very few studies have utilized the non-human primate monkey model for assessing insecticide toxicity and none have used respiratory exposure.

Two types of protection studies using different routes of delivery are currently being performed to assess the ability of BChE to protect against toxicity resulting from exposure to the insecticide paraoxon.


#### **4.4.1 Paraoxon**

The majority of OP insecticides are lipophilic, not ionised, and are absorbed rapidly following inhalation or ingestion (Vale, 1998). Dermal absorption is slower and can be prevented by removing clothes and bathing, but severe poisoning may still ensue if exposure is prolonged. Respiratory pesticide exposure by inhalation of powders, airborne droplets or vapours is particularly hazardous because pesticide particles can quickly enter the bloodstream via the lungs and cause serious damage. Under low pressure, droplet size is too large to remain airborne**.** However, when high pressure, ultra low volume application (ULV) or fogging equipment is used for agricultural purposes, respiratory exposure is increased due to the production of mist- or fog-size particles, which can be carried on air currents for a considerable distance (Armed Forces Pest Management Board Technical Guide No. 13). Small children are highly vulnerable because they breathe in greater volumes of air, relative to their body weight, than adults.

Fig. 6. Chemical structure of parathion and paraoxon.

Paraoxon is the active metabolite of the inactive parathion (Fig. 6) produced by a sulfur-foroxygen substitution carried out predominantly in the liver by the mixed-function oxidases (Dauterman, 1971). It was chosen for these studies because it inhibits AChE, BChE and carboxylesterase (Levine, 2006), it has a relatively low LD50, and low volatility and stability in aqueous solution. Parathion has probably been responsible for more cases of accidental

Many studies have demonstrated efficacy of native HuBChE, both pre-and post–exposure, in rodents and monkeys to protect against OP nerve agents delivered by sc injection, iv injection or vapour. (Lenz et al., 2005; Sun et al., 2008; Saxena et al., 2011; Mumford et al, 2010). Protection has also been shown in mice and guinea pigs using PEG-rBChE produced in goat and plants (Huang et al. 2007, Geyer et al., 2010). However, very few studies have utilized the non-human primate monkey model for assessing insecticide toxicity and none

Two types of protection studies using different routes of delivery are currently being performed to assess the ability of BChE to protect against toxicity resulting from exposure to

The majority of OP insecticides are lipophilic, not ionised, and are absorbed rapidly following inhalation or ingestion (Vale, 1998). Dermal absorption is slower and can be prevented by removing clothes and bathing, but severe poisoning may still ensue if exposure is prolonged. Respiratory pesticide exposure by inhalation of powders, airborne droplets or vapours is particularly hazardous because pesticide particles can quickly enter the bloodstream via the lungs and cause serious damage. Under low pressure, droplet size is too large to remain airborne**.** However, when high pressure, ultra low volume application (ULV) or fogging equipment is used for agricultural purposes, respiratory exposure is increased due to the production of mist- or fog-size particles, which can be carried on air currents for a considerable distance (Armed Forces Pest Management Board Technical Guide No. 13). Small children are highly vulnerable because they breathe in greater volumes

Paraoxon is the active metabolite of the inactive parathion (Fig. 6) produced by a sulfur-foroxygen substitution carried out predominantly in the liver by the mixed-function oxidases (Dauterman, 1971). It was chosen for these studies because it inhibits AChE, BChE and carboxylesterase (Levine, 2006), it has a relatively low LD50, and low volatility and stability in aqueous solution. Parathion has probably been responsible for more cases of accidental

1. Aerosolized PEG-rMaBChE 1 hr prior to aerosolized paraoxon exposure. 2. Intravenous delivery of PEG-rMaBChE 1 hr prior to sc delivery of paraoxon.

**4.4 Protection studies with PEG-rMaBChE** 

of air, relative to their body weight, than adults.

Fig. 6. Chemical structure of parathion and paraoxon.

have used respiratory exposure.

the insecticide paraoxon.

**4.4.1 Paraoxon** 

poisoning and death than any other OP insecticide (Lotti & Moretto, 2005) and was recently phased out of use in the US. In humans, parathion is absorbed via skin, mucous membranes, and orally and is rapidly metabolized to paraoxon which can result in headaches, convulsions, poor vision, vomiting, abdominal pain, severe diarrhea, unconsciousness, tremor, dyspnea and finally lung-edema as well as respiratory arrest. Symptoms of severe poisoning are known to last for extended periods of time, sometimes months. Additionally, peripheral neuropathy including paralysis is noticed as late sequelae after recovery from acute intoxication (http://extoxnet.orst.edu/pips/parathio.htm). Parathion has been extensively used for committing suicide and potentially for the deliberate killing of people.

#### **4.4.2 Aerosolized PEG-rMaBChE protection against aerosolized paraoxon exposure**

As an alternative to delivering high doses of a large molecule into the systemic circulation by sc or im routes, studies are currently being performed using aerosol therapy for delivering rBChE directly to the lung in order to create an effective "pulmonary bioshield" that will detoxify incoming inhaled insecticide in situ and prevent or reduce respiratory toxicity. This takes advantage of the large size of the molecule which will be retained in the lung due to its inability to pass through the lung endothelium into the blood. In this context, inhalation serves as a major means of intoxication because of rapid accesses of the OP to the blood. An efficient pre-exposure pulmonary therapeutic in the form of aerosolized PEGrBChE could be delivered before a known use/release of insecticides and prevent the lung damage and delayed neuropathy often associated with exposure, while reducing the need for post-exposure atropine and oximes.

Maxwell et al. (2006) have recently shown that for OP compounds (including the insecticides paraoxon, DFP and dichlorvos) the primary mechanism of in vivo toxicity is the inhibition of AChE and the residual unexplained variation in OP toxicity represents <10% of the total variation in toxicity. Almost all of the variation in the LD50 of OP compounds in rats was explained by the variation in their in vitro rate constants for inhibition of AChE. Thus, to develop a paraoxon/monkey animal model for aerosolized insecticide exposure and to avoid unnecessary stressing and killing of monkeys in developing the model, the dose of aerosolized paraoxon required to achieve a ~50% inhibition of RBC AChE and serum BChE has been used initially as a readout for toxicity and a basis from which to analyse protection by CHO-derived rMaBChE. Thus, paraoxon which is not neutralized in the lung will enter the blood and can be measured by the inhibition of AChE and BChE activity in lysed whole blood using using a modified assay (Ellman et al, 1961) with 5,5' dithiobis(2-nitrobenzoic acid), the substrate acetyl-thiocholine (ATC) and 20uM ethopropazine to inhibit BChE activity.

Initially, the dose of aerosolized paraoxon required to produce ~50% inhibition of red blood cell (RBC) AChE and BChE in the circulation was first determined in mice prior to the macaque studies. The LD50 of paraoxon in rodents has been established using oral, percutaneous (pc) and subcutaneous (sc) routes (mice: 760 ug/kg orally; 270 - 800 ug/kg sc and for rats: 1800 ug/kg orally and 200 - 430 sc (reviewed in Levine, 2006; Villa et al., 2007). Milatovic et al. (1996) showed that a single acute injection of 0.09, 0.12, or 0.19 mg/kg paraoxon in rats, representing 40% LD50, 52% LD50 and 83% LD50 respectively, did not produce signs of cholinergic hyperactively. In the present study, the effective dose of aerosolized paraoxon resulting in 50% inhibition in mice was found to be 150-180 ug/kg which is less toxic than the parenteral route. In addition, aerosolized BChE given 24 hr prior

Development of a Prophylactic Butyrylcholinesterase

*Chem*. 285(21):15923-30.

175(1-3):255-60.

231(3):423-9.

1155-61.

*Sci.* 116(2):623-31.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 95

Castilho, A., Strasser, R., Stadlmann, J., Grass, J., Jez, J., Gattinger, P., Kunert, R., Quendler,

Chilukuri, N., Sun, W., Naik, R. S., Parikh, K., Tang, L., Doctor, B. P., & Saxena, A. (2008a).

Chilukuri, N., Sun, W., Parikh, K., Naik, R. S., Tang, L., Doctor, B. P., & Saxena, A. (2008b). A

Chung, C. H., Mirakhur, B., Chan, E., Le, Q. T., Berlin, J., Morse, M., Murphy, B. A.,

specific for galactose-alpha-1,3-galactose. *N Engl J Med*. 358(11):1109-17. Cohen, O., Kronman, C., Chitlaru, T., Ordentlich, A., Velan, B., & Shafferman, A. (2001).

(Macaca mulatta). *Biochem J*. 378(Pt 1):117-28.

polyethylene glycol on its circulatory longevity. *Biochem J*. 357(Pt 3):795-802. Cohen, O., Kronman, C., Velan, B., & Shafferman, A. (2004). Amino acid domains control the

Colosio, C., Tiramani, M., Brambilla, G., Colombi, A., & Moretto, A. (2009).

Dauterman, W. C. (1971). Biological and nonbiological modifications of organophosphorus

Deshpande, L. S., Carter, D. S., Blair, R. E., & DeLorenzo, R.J. (2010). Development of a

Diaz, S. L., Padler-Karavani, V., Ghaderi, D., Hurtado-Ziola, N., Yu, H., Chen, X., Brinkman-

Dirnhuber, P., French, M. C., Green, D.M., Leadbeater, L., & Stratton, J. A. (1979). The

Doctor, B. P., Maxwell, D. M., Ashani, Y., Saxena, A., & Gordon, R. K. (2001). New

compounds. *Bull World Health Organ*. 44(1-3):133-50. Review.

and biotherapeutic products. *PLoS One*. 4(1):e4241.

pyridostigmine. J Pharm Pharmacol. 31(5):295-9.

SM and Romano, JA, Eds. CRC Press, New York, p191-214.

H., Pabst, M., Leonard, R., Altmann, F., & Steinkellner, H. (2010). In planta protein sialylation through overexpression of the respective mammalian pathway. *J Biol* 

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase. *Chem Biol Interact*.

repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice. *Toxicol Appl Pharmacol*.

Satinover, S.M., Hosen, J., Mauro, D., Slebos, R. J., Zhou, Q., Gold, D., Hatley, T., Hicklin, D. J., & Platts-Mills, T. A. (2008). Cetuximab-induced anaphylaxis and IgE

Effect of chemical modification of recombinant human acetylcholinesterase by

circulatory residence time of primate acetylcholinesterases in rhesus macaques

Neurobehavioural effects of pesticides with special focus on organophosphorus compounds: which is the real size of the problem? *Neurotoxicology*. 30(6):

prolonged calcium plateau in hippocampal neurons in rats surviving status epilepticus induced by the organophosphate diisopropylfluorophosphate. *Toxicol* 

Van der Linden, E. C., Varki, A., & Varki, N. M. (2009). Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues

protection of primates against soman poisoning by pretreatment with

approaches to Medical Protection against Chemical Warfare Nerve Agents. Somani,

to the paraoxon significantly reduced the AChE inhibition (our unpub. data). Rodents contain a high endogenous levels of CaE, another stoichiometric OP scavenger (Dirnhuber et al. 1979) and are known to be ~10-fold less sensitive to soman than non-human primates (Maxwell et al., 2006). Accordingly, a dose of 15 ug/kg of aerosolized paraoxon has been shown to result in 50-60% RBC AChE inhibition and preliminary data indicate that PEGrMaBChE , delivered as a pre-exposure aerosol one hour prior to exposure, can totally reduce this inhibition in a dose –dependent manner.

#### **4.4.3 Intravenous PEG-rMaBChE protection against subcutaneous paraoxon exposure**

These studies are being formed to compare routes of delivery with efficacy of protection and indicate that while paraoxon delivered sc is also more toxic than as an aerosol, complete protection can be achieved by PEG-rMaBChE pretreatment.

#### **5. References**


to the paraoxon significantly reduced the AChE inhibition (our unpub. data). Rodents contain a high endogenous levels of CaE, another stoichiometric OP scavenger (Dirnhuber et al. 1979) and are known to be ~10-fold less sensitive to soman than non-human primates (Maxwell et al., 2006). Accordingly, a dose of 15 ug/kg of aerosolized paraoxon has been shown to result in 50-60% RBC AChE inhibition and preliminary data indicate that PEGrMaBChE , delivered as a pre-exposure aerosol one hour prior to exposure, can totally

**4.4.3 Intravenous PEG-rMaBChE protection against subcutaneous paraoxon exposure**  These studies are being formed to compare routes of delivery with efficacy of protection and indicate that while paraoxon delivered sc is also more toxic than as an aerosol, complete

Alavanja, M. C. (2009). Pesticides Use and Exposure Extensive Worldwide. *Rev Environ* 

Altamirano, C. V., Lockridge, O. (1999). Association of tetramers of human

Altmann, F. (2007). The role of protein glycosylation in allergy. *Int Arch Allergy Immunol*.

Ashani, Y., & Pistinner, S. (2004). Estimation of the upper limit of human

butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy

butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model. *Toxicol Sci*. 77(2):358-67. Blong, R. M., Bedows, E., & Lockridge, O. (1997). Tetramerization domain of human butyrylcholinesterase is at the C-terminus. *Biochem J.* 327 ( Pt 3):747-57. Boeck, A. T., Schopfer, L. M., & Lockridge, O. (2002). DNA sequence of

butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase. *Biochem Pharmacol*. 63(12):2101-10. Boelaert, J. R., Schurgers, M. L., Matthys, E. G., Belpaire, F. M., Daneels, R. F., De Cre, M. J.,

& Bogaert, M. G. (1989). Comparative pharmacokinetics of recombinant erythropoietin administered by the intravenous, subcutaneous, and intraperitoneal routes in continuous ambulatory peritoneal dialysis (CAPD) patients. *Perit Dial Int*.

organophosphate pesticide poisoning. *Cochrane Database Syst Rev*. 16;(2):CD005085.

organophosphate induced plasticity of locus coeruleus neurons. *Nature Precedings*:

cholinesterases in severe organophosphorus poisioning. Our experience. *Minerva* 

Buckley, N. A., Eddleston, M., Li, Y., Bevan, M., & Robertson, J. (2011). Oximes for acute

Cao, J. L., Varnell, A. L., & Cooper, D. C. (2011). Gulf War Syndrome: A role for

Cascio, C., Comite Ghiara, M., Lanza, G., & Ponchione, A. (1988). Use of serum

hdl:10101/npre.2011.6057.1: Posted 23 Jun 2011.

reduce this inhibition in a dose –dependent manner.

**5. References** 

*Health*, 24(4):303-9.

142(2):99-115.

9(2):95-8.

Review.

*Anestesiol*. 54(7-8):337-8.

protection can be achieved by PEG-rMaBChE pretreatment.

terminus. *Chem Biol Interact.* 119-120:53-60.


Development of a Prophylactic Butyrylcholinesterase

40-6.

411(2):425-32.

Review.

*Rev.* 24(1):37-49. Review.

*Biol Interact*. 175(1-3):261-6.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 97

Kronman, C., Cohen, O., Raveh, L., Mazor, O., Ordentlich, A., & Shafferman, A. (2007).

Lenz, D. E., Broomfield, C. A., Maxwell, D. M., & Cerasoli, D. M. (2001). Nerve Agent

Lenz, D. E., Yeung, D., Smith, J. R., Sweeney, R. E., Lumley, L. A., & Cerasoli, D. M. (2007).

Levine, E. S. (2006). Nerve Agent Simulants: Can They Be Used as Substitutes for Nerve

Li, H., Schopfer, L. M., Masson, P., & Lockridge, O. (2008). Lamellipodin proline rich

Litchfield, M. H. (2005). Estimates of acute pesticide poisoning in agricultural workers in

Lockridge, O. (1990). Genetic variants of human serum cholinesterase influence metabolism

Lotti, M., & Moretto, A. (2005). Organophosphate-induced delayed polyneuropathy. *Toxicol* 

Luo, C., Tong, M., Maxwell, D. M., & Saxena, A. (2008). Comparison of oxime reactivation

Mager, D. E., Neuteboom, B., & Jusko, W. J. (2005). Pharmacokinetics and

Mager, D. E., Woo, S., & Jusko, W. J. (2009). Scaling pharmacodynamics from in vitro and

Matzke, S. M., Oubre, J. L. Caranto, G. R., Gentry, M. K., & Galbicka, G. (1999). Behavioral

Maxwell, D. M., Brecht, K. M., Koplovitz, I., & Sweeney, R. E. (2006). Acetylcholinesterase

McLennan, D. N., Porter, C. J., Edwards, G.A., Heatherington, A. C., Martin, S. W., &

and aging of nerve agent-inhibited monkey and human acetylcholinesterases. *Chem* 

pharmacodynamics of PEGylated IFN-beta 1a following subcutaneous

preclinical animal studies to humans. *Drug Metab Pharmacokinet*. 24(1):16-24.

and immunological effects of exogenous butyrylcholinesterase in rhesus monkeys.

inhibition: does it explain the toxicity of organophosphorus compounds? *Arch* 

Charman, S. A. (2006). The absorption of darbepoetin alfa occurs predominantly

Institute of Chemical Defense under Contract No. GS-23F-8006H.

less developed countries. *Toxicol Rev*., 24(4):271-8. Review.

administration in monkeys. *Pharm Res*. 22(1):58-61.

*Pharmacol Biochem Behav*. 62(3):523-30.

*Toxicol.* 80(11):756-60.

of the muscle relaxant succinylcholine.*Pharmacol Ther* 47: 35-60.

cynomolgus monkeys. *Chem Biol Interact*. 157-158:205-10.

mini review. *Toxicology.* 233(1-3):31-9. Review.

Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication. *Toxicology*. 233(1-3):

Bioscavengers: Protection against High- and Low- Dose Organophosphorus Exposure. Somani, SM and Romano, JA, Eds. CRC Press, New York, p215-243. Lenz, D. E., Maxwell, D. M., Koplovitz, I., Clark, C. R., Capacio, B. R., Cerasoli, D. M.,

Federko, J. M., Luo, C., Saxena, A., Doctor, B. P., & Olson, C. (2005). Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and

Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a

Agents in biomedical Research? Prepared for the U.S. Army Medical Research

peptides associated with native plasma butyrylcholinesterase tetramers. *Biochem J*.


Ellman, G. L., Courtney, K. D., Andres, V. Jr., & Feather-stone, R. M. (1961). A new and

Geyer, B. C., Kannan, L., Garnaud, P.E., Broomfield, C. A., Cadieux, C. L., Cherni, I.,

rodents against nerve agents. *Proc Natl Acad Sci U S A*. 107(47):20251-6. Gleba, Y., Klimyuk, V., & Marillonnet, S. (2005). Magnifecti on--a new platform for expressing recombinant vaccines in plants. *Vaccine*. 23(17-18):2042-8. Review. Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008). Nicotiana benthamiana: its

7:88-95.

*Interact*. 21(8):1015-26. Review.

J Cancer. 84 Suppl 1:11-6.

104(34):13603-8.

*Stat Q*., 43(3):139-44.

*Drugs*. 14(2):363-80. Review.

*Biol Chem*. 275(38):29488-502.

from human plasma. *Proteomics*. 8(2):254-63.

rapid colorimetric determination of acetylcholinesterase activity. *Biochem Pharmacol*.

Hodgins, S.M., Kasten, S.A., Kelley, K., Kilbourne, J., Oliver, Z. P., Otto, T. C., Puffenberger, I., Reeves, T. E., Robbins, N. 2nd., Woods, R. R., Soreq, H., Lenz, D. E., Cerasoli, D. M., & Mor, T. S. (2010). Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects

history and future as a model for plant-pathogen interactions. *Mol Plant Microbe* 

purification and long-term stability of human butyrylcholinesterase: a potential

plasma on cholinesterase levels and outcomes in patients with organophosphate

erythropoiesis stimulating protein (NESP) in cancer patients: preliminary report. Br

Bellemare, A., Côté, M., Herskovits, P., Touati, M., Turcotte, C., Valeanu, L., Lemée, N., Wilgus, H., Bégin, I., Bhatia, B., Rao, K., Neveu, N., Brochu, E., Pierson, J., Hockley, D. K., Cerasoli, D. M., Lenz, D. E., Karatzas, C. N., & Langermann, S. (2007). Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning. *Proc Natl Acad Sci U S A*.

Grunwald, J., Marcus, D., Papier, Y., Raveh, L., Pittel, Z., & Ashani, Y. (1997). Large-scale

Güven, M., Sungur, M,, Eser, B., Sari, I., & Altuntaş, F. (2004). The effects of fresh frozen

Heatherington, A. C., Schuller, J., & Mercer, A. J. (2001). Pharmacokinetics of novel

Huang, Y.J., Huang, Y., Baldassarre, H., Wang, B., Lazaris, A., Leduc, M., Bilodeau, A. S.,

Jenkins, T., Balinsky, D., & Patient, D. W. (1967). Cholinesterase in plasma: first reported absence in the Bantu; half-life determination. *Science*. 156(783):1748-50. Jeyaratnam, J. (1990). Acute pesticide poisoning: a major global health problem.*World Health* 

Kang, J. S., Deluca, P. P., & Lee, K. C. (2009). Emerging PEGylated drugs. *Expert Opin Emerg* 

Kolarich, D., Weber, A., Pabst, M., Stadlmann, J., Teschner, W., Ehrlich, H., Schwarz, H. P.,

Kronman, C., Chitlaru, T., Elhanany, E., Velan, B., & Shafferman, A. (2000). Hierarchy of

& Altmann, F. (2008). Glycoproteomic characterization of butyrylcholinesterase

post-translational modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of bovine acetylcholinesterase. *J* 

bioscavenger drug. *J Biochem Biophys Methods*. 34(2):123-35.

poisoning. *J Toxicol Clin Toxicol.* 42(5):617-23.


Development of a Prophylactic Butyrylcholinesterase

38(3):529-34.

86.

657-61.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 99

Raveh, L., Grauer, E., Grunwald, J., Cohen, E., & Ashani, Y. (1997). The stoichiometry of

Rosenberg, Y. J., Luo, C., Ashani, Y., Doctor, B. P., Fischer, R., Wo1fee, G., & Saxena, A.

Rosenberg, Y. J., Saxena, A., Sun, W., Jiang, X., Chilukuri, N., Luo, C., Doctor, B. P., & Lee, K.

Rosenberry, T. L., Mallender, W. D., Thomas, P. J., & Szegletes, T. (1999). A steric blockade

Sainsbury, F., Lavoie, P. O., D'Aoust, M. A., Vézina, L.P., & Lomonossoff, G. P. (2008).

Saxena, A., Ashani, Y., Raveh, L., Stevenson, D., Patel, T., & Doctor, B. P. (1998). Role of

Saxena, A., Sun, W., Fedorko, J. M., Koplovitz, I., & Doctor, B. P. (2011). Prophylaxis with

Stoger, E., Sack, M., Nicholson, L., Fischer, R., & Christou, P. (2005). Recent progress in

Strasser, R., Altmann, F., Mach, L., Glössl, J., & Steinkellner, H. (2004). Generation of

Strasser, R., Stadlmann, J., Svoboda, B., Altmann, F., Glössl, J., & Mach, L. (2005). Molecular

Sun, W., Doctor, B. P., & Saxena, A. (2005). Safety and pharmacokinetics of human serum butyrylcholinesterase in guinea pigs. Chem Biol Interact. 157-158:428-9. Sun, W., Doctor, B. P., Lenz, D. E., & Saxena, A. (2008). Long-term effects of human

Sun, W., Luo, C., Naik, R. S., Doctor, B. P., & Saxena, A. (2009). Pharmacokinetics

butyrylcholinesterase. *Toxicol Appl Pharmacol.* 145(1):43-53.

substrate. *Chem Biol Interact.* 119-120:85-97.

mosaic virus RNA-2. *Plant Biotechnol J*. 6(1):82-92.

engineered cholinesterases. *Mol Pharmacol*. 53(1):112-22.

doses of soman or VX. *Biochem Pharmacol*. 81(1):164-9.

plantibody technology. *Curr Pharm Des*. 11:2439-57.

and core alpha1,3-linked fucose. *FEBS Lett*. 561(1-3):132-6.

cynomolgus monkeys. *Chem Biol Interact*. 175(1-3):428-30.

plants lacking complex N-glycans. *Biochem J*. 387(Pt 2):385-91.

purified homologous butyrylcholinesterase. *Life Sci*. 72(2):125-34.

correlation between protection and blood-enzyme level in mice. *Biochem Pharmacol*.

protection against soman and VX toxicity in monkeys pretreated with human

(2002). Pharmacokinetics and immunologic consequences of exposing macaques to

D. (2010). Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model. *Chem Biol Interact.* 187(1-3):279-

model for inhibition of acetylcholinesterase by peripheral site ligands and

Expression of multiple proteins using full-length and deleted versions of cowpea

oligosaccharides in the pharmacokinetics of tissue-derived and genetically

human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal

Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose

basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana

butyrylcholinesterase pretreatment followed by acute soman challenge in

and immunologic consequences of repeated administrations of purified heterologous and homologous butyrylcholinesterase in mice. *Life Sci.* 85(17-18):

via the lymphatics following subcutaneous administration to sheep. *Pharm Res*. 23(9):2060-6.


Milatovic, D., & Dettbarn, W. D. (1996). Modification of acetylcholinesterase during

Millard, C. B., Kryger, G., Ordentlich, A., Greenblatt, H. M., Harel, M., Raves, M. L., Segall,

Mumford, H., Price, M. E., Cerasoli, D.M., Teschner, W., Ehrlich, H., Schwarz, H.P., & Lenz,

Nachon, F., Nicolet, Y., Viguié, N., Masson, P., Fontecilla-Camps, J. C., & Lockridge, O.

Ohayo-Mitoko, G. J., Kromhout, H., Simwa, J. M., Boleij, J. S., & Heederik, D. (2000). Self

Paccalet, T., Bardor, M., Rihouey, C., Delmas, F., Chevalier, C., D'Aoust, M. A., Faye, L.,

Park, E. I., Mi, Y., Unverzagt, C., Gabius, H. J., & Baenziger, J. U. (2005). The

Pepinsky, R. B., LePage, D. J., Gill, A., Chakraborty, A., Vaidyanathan, S., Green, M., Baker,

Porter, C. J., Edwards, G. A.,& Charman, S.A. (2001). Lymphatic transport of proteins after

Radwanski, E., Perentesis, G., Jacobs, S., Oden, E., Affrime, M., Symchowicz, S., &

Ramakrishnan, R., Cheung, W. K., Farrell, F., Joffee, L., & Jusko, W. J. (2003).

Raveh, L., Ashani, Y., Levy, D., De La Hoz, D., Wolfe, A. D., & Doctor, B.P. (1989).

agricultural workers. *Occup Environ Med.* 57(3):195-200.

alpha 2,6GalNAc. *Proc Natl Acad Sci U S A*. 102(47):17125-9.

preserved in vitro bioactivity. *J Pharmacol Exp Ther*. 297(3):1059-66.

cynomolgus monkeys. . J Pharmacol Exp Ther. 306(1):324-31.

23(9):2060-6.

136(1):20-8.

atomic level. *Biochemistry,* 38:7032-9.

*Chem Biol Interact*. 187(1-3):304-8.

enzymes. *Plant Biotechnol J*. 5(1):16-25.

volunteers. *J Clin Pharmacol*. 27(5):432-5.

*Eur J Biochem*. 269(2):630-7.

2):157-71. Review.

via the lymphatics following subcutaneous administration to sheep. *Pharm Res*.

adaptation to chronic, subacute paraoxon application in rat. *Toxicol Appl Pharmacol*.

Y., Barak, D., Shafferman, A., Silman, I., & Sussman, J. L. (1999). Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the

D. E. (2010). Efficacy and physiological effects of human butyrylcholinesterase as a post-exposure therapy against percutaneous poisoning by VX in the guinea-pig.

(2002). Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization.

reported symptoms and inhibition of acetylcholinesterase activity among Kenyan

Vézina, L., Gomord, V., & Lerouge, P. (2007). Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing

asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid

D. P., Whalley, E., Hochman, P. S., & Martin, P. (2001). Improved pharmacokinetic properties of a polyethylene glycol-modified form of interferon-beta-1a with

s.c. injection: implications of animal model selection. *Adv Drug Deliv Rev*. 50(1-

Zampaglione, N. (1987). Pharmacokinetics of interferon alpha-2b in healthy

Pharmacokinetic and pharmacodynamic modeling of recombinant human erythropoietin after intravenous and subcutaneous dose administration in

Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative

correlation between protection and blood-enzyme level in mice. *Biochem Pharmacol*. 38(3):529-34.


**6** 

*Iran* 

Mahdi Banaee

*Behbahan University of Technology,* 

**Adverse Effect of Insecticides on Various** 

**Aspects of Fish's Biology and Physiology** 

*Department of Aquaculture, Natural Resource and Environmental Faculty,* 

Today, water quality management faces greater problems than at any time in its history. In addition to natural pollutants, varied contaminants exist in surface waters including multiple chemical compounds and different products of industrial and agricultural revolution. The insecticides constitute one group of these pollutants, both synthetic and natural, which contribute to the environmental problems. At present, it seems that the problem is more conspicuous in developing countries, where lately there has been an increase in the use of insecticides as a means of increasing agricultural productivity, without much concern to the consequences of indiscriminate application. There are many pathways by which insecticides leave their sites of application and distribute throughout the environment and enter the aquatic ecosystem. The major route of insecticides to water ecosystems in urban areas is through rainfall runoff and atmospheric deposition. Another source of water contamination by insecticides is from municipal and industrial dischargers. Most insecticides ultimately find their way into rivers, lakes and ponds (Tarahi Tabrizi, 2001; Honarpajouh, 2003; Bagheri, 2007; Shayeghi *et al*., 2007; Vryzas *et al*., 2009; Werimo *et al*., 2009; Arjmandi *et al*., 2010) and have been found to be highly toxic to non-target organisms that inhabit natural environments close to agricultural fields. The contamination of surface waters by insecticides is known to have ill effects on the growth, survival and reproduction of aquatic animals. In the past few years, the increase of mortality among the fish in various streams, lakes and ponds of around the world has drawn scholars' attention to the problems caused by insecticides and pesticides runoff associated with intense agricultural practices. Different concentrations of insecticides are present in many types of wastewater and numerous studies have found them to be toxic to aquatic organisms especially fish species (Talebi, 1998; Üner *et al*., 2006; Banaee *et al*., 2008). Fishes are particularly sensitive to the environmental contamination of water. Hence, pollutants such as insecticides may significantly damage certain physiological and biochemical processes when they enter into the organs of fishes (John, 2007; Banaee *et al*., 2011). Authors found out that different kinds of insecticides can cause serious impairment to physiological and health status of fishes (Begum, 2004; Monteiro *et al*., 2006; Siang *et al*., 2007; Banaee *et al*., 2009). Since fishes are important sources of proteins and lipids for humans and domestic animals, so health of fishes is very important for human beings. Recently, many studies have been conducted to determine the mechanisms of insecticides in fishes, with the ultimate goal of

**1. Introduction** 


#### Mahdi Banaee

*Department of Aquaculture, Natural Resource and Environmental Faculty, Behbahan University of Technology, Iran* 

#### **1. Introduction**

100 Insecticides – Basic and Other Applications

Supersaxo, A., Hein, W. R., & Steffen, H. (1990). Effect of molecular weight on the lymphatic

Vale, J. (1998). Toxicokinetic and toxicodynamic aspects of organophosphorus (OP)

Varki, A. (2001). N-glycolylneuraminic acid deficiency in humans. *Biochimie*. 83(7):615-22.

Villa, A. F., Houze, P., Monier, C., Risède, P., Sarhan, H., Borron, S. W., Mégarbane, B.,

Wenderoth, I., & von Schaewen, A. (2000). Isolation and characterization of plant N-acetyl

Worek, F., Aurbek, N., Herkert, N. M., John, H., Eddleston, M., Eyer, P., & Thiermann, H.

Yang, M.X., Shenoy, B., Disttler, M., Patel, R., McGrath, M., Pechenov, S., & Margolin, A. L.

insecticide poisoning. Toxicol Lett. 102-103:649-52.

*Pharm Res*. 7(2):167-9.

*Interact*. 187(1-3):259-64.

*Sci U S A*. 100(12):6934-9.

Review.

37-49.

108.

absorption of water-soluble compounds following subcutaneous administration.

Garnier, R., & Baud, F. J. (2007). Toxic doses of paraoxon alter the respiratory pattern without causing respiratory failure in rats. *Toxicology*. 232(1-2):

glucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants. *Plant Physiol*. 123(3):1097-

(2010). Evaluation of medical countermeasures against organophosphorus compounds: the value of experimental data and computer simulations. *Chem Biol* 

(2003). Crystalline monoclonal antibodies for subcutaneous delivery. *Proc Natl Acad* 

Today, water quality management faces greater problems than at any time in its history. In addition to natural pollutants, varied contaminants exist in surface waters including multiple chemical compounds and different products of industrial and agricultural revolution. The insecticides constitute one group of these pollutants, both synthetic and natural, which contribute to the environmental problems. At present, it seems that the problem is more conspicuous in developing countries, where lately there has been an increase in the use of insecticides as a means of increasing agricultural productivity, without much concern to the consequences of indiscriminate application. There are many pathways by which insecticides leave their sites of application and distribute throughout the environment and enter the aquatic ecosystem. The major route of insecticides to water ecosystems in urban areas is through rainfall runoff and atmospheric deposition. Another source of water contamination by insecticides is from municipal and industrial dischargers. Most insecticides ultimately find their way into rivers, lakes and ponds (Tarahi Tabrizi, 2001; Honarpajouh, 2003; Bagheri, 2007; Shayeghi *et al*., 2007; Vryzas *et al*., 2009; Werimo *et al*., 2009; Arjmandi *et al*., 2010) and have been found to be highly toxic to non-target organisms that inhabit natural environments close to agricultural fields. The contamination of surface waters by insecticides is known to have ill effects on the growth, survival and reproduction of aquatic animals. In the past few years, the increase of mortality among the fish in various streams, lakes and ponds of around the world has drawn scholars' attention to the problems caused by insecticides and pesticides runoff associated with intense agricultural practices. Different concentrations of insecticides are present in many types of wastewater and numerous studies have found them to be toxic to aquatic organisms especially fish species (Talebi, 1998; Üner *et al*., 2006; Banaee *et al*., 2008). Fishes are particularly sensitive to the environmental contamination of water. Hence, pollutants such as insecticides may significantly damage certain physiological and biochemical processes when they enter into the organs of fishes (John, 2007; Banaee *et al*., 2011). Authors found out that different kinds of insecticides can cause serious impairment to physiological and health status of fishes (Begum, 2004; Monteiro *et al*., 2006; Siang *et al*., 2007; Banaee *et al*., 2009). Since fishes are important sources of proteins and lipids for humans and domestic animals, so health of fishes is very important for human beings. Recently, many studies have been conducted to determine the mechanisms of insecticides in fishes, with the ultimate goal of

glutathione, uridyl-diphosphate glucose (UDPG), uridyl-diphosphate-glucuronic acid (UDPGA), amino acid derivatives and sulfate derivatives and can readily excrete from the fish body (Iannelli *et al*., 1994; Keizer *et al*., 1995; Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; Nebbia, 2001). In other words, final metabolites may also be excreted from the body of fish through the skin, gills, genital products, urine as sulphated and glucuronidated metabolites and stool as glutathione conjugated metabolites (Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; McKim & Lein, 2001; Nebbia, 2001). Since metabolites produced during detoxification process may be more dangerous than parental compounds, these metabolites can cause serious damage in fish. Furthermore, the production of reactive oxygen species (ROS) during detoxification process can induce oxidative damage and may be a mechanism of toxicity for aquatic organisms living in environments receiving insecticides (Monteiro *et al*., 2009). ROS can indiscriminately attack and react with susceptible vital macromolecules -lipids, proteins and DNA- in living cells, inducing cytotoxicity and can result in serious disturbances in physiological cell processes (Dogan *et al*., 2011; Jin *et al*., 2011). Lipid peroxidation, the major contributor to the loss of cell function, DNA damage, enzyme inactivation, and hormone oxidation are bio-indicators of oxidative cell damage and examples of toxic mechanisms of insecticide induced ROS being involved in pathological processes and in the etiology of many fish diseases (Üner *et* 

In acute toxicity, sudden and intense mortality may be observed in a fish population exposed to toxic materials. The most apparent symptoms of insecticides' acute poisoning in fishes include lethargy, forward extension fins, pallor or blur parts of body, severe reaction to external stimuli and muscle spasms and sudden fast swimming in circles. The main clinical internal sings that can lead to death of fishes include neurological disorder and disruption of nerve functions, respiratory dysfunction and suffocation (Banaee *et al*., 2011). Acute toxicity testing is widely used in order to identify the dose or exposure concentration and the time associated with death of 50 percent of the fish exposed to toxic materials which is expressed as LC50 in parts per million (ppm) or milligrams per liter (mg/L). In addition, we can use the LC50 value in the classification of insecticides based on potential toxicity for fishes. Furthermore, the relative acute toxicity of chemicals to fish can be categorized as

**Toxicity rating 96 hr LC50** Slightly toxic 10-100 ppm Moderately toxic 1-10 ppm Highly toxic 0.1-1.0 ppm Extremely toxic Less than 0.1 ppm Our literature reviews demonstrate that different fish species, even from the same family, show differences in the sensitivity to high concentrations of insecticides in water. Acute toxicity of different insecticides is influenced by the age, sex, genetic properties and body size of fish, water quality and its physicochemical parameters, and purity and formulation

The eight tables, which give relative acute toxicity of some insecticides to fishes, can be used to determine the potential toxicity to fish of using these compounds in farms around surface waters and to select products which less likely to cause problems. The data are derived from

*al*., 2006; Dogan *et al*., 2011).

**3. Acute toxicity** 

follows:

of insecticides.

monitoring, controlling and possibly intervening in xenobiotics exposure and its effects on the aquatic ecosystem. This chapter presents further information concerning the toxic effects of different concentrations of insecticides on various aspects of fish's biology and physiology. In other words, this chapter depicts the effects of insecticides on the survival chance, blood biochemical parameters, tissues and organs, reproduction, development and growth, nervous system, behavior, genetic and immune system of fish. The information given in this part facilitates the evaluation of potential toxic hazard resulting from exposure to different levels of these compounds.

#### **2. Biokinetics and bioteransformation**

After exposure to difference concentrations of insecticides in water, the fish absorbs them in its gills, skin or gastrointestinal tract. In the other words, Due to their lipophilicity, most insecticides easily permeate the biological membranes and it increases the sensitivity of sh to aqueous insecticides. Then, these compounds are rapidly metabolized and extracted, and may be bio-concentrated in various tissues of fish. In other words, bio-accumulation occurs if the insecticides is slowly metabolized or excreted from the body. As the amount of insecticide increases, it becomes more harmful to the consumer or animal. Accumulated insecticide can cause death or long-term damage. Ballesteros *et al.* (2011) showed that during the initial 24 h of exposure, insecticides may be transformed in various tissues of fish. However, some differences exist among tissues relating to metabolism rates, leading to different distribution models of the original compounds and their metabolites. For example, the low biodegradation and the high lipid solubility of some insecticides such as organochlorine insecticides have led to problems with the bio-concentrations of these compounds in different tissues of fish. In addition, since some fish are lower on the food chain, bioaccumulation of insecticides may increase in tissues of their predators and consumers, such as humans and thus affecting their health and survival. So, the bioaccumulation of these contaminants in sh and the potential biomagnication in humans are perceived as threats (Favari *et al*., 2002). Bioaccumulation rate of insecticides in fish depends on the species, life stages, the amount of fat reservation in different tissues and diet of fish, chemical and physical properties of insecticides and the rate of water pollution.

In order to facilitate the elimination and detoxification of toxic compounds, fishes have developed partly complex detoxification mechanisms including the release of several enzymes collectively termed xenobiotic metabolizing enzymes. Enzymatic biotransformation of insecticides can potentially alter their activity and toxicity. Enzymes participating in the biotransformation of insecticides are classified into phase I and phase II enzymes. The phase I enzymes, cytochrome P450 enzymes including CYP1A and CYP3A, are generally involved in the biotransformation of exogenous and endogenous compounds; thereby creating a more polar and water soluble compound. A great diversity of cytochrome P450 enzymes in fish has been recognized (Stegeman and Hahn, 1994), and CYP1A, CYP2B, CYP2E1, CYP2K1 and CYP3A have been recently identified in liver of some freshwater fish (Nabb *et al*., 2006) which play an important role in the detoxification of organophosphate and carbamate insecticides (Ferrari *et al*., 2007). The common pathways of biotransformation of different kinds of insecticides include three cytochrome P450 (CYP) mediated reactions: *O*-dealkylation, hydroxylation, and epoxidation of insecticides (Soldano *et al*., 1992; Keizer *et al*., 1995; Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; Nebbia, 2001). In phase II reactions, metabolites produced in phase I detoxification often conjugate with glutathione, uridyl-diphosphate glucose (UDPG), uridyl-diphosphate-glucuronic acid (UDPGA), amino acid derivatives and sulfate derivatives and can readily excrete from the fish body (Iannelli *et al*., 1994; Keizer *et al*., 1995; Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; Nebbia, 2001). In other words, final metabolites may also be excreted from the body of fish through the skin, gills, genital products, urine as sulphated and glucuronidated metabolites and stool as glutathione conjugated metabolites (Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; McKim & Lein, 2001; Nebbia, 2001). Since metabolites produced during detoxification process may be more dangerous than parental compounds, these metabolites can cause serious damage in fish. Furthermore, the production of reactive oxygen species (ROS) during detoxification process can induce oxidative damage and may be a mechanism of toxicity for aquatic organisms living in environments receiving insecticides (Monteiro *et al*., 2009). ROS can indiscriminately attack and react with susceptible vital macromolecules -lipids, proteins and DNA- in living cells, inducing cytotoxicity and can result in serious disturbances in physiological cell processes (Dogan *et al*., 2011; Jin *et al*., 2011). Lipid peroxidation, the major contributor to the loss of cell function, DNA damage, enzyme inactivation, and hormone oxidation are bio-indicators of oxidative cell damage and examples of toxic mechanisms of insecticide induced ROS being involved in pathological processes and in the etiology of many fish diseases (Üner *et al*., 2006; Dogan *et al*., 2011).

#### **3. Acute toxicity**

102 Insecticides – Basic and Other Applications

monitoring, controlling and possibly intervening in xenobiotics exposure and its effects on the aquatic ecosystem. This chapter presents further information concerning the toxic effects of different concentrations of insecticides on various aspects of fish's biology and physiology. In other words, this chapter depicts the effects of insecticides on the survival chance, blood biochemical parameters, tissues and organs, reproduction, development and growth, nervous system, behavior, genetic and immune system of fish. The information given in this part facilitates the evaluation of potential toxic hazard resulting from exposure

After exposure to difference concentrations of insecticides in water, the fish absorbs them in its gills, skin or gastrointestinal tract. In the other words, Due to their lipophilicity, most insecticides easily permeate the biological membranes and it increases the sensitivity of sh to aqueous insecticides. Then, these compounds are rapidly metabolized and extracted, and may be bio-concentrated in various tissues of fish. In other words, bio-accumulation occurs if the insecticides is slowly metabolized or excreted from the body. As the amount of insecticide increases, it becomes more harmful to the consumer or animal. Accumulated insecticide can cause death or long-term damage. Ballesteros *et al.* (2011) showed that during the initial 24 h of exposure, insecticides may be transformed in various tissues of fish. However, some differences exist among tissues relating to metabolism rates, leading to different distribution models of the original compounds and their metabolites. For example, the low biodegradation and the high lipid solubility of some insecticides such as organochlorine insecticides have led to problems with the bio-concentrations of these compounds in different tissues of fish. In addition, since some fish are lower on the food chain, bioaccumulation of insecticides may increase in tissues of their predators and consumers, such as humans and thus affecting their health and survival. So, the bioaccumulation of these contaminants in sh and the potential biomagnication in humans are perceived as threats (Favari *et al*., 2002). Bioaccumulation rate of insecticides in fish depends on the species, life stages, the amount of fat reservation in different tissues and diet of fish, chemical and physical properties of insecticides and the rate of water pollution. In order to facilitate the elimination and detoxification of toxic compounds, fishes have developed partly complex detoxification mechanisms including the release of several enzymes collectively termed xenobiotic metabolizing enzymes. Enzymatic biotransformation of insecticides can potentially alter their activity and toxicity. Enzymes participating in the biotransformation of insecticides are classified into phase I and phase II enzymes. The phase I enzymes, cytochrome P450 enzymes including CYP1A and CYP3A, are generally involved in the biotransformation of exogenous and endogenous compounds; thereby creating a more polar and water soluble compound. A great diversity of cytochrome P450 enzymes in fish has been recognized (Stegeman and Hahn, 1994), and CYP1A, CYP2B, CYP2E1, CYP2K1 and CYP3A have been recently identified in liver of some freshwater fish (Nabb *et al*., 2006) which play an important role in the detoxification of organophosphate and carbamate insecticides (Ferrari *et al*., 2007). The common pathways of biotransformation of different kinds of insecticides include three cytochrome P450 (CYP) mediated reactions: *O*-dealkylation, hydroxylation, and epoxidation of insecticides (Soldano *et al*., 1992; Keizer *et al*., 1995; Kitamura *et al*., 2000; Straus *et al*., 2000; Behrens & Segner, 2001; Nebbia, 2001). In phase II reactions, metabolites produced in phase I detoxification often conjugate with

to different levels of these compounds.

**2. Biokinetics and bioteransformation** 

In acute toxicity, sudden and intense mortality may be observed in a fish population exposed to toxic materials. The most apparent symptoms of insecticides' acute poisoning in fishes include lethargy, forward extension fins, pallor or blur parts of body, severe reaction to external stimuli and muscle spasms and sudden fast swimming in circles. The main clinical internal sings that can lead to death of fishes include neurological disorder and disruption of nerve functions, respiratory dysfunction and suffocation (Banaee *et al*., 2011). Acute toxicity testing is widely used in order to identify the dose or exposure concentration and the time associated with death of 50 percent of the fish exposed to toxic materials which is expressed as LC50 in parts per million (ppm) or milligrams per liter (mg/L). In addition, we can use the LC50 value in the classification of insecticides based on potential toxicity for fishes. Furthermore, the relative acute toxicity of chemicals to fish can be categorized as follows:


Our literature reviews demonstrate that different fish species, even from the same family, show differences in the sensitivity to high concentrations of insecticides in water. Acute toxicity of different insecticides is influenced by the age, sex, genetic properties and body size of fish, water quality and its physicochemical parameters, and purity and formulation of insecticides.

The eight tables, which give relative acute toxicity of some insecticides to fishes, can be used to determine the potential toxicity to fish of using these compounds in farms around surface waters and to select products which less likely to cause problems. The data are derived from

0.1-30 ppm McAllister *et al*.,

9-348 ppb Jonson & Finley,

0.9-39 ppm Jonson & Finley,

0.22-23 ppm Dobšíková, 2003;

0.02-6 ppm Jonson & Finley,

2.4-280 ppb Yi *et al*., 2006;

3-115 ppb Jonson & Finley, 1980

0.72-11.9 ppm Jonson & Finley, 1980

0.34-1.2 ppm Jonson & Finley, 1980

0.57-3270 ppb Davey *et al*., 1976; Holcombe *et* 

*al*., 1982; Bowman, 1988a, b; Gül, 2005; Wang *et al*., 2009

6.5-14.7 ppb Rand, 2004

1986a, b;

1980

1980

Begum, 2004

Jonson & Finley,

1980

1980

**Insecticide species Range of 96h LC50 Reference** 

Bensulide Rainbow trout, Bluegill, Black

Carbaryl Coho salmon, Chinook salmon,

Carbofuran Walked catfish, *Poecilia reticulate*, Chubs

Carbophenthion Channel catfish, Bluegill, Green sunfish

Table 2. Summary of acute toxicity.

Chlordane Coho salmon, Cutthroat

Chlorfenapyr Bluegill, Rainbow trout,

Chlorpyrifos Mosquito fish, Bluegill,

bass

Coumaphos Cutthroat trout,

channel catfish

Fathead minnow, Rainbow trout, Nile tilapia, Goldfish

Rainbow trout, Lake trout, Channel catfish, Bluegill, Largemouth

Chlorethoxyphos Cutthroat trout,

Carbosulfan Bluegill, Cutthroat trout, Rainbow

trout, Rainbow trout, Brown trout, Fathead minnow, Channel catfish, Bluegill, Largemouth bass

Rainbow trout, Fathead minnow, Channel catfish, Bluegill, Largemouth bass

Benzene hexachloride

bullhead, Common eel, Guppy, Sheephead minnow, Crucian carp

Rainbow trout, Bluegill, Cutthroat trout, Goldfish, fathead minnow, Channel catfish, Largemouth bass

Cutthroat trout, Atlantic salmon, Brown trout, Brook trout, Lake trout, Carp, Channel Catfish, Fathead Minnow, Rainbow Trout, Bluegill, Goldfish, Black bullhead, Green sunfish, Largemouth bass, Black crappie, Yellow perch

trout, Lake trout, Channel catfish

**Insecticide species Range of 96h LC50 Reference** 

Cryolite Rainbow trout, Bluegill 47-400 ppm Jonson & Finley, 1980

laboratory studies and are given only as a guideline and not absolute data of the acute toxicity of the insecticides to different species of fish (Table 1-11).


Table 1. Summary of acute toxicity.


Table 2. Summary of acute toxicity.

104 Insecticides – Basic and Other Applications

laboratory studies and are given only as a guideline and not absolute data of the acute

**Insecticide species Range of 96h LC50 Reference** 

Aldicarb Fathead minnow 1.3-2.4 ppm Pant *et al*., 1987

Allethrin Rainbow trout, Bluegill 19-56 ppb Jonson & Finley,

Rainbow trout, Bluegill 1.1-20 ppb Jonson & Finley,

0.17-1370 ppb Jonson & Finley,

2.3-53 ppb Jonson & Finley,

3.1-31 ppm Jonson & Finley,

2.1-4270 ppb Arufe *et al*., 2007;

4.9-50 ppm Jonson & Finley,

1980

1980

1980

1980

1980

1980

1980

Jonson & Finley,

toxicity of the insecticides to different species of fish (Table 1-11).

Akton Channel catfish, Bluegill,

Aldrin Chinook salmon, Rainbow

Aminocrab Cutthroat trout, Rainbow

trout

perch

Azodrin Rainbow trout, Bluegill,

minnow

Table 1. Summary of acute toxicity.

Azinphos ethyl

Azinphos methyl

bullhead, Bluegill, Largemouth bass

trout, Bluegill, Atlantic salmon, Fathead minnow, Channel catfish, Largemouth bass, Yellow perch, Brook

Gilthead seabream, Coho salmon, Rainbow trout, Bluegill, Atlantic salmon, Brown trout, pike, Goldfish, Carp, Fathead minnow, Black bullhead, Channel catfish, Green sunfish, Largemouth bass, Black crappie, Yellow

Channel catfish. Fathead

minnow

Rainbow trout, Fathead

trout, Fathead minnow, Black


22-110 ppb Jonson & Finley, 1980

60-4700 ppb Jonson & Finley, 1980

2.6-66 ppb Jonson & Finley, 1980

0.15-1.8 ppb Jonson & Finley, 1980

110-420 ppb Jonson & Finley, 1980

0.17-7.6 ppm Jonson & Finley, 1980

0.4-3.52 ppm Jonson & Finley, 1980

1.7-12 ppm Johnson & Finley, 1980;

1.1-3.4 ppm Jonson & Finley, 1980

1980

Woodward & Mauck, 1980; Jonson & Finley,

0.1-20 ppb Mayer & Ellersieck, 1986; Siang *et al*.,

2007; Capkin *et al*., 2006; Magesh & Kumaraguru, 2006, Velasco-Santamaría *et al*., 2011

**Insecticide species Range of 96h LC50 Reference** 

Dioxathion Cutthroat trout, Rainbow

Disulfoton Rainbow trout, Fatheah

perch

Endosulfan Striped bass, Bluegill,

Endrin Rainbow trout, Goldfish,

perch, carp

EPN Cutthroat trout, Rainbow

Ethion Cutthroat trout, Rainbow

Table 5. Summary of acute toxicity.

perch

Fenitrothion Coho salmon, Cutthroat trout,

Fenthion Coho salmon, , Rainbow trout, Brown trout, Brook trout, Atlantic salmon, Goldfish, Yellow perch, Bluegill, Channel catfish, Green sunfish, Fathead minnow, Largemouth

bass, Carp

Walleye

d-Trans Allethrin

Ethyl Parathion trout, Largemouth bass

minnow, Channel catfish, Bluegill, Largemouth bass

Coho salmon, Steelhead, Lake trout, pike, Fathead minnow, Channal catfish, Largemouth bass, Yellow

Rainbow trout, Fathead minnows, Asian swamp eel, Milk fish, Zebra fish

fathead minnow, black bullhead, Channel catfish, Mosquito fish, Bluegill, Largemouth bass, Yellow

trout, Channel catfish, Bluegill, Largemouth bass,

trout, Channel catfish, Bluegill, Largemouth bass,

Coho salmon, Cutthroat trout, Rainbow trout, Brown trout, Goldfish, Carp, Fathead minnow, Channel catfish, Bluegill, Black bullhead, Largemouth bass, Yellow

Rainbow trout, Brown trout, Brook trout, Atlantic salmon, Goldfish, Bluegill, Channel catfish, Fathead minnow, Carp

**Insecticide species Range of 96h LC50 Reference** 

Fathead minnow


Table 3. Summary of acute toxicity.


Table 4. Summary of acute toxicity.



Table 5. Summary of acute toxicity.

106 Insecticides – Basic and Other Applications

0.39-0.95 ppb Jaber & Hawk, 1981; Sousa,

14-4400 ppb Jonson & Finley, 1980

32-240 ppb Jonson & Finley, 1980

1.5-21.5 ppb Jonson & Finley, 1980

2003

0.9-2.6 ppm Calmbacher, 1978a, b; Banaee *et* 

0.18-7.5 ppm Mayer & Ellersieck 1986; Jones & Davis, 1994

6.3-24.2 ppm Jonson & Finley, 1980

1.2-19 ppb Jonson & Finley, 1980

25-240 ppm Jonson & Finley, 1980

28-1275 ppb Jonson & Finley, 1980

*al*., 2011; Banaee *et al*., 2008; Jonson & Finley, 1980

1998; Mishra et al., 2005

Cypermethrin Sheepshead minnow,

DDD Rainbow trout, Fathead

DDE Rainbow trout, Atlantic

Table 3. Summary of acute toxicity.

DDT Coho samon, Rainbow trout,

perch

Diazinon Cutthroat trout, Rainbow

Dichlorvos Lake Trout, Sheephead minnow

Dicrotophos Bluegill, Rainbow trout, Channel catfish

Dieldrin Cutthroat trout, Rainbow

Diflubenzuron Cutthroat trout, Rainbow

Dimethrin Fathead minnow, Channel

Bluegill,

Table 4. Summary of acute toxicity.

Fathead minnow, Channel catfish, Bluegill, Largemouth bass, Black bullhead, Yellow

trout, Lake trout, Fathead minnow, Carp, Bluegill

trout, Goldfish, Fathead minnow, Channel catfish, Bluegill, Largemouth bass

trout, Brook trout, Fathead minnow, Channel catfish, Bluegill, Yellow perch

catfish, Yellow perch,

Rainbow trout, Bluegill, Freshwater catsh

minnow, Channel catfish, Largemouth bass, Walleye

salmon, Bluegill

**Insecticide species Range of 96h LC50 Reference** 

Deltamethrin Guppies, Channa punctatus, 1.5-5.13 ppb Viran et al., 2003; Sayeed et al.,

Dimethoate Rainbow trout, Bluegill 6-9.3 ppm Jonson & Finley, 1980

Dinitrocresol Rainbow trout, Bluegill 66-360 ppb Jonson & Finley, 1980


15-64 ppb Jonson & Finley, 1980

0.25-9 ppm Mayer & Ellersieck,1986;

0.76-2.8 ppm Jonson & Finley, 1980

0.32-23 ppm Jonson & Finley, 1980

100 < ppm Jonson & Finley, 1980

Monteiro et al., 2006; Jonson & Finley, 1980

Rao, 2007

0.13-3.3 ppm Jonson & Finley, 1980

13-31.5 ppm Jonson & Finley, 1980

2-110 ppb Jonson & Finley, 1980

**Insecticide species Range of 96h LC50 Reference** 

Methoxychlor Rainbow trout, Atlantic

Methyl Parathion

Methyl Trithion salmon, Cutthroat trout, Brook trout, Lake trout, pike, Goldfish, Largemouth bass, Bluegill, Yellow perch, Fathead minnow, Channel catfish

Freshwater characid fish, Coho salmon, Cutthroat trout, Rainbow trout, Brown trout, Lake trout, Goldfish, Carp, Fathead minnow, Channel catfish, Bluegill, Black bullhead, Green sunfish, Largemouth bass, Yellow perch

Cutthroat trout, Rainbow trout, Channel catfish, Bluegill, Largemouth bass

trout, Rainbow trout, Atlantic salmon, Lake trout, Carp, Fathead minnow, Channel catfish, Bluegill, Black bullhead, Largemouth bass, Yellow

trout, Fathead minnow, Channel catfish, Bluegill, Largemouth bass, Yellow

**Insecticide species Range of 96h LC50 Reference** 

Monocrotophos Tilapia, Mosquito fish 11.5-20.5 ppm Rao, 2006; Kavitha &

Permethrin Brook trout 1.4-7.9 ppb Jonson & Finley, 1980

perch, Walleye

Naled Cutthroat trout, Rainbow

Oxydemetonmethyl

trout, Lake trout, Fathead minnow, Channel catfish, Bluegill, Largemouth bass

Rainbow trout, Channel catfish, Bulegill, Largemouth

trout, Northern pike, Largemouth bass, Channel

bass, Walleye

catfish, Bluegill

Phorate Cutthroat trout, Rainbow

Mexacarbate Coho salmon, Cutthroat

perch

Mirex Rainbow trout, Brown

Table 8. Summary of acute toxicity.


Table 6. Summary of acute toxicity.


Table 7. Summary of acute toxicity.


Table 8. Summary of acute toxicity.

108 Insecticides – Basic and Other Applications

5.3-63 ppb Jonson & Finley, 1980

30-225 ppb Jonson & Finley, 1980

0.03-30 ppm Jonson & Finley, 1980

0.03-1.29 ppm Ferrando *et al*., 1988, Feltz,

4-12900 ppb Mayer & Ellersieck,1986;

1.6-100 ppm Jonson & Finley, 1980

0.3-6.8 ppm Jonson & Finley, 1980; Yi

et al., 2006

1971; Lawson *et al*., 2011; Jonson & Finley, 1980

Ellersieck,1986

Jonson & Finley, 1980

Fenvalerate Zebra fish 3.5-193 ppb Ma *et al*., 2009

Isoprocarb Goldfish 4.61 ppm Wang *et al*., 2009

**Insecticide species Range of 96h LC50 Reference** 

Linuron Bluegill, Rainbow trout 3-16.2 ppm Wetzel, 1986; Mayer &

Heptachlor Rainbow trout, Northern pike, Fathead minnow, Black bullhead, Channel catfish, Redear sunfish, Bluegill, Largemouth bass,

Table 6. Summary of acute toxicity.

Kepone Rainbow trout, Channel

sunfish

Lindane Eel, Tilapia, African

perch

Malathion Coho salmon, Cutthroat

Methamidophos Rainbow trout, Fathead

Bluegill

Methomyl Cutthroat trout, Rainbow

Table 7. Summary of acute toxicity.

Leptophos Rainbow trout, Lake trout,

catfish, Bluegill, redear

Fathead minnow, Bulegill

Catfish, Coho salmon, Rainbow trout, Brown trout, Goldfish, Carp, Fathead minnow, Black bullhead, Green sunfish, Largemouth bass, Yellow

trout, Rainbow trout, Brown trout, Lake trout, Goldfish, Carp, Fathead minnow, Black bullhead, Bluegill, Green sunfish, Largemouth bass, Yellow perch, Redear sunfish

minnow, Channel catfish,

trout, Atlantic salmon, Brook trout, Fathead minnow, Channel catfish, Bluegill, Largemouth bass


1.44-34 ppm Jonson & Finley, 1980

1.2-3.7 ppm Jonson & Finley, 1980

2-18 ppb Jonson & Finley, 1980

0.36-9.2 ppm Lopes *et al*., 2006;

Jonson & Finley, 1980

Insecticide species Range of 96h LC50 Reference

Thiodicarb Bluegill, Rainbow trout 1.4-3.3 ppm Yi *et al*., 2006

Sub-lethal toxicity testing was planned based on one tenth or more of LC50 dose in moderate periods. In sub-lethal toxicity, the organs or biological systems which may be affected at such exposure can be respiratory, hepatic, haematopoietic, nervous, cardiovascular, and reproductive and immune systems. Different biomarkers of fish exposed to insecticides are usually evaluated in these experiments. Insecticides may lead to changes in the blood biochemical parameters and haematological profile of fish which can be investigated as biomarker in pollution monitoring (Mushigeri & David, 2005; Banaee *et al*., 2008; Kavitha & Rao, 2009). In fact, these compounds may induce alterations in the activities or levels of a number of different enzyme systems, including those necessary for biochemical reactions in cells (Banaee *et al*., 2011). Decreased rate of growth, reproductive disorder, spinal deformities, histopathological changes (Benli & Özkul, 2010) in gills, liver, haematopoietic tissue such as spleen, head of kidney, and renal tubules, endocrine tissues as well as brain, neurological and behavioral disorder and genetic defects are other biological indicators of

exposure to insecticides which are described in details in the following sections.

Therefore, this experiment is important in insecticides toxicology.

Chronic toxicity tests commonly include the measurement of long term effects of low concentrations of insecticides on the survival, growth, reproduction, nervous system and other biological and physiological aspects of fishes. Type of injury to fish in chronic toxicity is similar to sub-lethal toxicity damage, but the frequency and intensity injury and lesion resulting from chronic toxicity may be more or even less than damage of sub-lethal toxicity.

Temephos Cutthroat trout, Rainbow trout,

Thanite Rainbow trout, Channel catfish,

Toxaphene Coho salmon, Rainbow trout,

Trichlorfon Eel, Rainbow trout, Cutthroat

Table 11. Summary of acute toxicity.

Brown trout, Goldfish, Carp, Fathead minnow, Black bullhead, Channel catfish, Bluegill, Largemouth bass, Yellow perch

trout, Atlantic salmon, Brown trout, Brook trout, Lake trout, Fathead minnow, Channel catfish, Bluegill, Largemouth

bass

bass

**4. Sub-lethal toxicity** 

**5. Chronic toxicity** 

Bluegill

Atlantic trout, Brook trout, Lake trout, Fathead minnow, Channel catfish, Bluegill, Largemouth


Table 9. Summary of acute toxicity.


Table 10. Summary of acute toxicity.


Table 11. Summary of acute toxicity.

#### **4. Sub-lethal toxicity**

110 Insecticides – Basic and Other Applications

**Insecticide species Range of 96h LC50 Reference** 

Strobane Bluegill, Rainbow trout 8.7-12 ppb Jonson & Finley,

0.15-10.6 ppm Jonson & Finley, 1980

3.4-100 ppm Jonson & Finley, 1980

0.11-2.9 ppm Jonson & Finley, 1980

4.8-36.2 ppm Wang *et al*., 2009;

13-65 ppb Jonson & Finley,

1.7-9.9 ppb Jonson & Finley,

0.6-1.6 ppm Jonson & Finley,

2.6-36 ppb Jonson & Finley,

0.3-28 ppb Jonson & Finley,

7.8-90 ppb Jonson & Finley,

1.5-5.7 ppm Jonson & Finley,

240-980 ppb Jonson & Finley,

Jonson & Finley, 1980

1980

1980

1980

1980

1980

1980

1980

1980

1980

Phosmet Coho salmon, Rainbow

Phosphamidon Rainbow trout, Fathead

Phoxim Coho salmon, Atlantic

Bluegill

bluegill

Pyrethrum Coho salmon, Atlantic salmon, Brown trout, Lake trout, Channel catfish, bluegill

catfish, bluegill

Ronnel Rainbow trout, Channel catfish,

Rotenone Rainbow trout, Channel catfish,

RU-1169 Coho salmon, Atlantic salmon,

SD-17250 Coho salmon, Rainbow trout, bluegill

TEPP Rainbow trout, Fathead minnow, Bluegill

Table 10. Summary of acute toxicity.

Fathead minnow, Channel

bluegill, Cutthroat trout, Lake

Lake trout, fathead minnow, White sucker, Bluegill

Fathead minnow, Channel catfish, Bluegill, Yellow perch

Resmethrin Cho salmon, Lake trout,

trout

S-

Bioallethrin

bluegill

Propoxur Goldfish, Rainbow trout,

Table 9. Summary of acute toxicity.

trout, Fathead minnow, Channel catfish, bluegill, Smallmouth bass, Largemouth bass

minnow, Channel catfish,

salmon, Rainbow trout, Brown trout, Brook trout, Northern pike, Fathead minnow, Channel catfish,

Fathead minnow, Bluegill

Sub-lethal toxicity testing was planned based on one tenth or more of LC50 dose in moderate periods. In sub-lethal toxicity, the organs or biological systems which may be affected at such exposure can be respiratory, hepatic, haematopoietic, nervous, cardiovascular, and reproductive and immune systems. Different biomarkers of fish exposed to insecticides are usually evaluated in these experiments. Insecticides may lead to changes in the blood biochemical parameters and haematological profile of fish which can be investigated as biomarker in pollution monitoring (Mushigeri & David, 2005; Banaee *et al*., 2008; Kavitha & Rao, 2009). In fact, these compounds may induce alterations in the activities or levels of a number of different enzyme systems, including those necessary for biochemical reactions in cells (Banaee *et al*., 2011). Decreased rate of growth, reproductive disorder, spinal deformities, histopathological changes (Benli & Özkul, 2010) in gills, liver, haematopoietic tissue such as spleen, head of kidney, and renal tubules, endocrine tissues as well as brain, neurological and behavioral disorder and genetic defects are other biological indicators of exposure to insecticides which are described in details in the following sections.

#### **5. Chronic toxicity**

Chronic toxicity tests commonly include the measurement of long term effects of low concentrations of insecticides on the survival, growth, reproduction, nervous system and other biological and physiological aspects of fishes. Type of injury to fish in chronic toxicity is similar to sub-lethal toxicity damage, but the frequency and intensity injury and lesion resulting from chronic toxicity may be more or even less than damage of sub-lethal toxicity. Therefore, this experiment is important in insecticides toxicology.

2003; Banaee *et al*., 2009), vitellogenesis process impairment (Haider and Upadhyaya, 1985; ), and disruption in steroidogenesis process (Zaheer Khan & Law, 2005), delay in gonads maturation (Skandhan *et al*., 2008), alter in reproductive and parental behavior (Jaensson *et al*., 2007), impairment in olfactory response and disorder in reproductive migrations (Scholz *et al*., 2000), as well as disruption in coordinating courtship behavior of male and female fish

Some insecticides are known as endocrine disrupting chemicals (EDC) which can interfere with the normal functioning of endocrine system in fish. Adverse effects of insecticides on the hypothalamus-pituitary-gonads axis can also play a significant role in causing reproductive failures in fish. In fishes, chronic toxicity of insecticides can change sex steroid hormone levels in plasma. While the mechanism is not exactly known, it is possible that insecticides and their metabolites disrupt reproductive systems through activation or inhibition of key enzymes which participated in the steroid hormone biosynthesis in fishes. For example, DDT, endosulfan, methoxychlor and some other insecticides possess estrogenic properties and are probably capable of disrupting functions of endocrine system and causing disorder in the reproductive system of fish (Arukwe, 2001). These compounds may directly or indirectly interact with natural hormones, changing the hormone functions and thus altering physiological cellular response or mutate the natural patterns of hormone synthesis and metabolism. Impact of organophosphate insecticides such as malathion, diazinon and fenitrothion on the hypothalamus-pituitary-gonads axis and also disturbance in hormones associated with reproductive systems were studied by Kapur & Toor, (1978);

Insecticides can also cause adverse effects on gonad histology, morphology and its growth. In addition, there are significant relationships between blood sex steroid hormone concentrations, sperm or oocytes quality, rate of fecundity and histopathological alterations in ovary and testis of fish exposed to different insecticides (Duttaa & Meijer, 2003; Maxwell & Dutta, 2005). Banaee *et al*., (2008) reported that diazinon inhibits steroidogenesis in testis of male carp by histopatological alterations. Research results showed that direct toxic effects of insecticides on seminiferous tubules or Leydig cells may be the most important parameter for the low quality of sperms in fish (Fadakar Masouleh *et al*., 2011). Similar results were reported in walking catfish (*Clarias batrachus*), freshwater eel (*Monopterus albus*), and Atlantic salmon (*Salmo salar*) that were exposed to different insecticides (Singh & Singh, 1987; Singh,

Exposure of fish eggs and milt to insecticides also reduced the level of fertilization, hatching rate and the larval survivability. Further studies on bluegills (*Lepomis macrochirus*), atlantic salmon (*Salmo salar* L.) demonstrated that the gametes and fertilized eggs were sensitive to the insecticides (Tanner & Knuth, 1996; Moore & Waring, 2001) suggesting a further toxic impact of these toxicants on the fish reproduction. In addition, the waste of energy in the

Study of development disorders caused by insecticides is to emphasize the links between the concentrations of toxins and dysfunction in normal development from embryonic to puberty periods. So, impairment in the normal development and the growth may reduce the

Singh and Singh, (1987); Maxwell & Dutta, (2005); Skandhan *et al*. (2008).

fish exposed to insecticides reduces their reproductive ability.

and time of spawning (Jaensson *et al*., 2007).

1989; Moore & Waring, 1996; 2001).

**6.4 Development disorders** 

fish's survival chance.

#### **6. Side effect of insecticides on various aspects of fish's biology and physiology**

#### **6.1 Alterations in blood biochemical parameters**

Insecticides can cause serious impairment to physiological and health status of fish. Therefore, biochemical tests are routine laboratory tests useful in recognizing acute or chronic toxicity of insecticides (Banaee *et al*., 2008; Al-Kahtani, 2011) and can be a practical tool to diagnose toxicity effects in target organs and to determine the physiological status in fish. Blood biochemistry test gives indicates what is happening in the body of fish exposed to insecticides. When different tissues are injured, the damaged cells release specific enzymes into plasma and we can recognize their abnormality levels in blood. Then it helps locate the lesions. Also, if certain organs are not eliminating certain waste products or not synthesizing certain important materials, this can tell us they are not functioning properly. In some cases due to the severity of the damage to tissues, particularly liver, synthesis of many biochemical parameters may reduce significantly in cells, which can decrease some biochemical factors in blood of fish exposed to insecticides. These changes were observed in *Channa punctatus* (Agrahari *et al*., 2007), *Oreochromis niloticus* (Velisek *et al*., 2004), *O.mossambicus* (Arockia and Mitton, 2006; Matos *et al*., 2007), *Heteropneustes fossilis* (Saha & Kaviraj, 2009), *Cirrhinus mrigala* (Prashanth & Neelagund, 2008) *Clarias batrachus* (Begum, 2005; Ptnaik, 2010), *Cyprinus carpio* (Banaee *et al*., 2008), *Oncorhynchus mykiss* (Banaee *et al*., 2011), *Colisa fasciatus* (Singh *et al*., 2004) which were exposed to monocrotophos, bifenthrin, carbaryl, dimethoate, cypermethrin, sevin, diazinon, and malathion respectively.

#### **6.2 Tissue and organ damage**

Histopathological investigations on different tissues of fish are valuable tools for toxicology studies and monitoring water pollutions. In histopathology, we can provide information about the health and functionality of organs. Tissues injuries and damages in organs can result in the reduced survival, growth and fitness, the low reproductive success or increase of susceptibility to pathological agents. Frequency and intensity of tissue lesions depend on the concentrations of insecticides and the length of the period fish are exposed to toxins. Nevertheless, many insecticides cause specific or non-specific histopathological damage (Fanta *et al*., 2003). For example, histopathological lesions in the liver tissue of freshwater fish (*Cirrhinus mrigala*) (Velmurugan *et al*., 2009) and common carp carp (*Cyprinus carpio*) (Banaee *et al*., In press) were observed after 10 and 30 days exposure to sublethal concentrations of dichlorvos and diazinon insecticides, respectively. Other researchers reported the same histopathological alterations in different tissues of fish treated with diazinon (Duttaa & Meijer, 2003; Banaee *et al*., 2011), deltamethrin (Cengiz, 2006; Cengiz & Unlu, 2006), fenitrothion (Benli & Özkul, 2010).

#### **6.3 Reproductive dysfunction**

Reproduction guarantees the survival of fish population. Any changes in environmental parameters or physiological conditions of fish can affect its reproductive success. Since fish may be exposed to environmental pollutants, including insecticides, herbicides, heavy metals and other xenobiotics, disorders may occur in their natural reproductive process. Recent researches showed the dysfunction in the reproductive systems of fishes exposed to insecticides. Insecticides' effects on reproductive biology of fishes are numerous, and include decreased fecundity, testicular and ovarian histological damage (Duttaa & Meijer,

Insecticides can cause serious impairment to physiological and health status of fish. Therefore, biochemical tests are routine laboratory tests useful in recognizing acute or chronic toxicity of insecticides (Banaee *et al*., 2008; Al-Kahtani, 2011) and can be a practical tool to diagnose toxicity effects in target organs and to determine the physiological status in fish. Blood biochemistry test gives indicates what is happening in the body of fish exposed to insecticides. When different tissues are injured, the damaged cells release specific enzymes into plasma and we can recognize their abnormality levels in blood. Then it helps locate the lesions. Also, if certain organs are not eliminating certain waste products or not synthesizing certain important materials, this can tell us they are not functioning properly. In some cases due to the severity of the damage to tissues, particularly liver, synthesis of many biochemical parameters may reduce significantly in cells, which can decrease some biochemical factors in blood of fish exposed to insecticides. These changes were observed in *Channa punctatus* (Agrahari *et al*., 2007), *Oreochromis niloticus* (Velisek *et al*., 2004), *O.mossambicus* (Arockia and Mitton, 2006; Matos *et al*., 2007), *Heteropneustes fossilis* (Saha & Kaviraj, 2009), *Cirrhinus mrigala* (Prashanth & Neelagund, 2008) *Clarias batrachus* (Begum, 2005; Ptnaik, 2010), *Cyprinus carpio* (Banaee *et al*., 2008), *Oncorhynchus mykiss* (Banaee *et al*., 2011), *Colisa fasciatus* (Singh *et al*., 2004) which were exposed to monocrotophos, bifenthrin,

**6. Side effect of insecticides on various aspects of fish's biology and** 

carbaryl, dimethoate, cypermethrin, sevin, diazinon, and malathion respectively.

Histopathological investigations on different tissues of fish are valuable tools for toxicology studies and monitoring water pollutions. In histopathology, we can provide information about the health and functionality of organs. Tissues injuries and damages in organs can result in the reduced survival, growth and fitness, the low reproductive success or increase of susceptibility to pathological agents. Frequency and intensity of tissue lesions depend on the concentrations of insecticides and the length of the period fish are exposed to toxins. Nevertheless, many insecticides cause specific or non-specific histopathological damage (Fanta *et al*., 2003). For example, histopathological lesions in the liver tissue of freshwater fish (*Cirrhinus mrigala*) (Velmurugan *et al*., 2009) and common carp carp (*Cyprinus carpio*) (Banaee *et al*., In press) were observed after 10 and 30 days exposure to sublethal concentrations of dichlorvos and diazinon insecticides, respectively. Other researchers reported the same histopathological alterations in different tissues of fish treated with diazinon (Duttaa & Meijer, 2003; Banaee *et al*., 2011), deltamethrin (Cengiz, 2006; Cengiz &

Reproduction guarantees the survival of fish population. Any changes in environmental parameters or physiological conditions of fish can affect its reproductive success. Since fish may be exposed to environmental pollutants, including insecticides, herbicides, heavy metals and other xenobiotics, disorders may occur in their natural reproductive process. Recent researches showed the dysfunction in the reproductive systems of fishes exposed to insecticides. Insecticides' effects on reproductive biology of fishes are numerous, and include decreased fecundity, testicular and ovarian histological damage (Duttaa & Meijer,

**6.1 Alterations in blood biochemical parameters** 

**6.2 Tissue and organ damage** 

Unlu, 2006), fenitrothion (Benli & Özkul, 2010).

**6.3 Reproductive dysfunction** 

**physiology** 

2003; Banaee *et al*., 2009), vitellogenesis process impairment (Haider and Upadhyaya, 1985; ), and disruption in steroidogenesis process (Zaheer Khan & Law, 2005), delay in gonads maturation (Skandhan *et al*., 2008), alter in reproductive and parental behavior (Jaensson *et al*., 2007), impairment in olfactory response and disorder in reproductive migrations (Scholz *et al*., 2000), as well as disruption in coordinating courtship behavior of male and female fish and time of spawning (Jaensson *et al*., 2007).

Some insecticides are known as endocrine disrupting chemicals (EDC) which can interfere with the normal functioning of endocrine system in fish. Adverse effects of insecticides on the hypothalamus-pituitary-gonads axis can also play a significant role in causing reproductive failures in fish. In fishes, chronic toxicity of insecticides can change sex steroid hormone levels in plasma. While the mechanism is not exactly known, it is possible that insecticides and their metabolites disrupt reproductive systems through activation or inhibition of key enzymes which participated in the steroid hormone biosynthesis in fishes. For example, DDT, endosulfan, methoxychlor and some other insecticides possess estrogenic properties and are probably capable of disrupting functions of endocrine system and causing disorder in the reproductive system of fish (Arukwe, 2001). These compounds may directly or indirectly interact with natural hormones, changing the hormone functions and thus altering physiological cellular response or mutate the natural patterns of hormone synthesis and metabolism. Impact of organophosphate insecticides such as malathion, diazinon and fenitrothion on the hypothalamus-pituitary-gonads axis and also disturbance in hormones associated with reproductive systems were studied by Kapur & Toor, (1978); Singh and Singh, (1987); Maxwell & Dutta, (2005); Skandhan *et al*. (2008).

Insecticides can also cause adverse effects on gonad histology, morphology and its growth. In addition, there are significant relationships between blood sex steroid hormone concentrations, sperm or oocytes quality, rate of fecundity and histopathological alterations in ovary and testis of fish exposed to different insecticides (Duttaa & Meijer, 2003; Maxwell & Dutta, 2005). Banaee *et al*., (2008) reported that diazinon inhibits steroidogenesis in testis of male carp by histopatological alterations. Research results showed that direct toxic effects of insecticides on seminiferous tubules or Leydig cells may be the most important parameter for the low quality of sperms in fish (Fadakar Masouleh *et al*., 2011). Similar results were reported in walking catfish (*Clarias batrachus*), freshwater eel (*Monopterus albus*), and Atlantic salmon (*Salmo salar*) that were exposed to different insecticides (Singh & Singh, 1987; Singh, 1989; Moore & Waring, 1996; 2001).

Exposure of fish eggs and milt to insecticides also reduced the level of fertilization, hatching rate and the larval survivability. Further studies on bluegills (*Lepomis macrochirus*), atlantic salmon (*Salmo salar* L.) demonstrated that the gametes and fertilized eggs were sensitive to the insecticides (Tanner & Knuth, 1996; Moore & Waring, 2001) suggesting a further toxic impact of these toxicants on the fish reproduction. In addition, the waste of energy in the fish exposed to insecticides reduces their reproductive ability.

#### **6.4 Development disorders**

Study of development disorders caused by insecticides is to emphasize the links between the concentrations of toxins and dysfunction in normal development from embryonic to puberty periods. So, impairment in the normal development and the growth may reduce the fish's survival chance.

*et al*., 2009; Banaee *et al*., 2011;). Similar results have been observed for pyrethroids insecticide toxicity (Koprucu *et al*., 2006). Disorder in γ-aminobutyrate (GABA) system in brain of rainbow trout exposed to sub-lethal lindane was reported by Aldegunde *et al*., (1999). GABA receptors inhibit the transmission of nerve impulses; thus disturbances in this

In addition, nervous tissue has weaker antioxidant defense system than other kinds of tissue. On the other hand, the brain as center of the nervous system in fish contains low levels of enzymatic and non-enzymatic antioxidant and higher levels of oxidizable unsaturated lipids and catecholamines. So, nerve tissue is very sensitive to oxidative stress damage induced insecticide toxicity compared with other tissues (Üner *et al*., 2006; Li *et al*.,

Behavioral changes are the most sensitive indicators of potential toxic effects. The behavioral and the swimming patterns of the fish exposed to different insecticides include changes in swimming behavior, feeding activities, predation, competition, reproduction and speciesspecies social interactions such as aggression (Cong *et al*., 2008; 2009; Werner and Oram, 2008). Banaee *et al*. (2008; 2011) reported similar behavioral responses in common carp and rainbow trout exposed to sub-lethal levels of diazinon. In fact, most insecticides influence the behavior patterns of fish by interfering with the nervous systems and sensory receptors (Keizer *et al*., 1995; Pan & Dutta, 1998; Cong *et al*., 2008; 2009); and this incident may impair the identification of situation and development of appropriate response by the fish exposed to insecticide. The effect of certain insecticides on the activity of acetylcholinestrase may lead to a decreased mobility in sh (Bretaud *et al*., 2000). Researchers have reported the same alterations in *Oryzias latipes*, *Cyprinus carpio*, *Labeo rohita*, *Oncorhynchus tshawytscha*, *O.latipes*, *Cirrhinus mrigala*, *Oreochromis niloticus*, *Clarias gariepinus* treated with chlorpyrifos (Rice *et al*., 1997; Halappa & David, 2009), malathion (Patil & David, 2008), diazinon (Scholz *et al*., 2000), endosulfan (Gormley & Teather, 2003), Fenvalerate (Mushigeri & David, 2005), fenitrothion

In genetic-toxicology any heritable damage or DNA inactivation resulting from the animal's exposure to xenobiotics is studied. Genotoxic chemicals such as insecticides have common chemicals and physical properties that enable them interact with genetic materials (Campana *et al*., 1999; Çava & Ergene-Gözükara, 2003; Candioti *et al*., 2010; Dogan et al., 2011). The mutation that may result from an interaction between a chemical and the genetic material is a heritable change in the cell genotype, and thus the error may be transferred to the daughter cell or the next generation. Carcinogenesis and the formation of some tumors in different tissues of fish exposed to insecticides may also be caused by genotoxic properties of these xenobiotics. One of the ill effects of insecticides' arrival into surface waters may be an induction of chromosomal damage in eggs and larvae of fishes in different

Some insecticides that behave as endocrine active compounds can change the expression of vital genes resulting in unusual concentrations of plasma steroid hormones and

2010). These results have been reported by other scientists (Senger *et al*., 2005).

receptor would also lead to an over stimulation of the nerves.

(Benli & Özkul, 2010), dimethoate (Auta *et al*., 2002), respectively.

reproductive dysfunction or immuosuppression (Jin *et al*., 2010).

**6.6 Behavioral alterations** 

**6.7 Genotoxicity** 

stages of development.

Embryos and larvae may be directly exposed to insecticides, through the yolk or via parental transfer in viviparous fish (Viant *et al*., 2006). Spinal deformities, mostly scoliosis and lordosis, and morphological abnormalities were among the more adverse effects registered for insecticides toxicity. Other alterations in the embryo of fish exposed to insecticides also consist of yolk sac edema, pericardial edema and crooked body of larvae (Xu *et al.*, 2010). Teratogenic effects of carbaryl insecticides on the embryo of fish have been proved (Todd and Leeuwen, 2002). Similar results were reported in silversides *Menidia beryllina* exposed to tebufos during embryogenesis (Middaugh *et al*., 1990; Hemmer *et al*., 1990).

The most important factors decreasing fish growth consist of disorder in feeding behaviors, decrease in feeding rate, dysfunction in metabolism process and waste of energy to overcome the stress caused by insecticide exposure (Tripathi *et al*., 2003). For example, disorder in the metabolism of carbohydrates, proteins and lipids in various tissues, particularly liver of fish exposed to insecticides, may reduce their growth rates. Begum (2004) found out that protein and carbohydrate metabolism in the liver and muscle tissue is disrupted on the exposure to a carbofuran insecticide. In addition, exposure during embryonic or larval stage can result in behavioral abnormalities, such as decreased ability to capture prey after hatching, functional deficiencies or slowing of growth and finally death (Kuster, 2005; Viant *et al*., 2006; Arufe *et al*., 2007). These changes were observed in larvae and embryo of zebra fish (*Danio rerio*) in contact with endosulfan (Velasco-Santamaria *et al*., 2011), beta-cyprmethrin (Xu *et al*., 2010); paraoxon-methyl (Küster, 2005) and sevin (carbaryl insecticide) (Todd and Leeuwen, 2002).

#### **6.5 Neurotoxicity**

The primary mechanism of organophosphate and carbamate insecticides toxicity is well known – they function as inhibitors of acetycholinestrase enzyme (AChE) or and butyrylcholinesterase (BChE), as well as disturbing the metabolism of other neurotransmitters such as γ-aminobutyrate (GABA). The synthetic pyrethroids change normal neuronal function by interfering in the function of ion channels in the nerve cell membrane, alterations in intracellular calcium ion concentrations and possibly by blocking GABA receptors. Organochlorine insecticides act primarily by changing the transport of ions across the nerve cell membranes, thus altering the ability of nerve to stimulate.

Fish exposure to these insecticides is frequently assessed by determining the alterations in AChE in brain, muscle, plasma and other tissues or probably GABA activity in brain (Banaee, 2010). AChE is an enzyme responsible for inactivating the neurotransmitter acetylcholine (Fulton & Key, 2001). AChE inactivation results in the accumulation of the neurotransmitter acetylcholine in cholinergic synapses space, leading to synaptic blockage and disruption of signal transmission (Ferrari *et al*., 2004; 2007a, b). Inhibition of AChE induces alteration in the swimming behavior, shaking palsy, spasms and other undesirable effects (Sharbide *et al*., 2011). Disturbances in AChE activity can also impair feeding, identification and avoidance and escaping from predators, spatial orientation of the species, and reproductive behavior (Bretaud *et al*., 2000). Thus, AChE inhibition is considered to be a specific biomarker of exposure to organophosphorus and carbamate insecticides like diazinon, chlorpyrifos, propoxur, isoprocarb, (Üner *et al*., 2006; Cong *et al*., 2008; 2009; Wang *et al*., 2009; Banaee *et al*., 2011;). Similar results have been observed for pyrethroids insecticide toxicity (Koprucu *et al*., 2006). Disorder in γ-aminobutyrate (GABA) system in brain of rainbow trout exposed to sub-lethal lindane was reported by Aldegunde *et al*., (1999). GABA receptors inhibit the transmission of nerve impulses; thus disturbances in this receptor would also lead to an over stimulation of the nerves.

In addition, nervous tissue has weaker antioxidant defense system than other kinds of tissue. On the other hand, the brain as center of the nervous system in fish contains low levels of enzymatic and non-enzymatic antioxidant and higher levels of oxidizable unsaturated lipids and catecholamines. So, nerve tissue is very sensitive to oxidative stress damage induced insecticide toxicity compared with other tissues (Üner *et al*., 2006; Li *et al*., 2010). These results have been reported by other scientists (Senger *et al*., 2005).

#### **6.6 Behavioral alterations**

114 Insecticides – Basic and Other Applications

Embryos and larvae may be directly exposed to insecticides, through the yolk or via parental transfer in viviparous fish (Viant *et al*., 2006). Spinal deformities, mostly scoliosis and lordosis, and morphological abnormalities were among the more adverse effects registered for insecticides toxicity. Other alterations in the embryo of fish exposed to insecticides also consist of yolk sac edema, pericardial edema and crooked body of larvae (Xu *et al.*, 2010). Teratogenic effects of carbaryl insecticides on the embryo of fish have been proved (Todd and Leeuwen, 2002). Similar results were reported in silversides *Menidia beryllina* exposed to tebufos during embryogenesis (Middaugh *et al*., 1990; Hemmer *et al*.,

The most important factors decreasing fish growth consist of disorder in feeding behaviors, decrease in feeding rate, dysfunction in metabolism process and waste of energy to overcome the stress caused by insecticide exposure (Tripathi *et al*., 2003). For example, disorder in the metabolism of carbohydrates, proteins and lipids in various tissues, particularly liver of fish exposed to insecticides, may reduce their growth rates. Begum (2004) found out that protein and carbohydrate metabolism in the liver and muscle tissue is disrupted on the exposure to a carbofuran insecticide. In addition, exposure during embryonic or larval stage can result in behavioral abnormalities, such as decreased ability to capture prey after hatching, functional deficiencies or slowing of growth and finally death (Kuster, 2005; Viant *et al*., 2006; Arufe *et al*., 2007). These changes were observed in larvae and embryo of zebra fish (*Danio rerio*) in contact with endosulfan (Velasco-Santamaria *et al*., 2011), beta-cyprmethrin (Xu *et al*., 2010); paraoxon-methyl (Küster, 2005) and sevin (carbaryl

The primary mechanism of organophosphate and carbamate insecticides toxicity is well known – they function as inhibitors of acetycholinestrase enzyme (AChE) or and butyrylcholinesterase (BChE), as well as disturbing the metabolism of other neurotransmitters such as γ-aminobutyrate (GABA). The synthetic pyrethroids change normal neuronal function by interfering in the function of ion channels in the nerve cell membrane, alterations in intracellular calcium ion concentrations and possibly by blocking GABA receptors. Organochlorine insecticides act primarily by changing the transport of ions across the nerve cell membranes, thus altering the ability of nerve to

Fish exposure to these insecticides is frequently assessed by determining the alterations in AChE in brain, muscle, plasma and other tissues or probably GABA activity in brain (Banaee, 2010). AChE is an enzyme responsible for inactivating the neurotransmitter acetylcholine (Fulton & Key, 2001). AChE inactivation results in the accumulation of the neurotransmitter acetylcholine in cholinergic synapses space, leading to synaptic blockage and disruption of signal transmission (Ferrari *et al*., 2004; 2007a, b). Inhibition of AChE induces alteration in the swimming behavior, shaking palsy, spasms and other undesirable effects (Sharbide *et al*., 2011). Disturbances in AChE activity can also impair feeding, identification and avoidance and escaping from predators, spatial orientation of the species, and reproductive behavior (Bretaud *et al*., 2000). Thus, AChE inhibition is considered to be a specific biomarker of exposure to organophosphorus and carbamate insecticides like diazinon, chlorpyrifos, propoxur, isoprocarb, (Üner *et al*., 2006; Cong *et al*., 2008; 2009; Wang

1990).

insecticide) (Todd and Leeuwen, 2002).

**6.5 Neurotoxicity** 

stimulate.

Behavioral changes are the most sensitive indicators of potential toxic effects. The behavioral and the swimming patterns of the fish exposed to different insecticides include changes in swimming behavior, feeding activities, predation, competition, reproduction and speciesspecies social interactions such as aggression (Cong *et al*., 2008; 2009; Werner and Oram, 2008). Banaee *et al*. (2008; 2011) reported similar behavioral responses in common carp and rainbow trout exposed to sub-lethal levels of diazinon. In fact, most insecticides influence the behavior patterns of fish by interfering with the nervous systems and sensory receptors (Keizer *et al*., 1995; Pan & Dutta, 1998; Cong *et al*., 2008; 2009); and this incident may impair the identification of situation and development of appropriate response by the fish exposed to insecticide. The effect of certain insecticides on the activity of acetylcholinestrase may lead to a decreased mobility in sh (Bretaud *et al*., 2000). Researchers have reported the same alterations in *Oryzias latipes*, *Cyprinus carpio*, *Labeo rohita*, *Oncorhynchus tshawytscha*, *O.latipes*, *Cirrhinus mrigala*, *Oreochromis niloticus*, *Clarias gariepinus* treated with chlorpyrifos (Rice *et al*., 1997; Halappa & David, 2009), malathion (Patil & David, 2008), diazinon (Scholz *et al*., 2000), endosulfan (Gormley & Teather, 2003), Fenvalerate (Mushigeri & David, 2005), fenitrothion (Benli & Özkul, 2010), dimethoate (Auta *et al*., 2002), respectively.

#### **6.7 Genotoxicity**

In genetic-toxicology any heritable damage or DNA inactivation resulting from the animal's exposure to xenobiotics is studied. Genotoxic chemicals such as insecticides have common chemicals and physical properties that enable them interact with genetic materials (Campana *et al*., 1999; Çava & Ergene-Gözükara, 2003; Candioti *et al*., 2010; Dogan et al., 2011). The mutation that may result from an interaction between a chemical and the genetic material is a heritable change in the cell genotype, and thus the error may be transferred to the daughter cell or the next generation. Carcinogenesis and the formation of some tumors in different tissues of fish exposed to insecticides may also be caused by genotoxic properties of these xenobiotics. One of the ill effects of insecticides' arrival into surface waters may be an induction of chromosomal damage in eggs and larvae of fishes in different stages of development.

Some insecticides that behave as endocrine active compounds can change the expression of vital genes resulting in unusual concentrations of plasma steroid hormones and reproductive dysfunction or immuosuppression (Jin *et al*., 2010).

Arukwe, A. 2001. Cellular and molecular responses to endocrine-modulators and the impact

Auta, J. Balogun, J.K. Lawal, F.A. & Ipinjolu, J.K. (2002). Short-term effect of dimethoate on

Bagheri, F. (2007) Study of pesticide residues (Diazinon, Azinphosmethyl) in the rivers of

Ballesteros, M.L. Gonzalez, M.Wunderlin, D.A. Bistoni, M.A. & Miglioranza, K.S.B. (2011).

Banaee, M. (2010). Influence of silymarin in decline of sub-lethal diazinon-induced oxidative

Department, Natural Resource Faculty, University of Tehran, Iran, pp. 149. Banaee, M. Mirvagefei, A. R. Rafei, G. R. & Majazi Amiri, B. (2008). Effect of sub-lethal

Banaee, M. Mirvaghefei, A. R. Majazi Amiri, B. & Rafei, G.R. (2012). Biochemical Blood and

Banaee, M. Mirvaghefei, A. R. Majazi Amiri, B. Rafei, G.R. & Nematdost, B. (2011).

Banaee, M. Mirvaghefi, A.R. Ahmadi, K. & Ashori, R. (2009). The effect of diazinon on

Banaee, M. Mirvaghefi, A.R. Ahmadi, K. & Banaee, S. (2008). determination of LC50 and

Banaee, M. Sureda, A. Mirvaghe, A.R. & Ahmadi, K. (2011). Effects of diazinon on

Begum, G. (2004). Carbofuran insecticide induced biochemical alterations in liver and

Begum, G. (2005). In vivo biochemical changes in liver and gill of *Clarias batrachus* during

behavior of juveniles of *Oreochromis niloticus* (Trewavas) and *Clarias gariepinus*

Golestan province (GorganRoud and Gharehsou), M.Sc. Thesis, Tehran University

Uptake, tissue distribution and metabolism of the insecticide endosulfan in *Jenynsia multidentata* (Anablepidae, Cyprinodontiformes). Environmental Pollution, 159:

stress in rainbow trout (*Oncorhynchus mykiss*). Ph.D. Thesis, Aquaculture

Diazinon Concentrations on Blood Plasma Biochemistry. Int. J. Environ. Res., 2(2):

Histopathological Study of Experimental Diazinon Poisoning in common carp fish (*Cyprinus carpio*). Journal of Fisheries (Iranian Journal of Natural Resources),

Hematological and Histopathological Study of Experimental Diazinon Poisoning in common carp fish (*Cyprinus carpio*). Journal of Fisheries (Iranian Journal of Natural

histophatological changes of testis and ovaries of common carp (*Cyprinus carpio*).

investigation of acute toxicity effects of diazinon on hematology and serology indices of common carp (*Cyprinus carpio*). Journal of Marine Science and

biochemical parameters of blood in rainbow trout (Oncorhynchus mykiss).

muscle tissues of the sh *Clarias batrachus* (linn) and recovery response. Aquatic

cypermethrin exposure and following cessation of exposure. Pesticide Biochemistry

on fish reproduction. Marine Pollution Bulletin, 42(8): 643-655.

(Teugels). Journal of Tropical Biosciences, 2(1): 55-59.

of Medical Science. Tehran, Iran, 1-125.

1709-1714.

189-198.

(Article in press).

Resources), 64(1): 1-14.

Toxicology, 66: 83–92.

and Physiology, 82: 185–196.

Technology Research, 3(2): 1-10.

Scientific Journal of Marine Biology, 1(2): 25-35.

Pesticide Biochemistry and Physiology, 99: 1–6.

#### **6.8 Immunosuppression**

The immune system of fish is important for defense against a variety of pathogens. The system is very sensitive to homeostatic adjustments via endocrine regulation and is influenced by the biochemical status of the nervous system. Thus, any impairment in the nervous system and disturbance in the biochemical homeostasis can weaken the immune system of fish. On the other hand, insecticides may alter the function of the immune system and result in immunodepression, uncontrolled cell proliferation, and alterations of the host defense mechanisms including innate immunity and acquire immunity against pathogens.

Different insecticides at sub-lethal levels have been recognized as stressors causing immunesuppression in fish (Werner and Oram, 2008). In addition, some insecticides may exert immunotoxic effects by altering the transcription of important mediators of the sh's immune system (Eder *et al.*, 2009). Effects of insecticides like P,P'-DDE, lindane, cypermethrin, chlorpyrifos, diazinon on the immune factors of fish such as Interleukin-1β (IL-1β), IL-1β receptor (IL-1R1), Interferon gamma (IFN-γ2b), TNFα, MHCIα, MHCIIα, Mx, TLR9, IγML and C- reactive protein (CRP), TCRα in head- kidney leucocytes, Lysozyme activity, chemiluminuscence (CL) response and immunocompetent cells population size, IgM levels, value of white blood cells (WBC) and respiratory burst activity, head kidney phagocytes and peripheral blood leucocytes, etc., have been reported by scholars (Betoulle *et al*., 2000; Khoshbavar-Rostami *et al*., 2006; Banaee *et al*., 2008; Cuesta *et al*., 2008; Girón-Pérez *et al*., 2009; Shelley *et al*., 2009; Ahmadi *et al*., 2011; Jin *et al*., 2011, Wang *et al*., 2011). The exposure to sub-lethal concentrations of insecticides is what probably makes fish vulnerable to infectious diseases because of their immune-depressive effects (Zelikoff *et al*., 2000). For example, the susceptibility of juvenile chinoock salmon (*O.tshawytscha*) to infectious hematopoietic necrosis virus was significantly increased in fish exposed to sub-lethal concentrations of esfenvalerate (Clifford *et al*., 2005). Similar results were reported in goldfish and common carp that were exposed to carbaryl and lindane respectively (Shea, 1983; Shea & Berry, 1984; Cossarini-dunier & Hattenberger, 1988).

#### **7. References**


The immune system of fish is important for defense against a variety of pathogens. The system is very sensitive to homeostatic adjustments via endocrine regulation and is influenced by the biochemical status of the nervous system. Thus, any impairment in the nervous system and disturbance in the biochemical homeostasis can weaken the immune system of fish. On the other hand, insecticides may alter the function of the immune system and result in immunodepression, uncontrolled cell proliferation, and alterations of the host defense mechanisms including innate immunity and acquire immunity against pathogens. Different insecticides at sub-lethal levels have been recognized as stressors causing immunesuppression in fish (Werner and Oram, 2008). In addition, some insecticides may exert immunotoxic effects by altering the transcription of important mediators of the sh's immune system (Eder *et al.*, 2009). Effects of insecticides like P,P'-DDE, lindane, cypermethrin, chlorpyrifos, diazinon on the immune factors of fish such as Interleukin-1β (IL-1β), IL-1β receptor (IL-1R1), Interferon gamma (IFN-γ2b), TNFα, MHCIα, MHCIIα, Mx, TLR9, IγML and C- reactive protein (CRP), TCRα in head- kidney leucocytes, Lysozyme activity, chemiluminuscence (CL) response and immunocompetent cells population size, IgM levels, value of white blood cells (WBC) and respiratory burst activity, head kidney phagocytes and peripheral blood leucocytes, etc., have been reported by scholars (Betoulle *et al*., 2000; Khoshbavar-Rostami *et al*., 2006; Banaee *et al*., 2008; Cuesta *et al*., 2008; Girón-Pérez *et al*., 2009; Shelley *et al*., 2009; Ahmadi *et al*., 2011; Jin *et al*., 2011, Wang *et al*., 2011). The exposure to sub-lethal concentrations of insecticides is what probably makes fish vulnerable to infectious diseases because of their immune-depressive effects (Zelikoff *et al*., 2000). For example, the susceptibility of juvenile chinoock salmon (*O.tshawytscha*) to infectious hematopoietic necrosis virus was significantly increased in fish exposed to sub-lethal concentrations of esfenvalerate (Clifford *et al*., 2005). Similar results were reported in goldfish and common carp that were exposed to carbaryl and lindane respectively (Shea,

1983; Shea & Berry, 1984; Cossarini-dunier & Hattenberger, 1988).

Biochemistry and Physiology 88 268–272.

Agricultural and Biological Sciences, 6 (1): 62-68.

Biochemistry, 20: 325-330.

Science Technology, 7 (1): 175-182.

Agrahari, S. Pandey, K.C. & Gopal, K. (2007). Biochemical alteration induced by

Aldegunde, M. Soengas, J.L. Ruibal, C. & Andrés, M.D. (1999). Effects of chronic exposure to

Al-Kahtani, M.A. (2011). Effect of an Insecticide Abamectin on Some Biochemical

Arjmandi, R.Tavakol, M. & Shayeghi, M. (2010). Determination of organophosphorus

Arufe, M.I. Arellano, J.M. García, L. Albendín, G. & Sarasquete, C. (2007). Cholinesterase

to the organophosphate azinphosmethyl. Aquatic Toxicology, 84: 328–336.

monocrotophos in the blood plasma of fish, *Channa punctatus* (Bloch). Pesticide

γ-HCH (lindane) on brain serotonergic and gabaergic systems, and serum cortisol and thyroxine levels of rainbow trout, *Oncorhynchus mykiss.* Fish Physiology and

Characteristics of Tilapia Fish (*Oreochromis Niloticus*). American Journal of

insecticide residues in the rice paddies. International Journal Environmental

activity in gilthead seabream (*Sparus aurata*) larvae: Characterization and sensitivity

**6.8 Immunosuppression** 

**7. References** 


Cong, N.V. Phuong, N.T. & Bayley, M. (2008). Brain cholinesterase response in the

Cong, N.V. Phuong, N.T. & Bayley, M. (2009). Effects of repeated exposure of diazinon on

Cossarini-dunier, M. & Hattenberger, A.M. (1988). Effect of pesticides (atrazine and lindane)

Cuesta, A. Meseguer, J. & Esteban, M.Á. (2008). Effects of the organochlorines p, p'-DDE and

Davey, R.B. Meisch, M.V. & Carter, F.L. (1976). Toxicity of five rice field pesticides to the

Dobšíková, R. (2003). Acute Toxicity of Carbofuran to Selected Species of Aquatic and

Dogan, D. Can, C. Kocyigit, A. Dikilitas, M. Taskin, A. & Bilinc, H. (2011). Dimethoate-

Duttaa, H.M. & Meijer, H.J.M. (2003). Sublethal effects of diazinon on the structure of the

Eder, K.J. Leutenegger, C.M. Köhler, H.R. & Werner, I. (2009). Effects of neurotoxic

Fanta, E. Sant'Anna Rios, F. Romao, S. Vianna, A.C.C. & Freiberger, S. (2003).

Favari, L. Lopez, E. Martnez-Tabche, L. & Daz-Pardo, E. (2002). Effect of Insecticides on

Ferrari, A. Venturino, A. & de D'Angelo, A.M.P. (2004). Time course of brain cholinesterase

Insecticide Biochemistry and Physiology, 88: 134-142.

submitted by FMC Corp., Philadelphia, Pa. CDL:229241-Q).

Terrestrial Organisms. Plant Protect. Sci. 39(3): 103–108.

Environmental Safety, 71: 314– 318.

Fish Shellsh Immunol.25:682-8.

(Article in press).

108-116.

119–130.

Pollution 125: 355–360.

211.

and Environmental Safety, 72: 699–703.

snakehead sh (*Channa striata*) after eld exposure to diazinon. Ecotoxicology and

cholinesterase activity and growth in snakehead sh (*Channa striata*). Ecotoxicology

on the replication of spring viremia of carp virus *in vitro*. Ann. Rech. Vet., 19: 209-

lindane on gilthead seabream leucocyte immune parameters and gene expression.

mosquitofish, *Gambusia affinis*, and green sunfish, *Lepomis cyanellus*, under laboratory and field conditions in Arkansas. Environmental Entomology 5(6):1053- 1056. (Also In unpublished submission received Mar 30, 1977 under 279-2712.

induced oxidative stress and DNA damage in *Oncorhynchus mykiss*. Chemosphere

testis of bluegill, Lepomis macrochirus: a microscopic analysis. Environmental

insecticides on heat-shock proteins and cytokine transcription in Chinook salmon (*Oncorhynchus tshawytscha*). Ecotoxicology and Environmental Safety 72: 182– 190. Fadakar Masouleh, F. Mojazi Amiri, B. Mirvaghefi, A.R. & Nematollahi, M.A. (2011). *In* 

*vitro* effects of diazinon on male reproductive tissue and sperm motility of Caspian Kutum (*Rutilus frisii kutum*). Research Journal of Environmental Toxicology, 5(2):

Histopathology of the sh *Corydoras paleatus* contaminated with sublethal levels of organophosphorus in water and food. Ecotoxicology and Environmental Safety, 54:

Plankton and Fish of Ignacio Ramirez Reservoir (Mexico): A Biochemical and Biomagnification Study. Ecotoxicology and Environmental Safety, 51: 177–186. Ferrari, A. Venturino, A .& de D'Angelo, A.M.P. (2007a). Effects of carbaryl and azinphos

methyl on juvenile rainbow trout (*Onchorhynchus mykiss*) detoxifying enzymes.

inhibition and recovery following acute and subacute azinphosmethyl, parathion


Behrens, A. & Segner, H. (2001). Hepatic biotransformation enzymes of sh exposed to non-

Benli, A.Ç.K. & Özkul, A. (2010). Acute toxicity and histopathological effects of sublethal

Betoulle, S. Duchiron, C. & Deschaux, P. (2000). Lindane differently modulates intracellular

Bowman, J. (1988a) Acute Flow-Through Toxicity of Chlorpyrifos Technical to Bluegill

Bowman, J. (1988b) Acute Flow-Through Toxicity of Chlorpyrifos to Rainbow Trout (*Salmo* 

Bretaud, S. Toutant, J.P. & Saglio, P. (2000). Effects of carbofuran, diuron, and nicosulfuron

Calmbacher, C. (1978) Acute Toxicity of San 3261 Lot #7801 to Bluegill, Rafinesque: UCES

Campana, M.A. Panzeri, A.M. Moreno, F V.J. & Dulout, F.N. (1999). Genotoxic evaluation of

Capkin, E. Altinok, I. & Karahan, S. (2006). Water quality and sh size affect toxicity of

Çava, T. & Ergene-Gözükara, S. (2003). Evaluation of the genotoxic potential of lambda-

Cengiz, E.I. & Unlu, E. (2006). Sublethal effects of commercial deltamethrin on the structure

Cengiz, E.I. (2006). Gill and kidney histopathology in the freshwater sh *Cyprinus carpio*

Clifford, M.A. Eder, K.J. Werner, I. & Hedrick, R.P. (2005). Synergistic effects of

study. Environmental Toxicology and Pharmacology, 21: 246–253.

salmon mortality. Environ. Toxicol. Chem., 24 (7): 1766–1772.

the sh Cheirodon interruptus interruptus, Mutat. Res. 438: 155–161. Candioti, J.V. Soloneski, S. & Larramendy, M.L. (2010). Genotoxic and cytotoxic effects of

(Pisces: Poeciliidae). Mutation Research, 703: 180–186.

Recovery 8: 281–297.

Physiology, 97: 32–35.

Bio-Chemistry Laboratories, Inc. 188 p.

Chemistry Laboratories, Inc. 174 p.

San Diego, CA. CDL:097841-AD).

203–215.

Saf., 47: 117–124.

1800.

Research, 534: 93–99.

Pharmacology, 22: 200–204.

point source pollution in small streams. Journal of Aquatic Ecosystem Stress and

fenitrothion on Nile tilapia, *Oreochromis niloticus*. Pesticide Biochemistry and

calcium levels in two populations of rainbow trout (*Oncorhynchus mykiss*) immune cells: head kidney phagocytes and peripheral blood leucocytes. Toxicology, 145:

(*Lepomis macrochirus*): Project ID:37189. Unpublished study prepared by Analytical

*gairdneri*): Project ID: 37188. Unpublished study prepared by Analytical Bio-

on acetylcholinesterase activity in goldsh (*Carassius auratus*). Ecotoxicol. Environ.

Project # 11506-16-03. (Unpublished study received Mar 1, 1979 under 11273-EX-15. prepared by Union Carbide Corp., submitted by Sandoz, Inc., Crop Protection,

the pyrethroid lambda-cyhalothrin using themicronucleus test in erythrocytes of

the formulated insecticide Acida on *Cnesterodon decemmaculatus* (Jenyns, 1842)

endosulfan, an organochlorine pesticide, to rainbow trout. Chemosphere, 64:1793–

cyhalothrin using nuclear and nucleolar biomarkers on sh cells. Mutation

of the gill, liver and gut tissues of mosquitosh, Gambusia afnis: A microscopic

after acute exposure to deltamethrin. Environmental Toxicology and

esfenvalerate and infectious hematopoietic necrosis virus on juvenile Chinook


Jin, Y.X. Zheng, S.S. Pu, Y. Shu, L.J. Sun, L.W. & Liu, W.P. (2011). Cypermethrin has the

John, P.J. (2007). Alteration of certain blood parameters of freshwater teleost *Mystus vittatus*

Johnson, W. & Finley, M. (1980). Handbook of Acute Toxicity of Chemicals to Fish and

Jones, F. & Davis, J. (1994). DDVP Technical Grade: Acute Toxicity to Sheepshead Minnow

Kapur, K & Toor, H.S. (1978). The effect of fenitrothion on reproduction of a teleost fish,

Kavitha, P. & Rao, J. V. (2007). Oxidative stress and locomotor behaviour response as

Kavitha, P. & Rao, V.J. (2009). Sub-lethal effects of profenofos on tissue-specic antioxidative

Keizer, J. D'Agostino, G. Nagel, R. Volpe, T. Gnemid, P. & Vittozzi, L. (1995).

Khoshbavar-Rostami, H.A. Soltani, M. & Hassan, H.M.D. (2006). Immune response of great

Kitamura, S. Kadota, T. Yoshida, M. Jinno, N. & Ohta, S. (2000). Whole-body metabolism

Koprucu, S.S. Koprucu, K. & Urail, M.S. (2006). Acute toxicology of synthetic pyrethroid

Kumar, K. & Ansari, B.A. (1984). Malathion toxicity: skelet deformities in zebrafish

Küster, E. (2005). Cholin- and carboxylesterase activities in developing zebrash embryos

Lawson, E.O. Ndimele, P.E. Jimoh, A.A. & Whenu, O.O. (2011). Acute Toxicity of Lindane

1822). International Journal of Animal and Veterinary Advances, 3(2): 63-68.

zebrash (*Danio rerio*). Chemosphere 82:398-404.

Biochemistry and Physiology, 87: 182–188.

The Science of the Total Environment 171: 213-220.

Comp. Biochem. Physiol. C 126: 259-266.

Toxicology 75: 76–85.

the organophosphate, diazinon. Aquaculture, 256: 88–94.

Environmental Contamination and Toxicology, 76: 59-65.

(*Brachydanio rerio*, Cyprinidae). Pestic. Sci. 15: 107-111.

Environmental Safety 72: 1727–1733.

15-20.

Washington, D.C. 98 p.

Sciences, 59 p.

20: 438-442.

potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult

after chronic exposure to metasystox and sevin. Fish Physiology Biochemistry, 33:

Aquatic Invertebrates: resource Publication 137. USDI, Fish and wildlife service,

(*Cyprinodon variegatus*) Under Flow-through Test Conditions: Lab Project Numbers: J9403007F: J9403007B. Unpublished study prepared by Toxikon Environmental

*Cyprinus carpio communis* Lim: a biochemical study. Bull. Environ. Contam. Toxicol.

biomarkers for assessing recovery status of mosquito fish, *Gambusia affinis* after lethal effect of an organophosphate pesticide, monocrotophos. Pesticide

responses in a Euryhyaline sh, *Oreochromis mossambicus*. Ecotoxicology and

Enzymological differences of AChE and diazinon hepatic metabolism: correlation of *in vitro* data with the selective toxicity of diazinon to fish species.

sturgeon (*Huso huso*) subjected to long-term exposure to sublethal concentration of

of the organophosphorus pesticide, fenthion, in goldfish, Carassius auratus.

deltamethrin to fingerling European catfish (*Silirus glanis* L.). Bulletin of

(*Danio rerio*) and their potential use for insecticide hazard assessment. Aquatic

(Gamma Hexachloro-Cyclohexane) to African Catfish (*Clarias gariepinus*, Burchell,

and carbaryl exposure in the goldsh (*Carassius auratus*). Ecotoxicology and Environmental Safety, 57: 420–425.


Ferrari, A. Venturino, A. & de D'Angelo, A.M.P. (2007b). Muscular and brain cholinesterase

Fulton, M.H. & Key, P.B. (2001). Acetylcholinesterase inhibition in estuarine fish and

Ganai, F.A. Malik, M. & Nisar, Z. (2011). Genotoxic effects of organophosphate pesticide phorate in some exotic fishes of Kashmir. Journal of American Science, 7(4): 46-50. Girón-Pérez, M.I. Velázquez-Fernández, J. Díaz-Resendiz, K. Díaz-Salas, F.Canto-Montero,

Haider, S. & Upadhyaya, N. (1985). Effect of commercial formulation of four

Halappa, R. & David, M. (2009). Behavioural responses of the fresh water fish, *Cyprinus* 

Hemmer, M.J. Middaugh, D.P. & Moore, J.C. (1990). Effects of temperature and salinity on

Holcombe, G. Phipps, G. & Tanner, D. (1982) The acute toxicity of kelthane, dursban,

Honarpajouh, K. (2003). Study and Identification of OP pesticides residues (Azinphosmethyl

Jaensson, A. Scott, A.P. Moore, A. Kylin, H. & Olsén, K.H. (2007). Effects of a pyrethroid

male parr of brown trout (*Salmo trutta* L.). Aquatic Toxicology, 81: 1–9. Jin, Y.X Chen, R.J. Liu, W.P. & Fu, Z.W. (2010). Effect of endocrine disrupting chemicals on

rainbow trout Salmo gairdneri. Environ. Pollution 29:167-178.

University of Medical Science. Tehran, Iran, pp. 95.

Environmental Safety, 57: 420–425.

Environmental Toxicology & Chemistry 20 37-45.

(*Oreochromis niloticus* L.) larvae. Chemosphere, 59: 163–166.

Fisheries and Aquatic Sciences, 9: 233-238.

313.

136.

1049-1054.

and carbaryl exposure in the goldsh (*Carassius auratus*). Ecotoxicology and

sensitivities to azinphos methyl and carbaryl in the juvenile rainbow trout *Oncorhynchus mykiss*. Comparative Biochemistry and Physiology, Part C 146: 308–

invertebrates as an indicator of organophosphorus insecticide exposure and effects.

C. Medina-Díaz, I. Robledo-Marenco, M. Rojas-García, A. & Zaitseva, G. (2009). Immunologic parameters evaluations in Nile tilapia (*Oreochromis niloticus*) exposed to sublethal concentrations of diazinon. Fish & Shellsh Immunology 27 383–385. Gormley, K.L. & Teather, K.L. (2003). Developmental, behavioral, and reproductive effects

experienced by Japanese medaka (*Oryzias latipes*) in response to short-term exposure to endosulfan. Ecotoxicology and Environmental Safety, 54: 330–338. Gül, A. (2005). Investigation of acute toxicity of chlorpyrifos-methyl on Nile tilapia

organophosphate insecticides on the ovaries of a freshwater teleost, *Mystus vitattus* (Bloch): a histological and histochemical study. J. Environ. Sci. Health. B20: 321-340.

*carpio* (Limmaeus) following sublethal exposure to chlorpyrifos. Turkish Journal of

*Menidia berllina* embryos exposed to terbufos. Diseases Aquatic Organisms, 8: 127-

disulfoton, pydrin, and permethrin to fathead minnows Pimephales promelas and

and Diazinon) in the Mahabad and Siminerood Rivers, M.Sc. Thesis, Tehran

pesticide on endocrine responses to female odours and reproductive behaviour in

the transcription of genes related to the innate immune system in the early developmental stage of zebrash (*Danio Rerio*). Fish Shellsh Immunol. 28:854-61. Jin, Y.X. Zheng, S. & Fu, Z. (2011b). Embryonic exposure to cypermethrin induces apoptosis

and immunotoxicity in zebrash (Danio rerio). Fish & Shellsh Immunology, 30:


Monteiro, D.A. Rantin, F.T. & Kalinin, A.L. (2009). The effects of selenium on oxidative

Moore, A. & waring, C.P. (1996). Sublethal effect of the pesticide diazinon on olfactory

Moore, A. & Waring, C.P. (2001). The effects of a synthetic pyrethroid pesticide on some

Mushigeri, S.B. & David, M. (2005). Fenvalerate induced changes in the Ach and associated

Nabb, D.L. Mingoia, R.T. Yang, Ch. & Han, X. (2006). Comparison of basal level metabolic

Nebbia, C. (2001). Biotransformation Enzymes as Determinants of Xenobiotic Toxicity in

Pan, G. & Dutta, H.M. (1998). The inhibition of brain acetylcholinesterase activity of juvenile

Pant, J. Tewari, H. & Gill, T.S. (1987). Effects of aldicarb on the blood and tissues of a

Patil, V.K. & David, M. (2008). Behaviour and respiratory dysfunction as an index of

Patnaik, L. (2010). Biochemical Alterations Induced by Sevin in *Clarias batrachus*. ASIAN J.

Peddie, S. Zou, J. Cunningham, C. & Secombes, C.J. (2001). Rainbow trout (*Oncorhynchus* 

Rand, G.M. (2004). Fate and effects of the insecticide–miticide chlorfenapyr in outdoor aquatic microcosms. Ecotoxicology and Environmental Safety, 58: 50–60. Rao, J.V. (2006a). Sublethal effects of an organophosphorus insecticide (RPR-II) on

Rao, J.V. (2006b). Biochemical alterations in euryhaline sh, *Oreochromis mossambicus*

Domestic Animals. The Veterinary Journal 2001, 161, 238–252

freshwater fish. Bull. Environ. Contam. Toxicol. 38: 36-41.

kidney leucocytes in vitro. Fish Shellsh Immun. 11, 697–709.

Biochemistry and Physiology, Part C 143: 492–498.

monocrotophos. Chemosphere, 65: 1814–1820.

function in mature atlantic salmon parr. J. Fish. Biol. 48: 758-775.

Biochemistry and Physiology, Part C 143: 141–149.

*mykiss*) and rat. Aquat. Toxicol., 80: 52-59.

Environmental Research Section A, 79: 133-137.

Fisheries and Aquatic Sciences, 8: 233-237.

EXP. BIOL. SCI., 1(1):124 – 127.

40–49.

1–12.

65–72.

organophosphorus insecticide Folisuper 600 (methyl parathion). Comparative

stress biomarkers in the freshwater characid sh matrinxã, *Brycon cephalus* (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR® (methyl parathion). Comparative Biochemistry and Physiology, Part C 149:

aspects of reproduction in Atlantic salmon (*Salmo salar* L.). Aquatic Toxicology, 52:

AchE activity in different tissues of sh *Cirrhinus mrigala* (Hamilton) under lethal and sub-lethal exposure period. Environmental Toxicology and Pharmacology, 20:

enzyme activities of freshly isolated hepatocytes from rainbow trout (*Oncorhynchus* 

largemouth bass, *Micropterus salmoides* by sub-lethal concentrations of diazinon.

malathion toxicity in the freshwater fish, *Labeo rohita* (Hamilton). Turkish Journal of

*mykiss*) recombinant IL-1beta and derived peptides induce migration of head-

biochemical parameters of tilapia, *Oreochromis mossambicus*. Comparative

exposed to sub-lethal concentrations of an organophosphorus insecticide,


Li, Z.H. Zlabek, V. Velisek, J. Grabic, R. Machova, J. & Randak, T. (2010). Modulation of

Lopes, R.B. Paraiba, L.C. Ceccarelli, P.S. & Tornisielo, V.L. (2006). Bioconcentration of

Ma, Y. Chen, L. Lu, X. Chu, H. Xu, C. & Liu, W. (2009). Enantioselectivity in aquatic toxicity

Magesh, S. & Kumaraguru, A.K. (2006). Acute toxicity of endosulfan to the milk sh, *Chanos* 

Matos, P. Fontanhas-Fernandes, A. Peixoto, F. Carrola, J. & Rocha, E. (2007). Biochemical

Maxwell, L.B. & Dutta, H.M. (2005). Diazinon-induced endocrine disruption in bluegill

Mayer, F. L. & Ellersieck, M. R. (1986). Manual of acute toxicity: interpretation and data

(INZ-326118) to Rainbow Trout (*Salmo gairdneri*): Rept. No. HLR 525-86. McAllister, W. Swigert, J. & Bowman, J. (1986a). Acute Toxicity of Betasan Technical to

carbaryl. Pesticide Biochemistry and Physiology, 89: 73–80.

C, 151: 137-141.

Safety, 72: 1913–1918.

56–62.

21–27.

51 p.

antioxidant defence system in brain of rainbow trout (*Oncorhynchus mykiss*) after chronic carbamazepine treatment. Comparative Biochemistry and Physiology, Part

trichlorfon insecticide in pacu (*Piaractus mesopotamicus*). Chemosphere, 64:

of synthetic pyrethroid insecticide fenvalerate. Ecotoxicology and Environmental

*chanos*, of the southeast coast of India. Bull. Environ. Contam. Toxicol., 76: 622–628.

and histological hepatic changes of Nile tilapia *Oreochromis niloticus* exposed to

sunsh, *Lepomis macrochirus*. Ecotoxicology and Environmental Safety, 60:

base for 410 chemicals and 66 species of freshwater animals. U.S. Department of the Interior, Fish and Wildlife Service, Resource Publication 160. 579 p. Author: Mayer, F. Ellersieck, M. Wetzel, J. (1986) Static Acute 96-hour LC50 of Linuron

Bluegill (*Lepomis macrochirus*): Static Acute Toxicity Report #34027: T-12394. Unpublished study prepared by Analytical Bio-Chemistry Laboratories, Inc. 47 p. McAllister, W. Swigert, J. & Bowman, J. (1986b). Acute Toxicity of Betasan Technical to

Rainbow Trout (Salmo gairdneri): Static Acute Toxicity Report #34028: T-12395. Unpublished study prepared by Analytical Bio-Chemistry Laboratories, Inc.

in Marine and Freshwater Teleosts, edited by D. Schlenk and W.H. Benson,

inland silversides *Menidia beryllina* exposed to tebufos during embryogenesis.

plasma calcium and ultimobranchial gland of a teleost, *Heteropneustes fossilis*.

inhibits acetylcholinesterase in muscle and brain of the sh *Prochilodus lineatus*.

biomarkers in the freshwater characid fish, *Brycon cephalus*, exposed to

McKim, J.M. & Lein, G.L. (2001). Toxic responses of the skin. In: Target Organ Toxicity

Middaugh, D.P. Fournie, J.W. & Hemmer, M.J. (1990). Vertebral abnormalities in Juvenile

Mishra, D. Srivastav, S.K. & Srivastav, A.K. (2005). Effects of the insecticide cypermethrin on

Modesto, K.A. & Martinez, C.B.R. (2010). Roundup causes oxidative stress in liver and

Monteiro, D.A. Alves de Almeida, J. Rantin, F.T. & Kalinin, A.L. (2006). Oxidative stress

London, UK, Taylor & Francis, pp. 151-223.

Ecotoxicology and Environmental Safety, 60: 193–197.

Diseases Aquatic Organisms, 9: 109-116.

Chemosphere 78: 294–299.

organophosphorus insecticide Folisuper 600 (methyl parathion). Comparative Biochemistry and Physiology, Part C 143: 141–149.


Singh, S.K. Tripathi, P.K. Yadav, R.P. Singh, D. & Singh, A. (2004). Toxicity of malathion and

Skandhan, K.P. Sahab Khan, P. & Sumangala, B. (2001). DDT and male reproductive system.

Stegeman, J.J. & Hahn, M.E. (1994). Biochemistry and molecular biology of

Straus, D.L. Schlenk, D. & Chambers, J.E. (2000). Hepatic microsomal desulfuration and

Talebi, K. (1998). Diazinon Residues in the Basins of Anzali Lagoon, Iran. Bulletin

Tarahi Tabrizi, S. (2001). Study of pesticide residues (diazinon, malathion, metasytoux) in

Todd, N.E. & Leeuwen, M.V. (2002). Effects of Sevin (Carbaryl Insecticide) on Early Life

Tripathi, P.K. Srivastava, V.K. & Singh, A. (2003). Toxic effects of dimethoate

Üner, N. Oruç, E.Ö. Sevgiler, Y. Şahin, N. Durmaz, H. & Usta, D. (2006). Effects of diazinon

Velisek, J. Svobodova, Z. & Piackova, V. (2009). Effects of acute exposure to bifenthrin on

Velmurugan, B. Selvanayagam, M. Cengiz, E.I.& Unlu, E. (2009). Histopathological Changes

Viant, M.R. Pincetich, C.A. & Tjeerdema, R.S. (2006). Metabolic effects of dinoseb, diazinon

*niloticus*. Environmental Toxicology and Pharmacology, 21: 241-245. Velasco-Santamaría, Y.M. Handy, R.D. & Sloman, K.A. (2011). Endosulfan affects health

trout (*Oncorhynchus mykiss*). Veterinarni Medicina, 54, (3): 131–137.

*Colisa fasciatus*. Bull. Environ. Conram. Toxicol. 72: 592-599.

Research Journal of Environmental Toxicology, 5(2): 76-80.

effect from Aroclor 1254. Aquat. Toxicol. 50: 141-149.

Environmental Contamination Toxicology, 61: 477-483.

*Channa punctatus*. Asian Fisheries Science, 16: 349-359.

Florida, pp. 87-206.

Tehran, Iran, 1-88.

272.

380.

371.

Technology, 52(5): 1291-1296.

carbaryl pesticides: effects on some biochemical profiles of the freshwater fish

monooxygenases: current perspectives on forms, functions and regulation of cytochrome P450 in aquatic species, in: Malins, D.C. Ostrander, G.K. (Eds.), Aquatic Toxicology: Molecular, Biochemical Cellular Perspectives, CRC Press, Boca Raton,

dearylation of chlorpyrifos and parathion in fingerling channel catfish: lack of

the Tabriz Nahand River, M.Sc. Thesis, Tehran University of Medical Science,

Stages of Zebrafish (*Danio rerio*). Ecotoxicology and Environmental Safety, 53: 267-

(organophosphate) on metabolism and enzyme system of freshwater teleost fish

on acetylcholinesterase activity and lipid peroxidation in the brain of *Oreochromis* 

variables in adult zebrash (*Danio rerio*) and induces alterations in larvae development. Comparative Biochemistry and Physiology, Part C 153: 372–

some haematological, biochemical and histopathological parameters of rainbow

in the Gill and Liver Tissues of Freshwater Fish, *Cirrhinus mrigala* Exposed to Dichlorvos. An International Journal Brazilian Archives of Biology and

and esfenvalerate in eyed eggs and alevins of Chinook salmon (*Oncorhynchus tshawytscha*) determined by H NMR metabolomics. Aquatic Toxicology, 77: 359–


Rao, J.V. (2006c). Toxic effects of novel organophosphorus insecticide (RPR-V) on certain

Rice, J.P. Drews, C.D. Klubertans, T.M. Bradbury, S.P. & Coats, J.R. (1997). Acute toxicity

Saha, S. & Kaviraj, A. (2009). Effects of cypermethrin on some biochemical parameters and

Sayeed, I. Parvez, S. Pandey, S. Bin-Hafeez, B. Haque, R. & Raisuddin, S. (2003). Oxidative

Scholz, N.L. Truelove, N.K. French, B.L. Berejikian, B.A. Quinn, T.P. Casillas, E. Collier, T.K.

Senger, M.R. Rico, E.P. de Bem Arizi, M. Rosemberg, D.B. Dias, R.D. Bogo, M.R. & Bonan,

Sharbide, A.A. Metkari, V. & Patode, P. (2011). Effect of diazinon on acetylcholinestrase

Shayeghi, M. Darabi, H. Abtahi, H. Sadeghi, M. Pakbaz, F. & Golestaneh., S.R. (2007).

Shea, T.b. & Berry, E.S. (1984). Suppression of interferon synthesis by the pesticide carbaryl

Shea, T.B. (1983). Enhancement of goldfish virus-2 *in vitro* replication by the pesticides

Shelley, L.K. Balfry, S.K. Ross, P.S. & Kennedya, C.J. (2009). Immunotoxicological effects of a

Siang, H.Y. Yee, L.M. & Seng, C.T. (2007). Acute toxicity of organochlorine insecticide

Singh, H. (1989). Interaction of xenobiotics with reproductive endocrine functions in a protogynous teleost, *Monopterus albus.* Mar. Environ. Res., 28: 285-289. Singh, S. & Singh, T.P. (1987). Evaluation of toxicity limit and sex hormone production in

carbaryl and toxaphane. Appl. Environ. Microbiol., 45: 1859-1864.

(*Oncorhynchus mykiss*). Aquatic Toxicology, 92: 95–103.

catsh *Heteropneustes fossilis*. Chemosphere, 74: 1254–1259.

Bloch. Ecotoxicology and Environmental Safety 56: 295–301.

(*Danio rerio*) brain membranes. Toxicology, 212: 107–115.

Environmental Toxicology, 5(2): 152-161.

South Medical Journals 10(1) 54-60.

Microbiol., 47: 250-252.

Physiology, 89: 46–53.

Res., 42: 428-488.

(*Oncorhynchus tshawytscha*). Can. J. Fish. Aquat. Sci. 57: 1911–1918.

Biochemistry and Physiology, 86: 78–84.

Toxicology and Chemistry, 16: 696-704.

biochemical parameters of euryhaline fish, *Oreochromis mossambicus*. Pesticide

and behavioural effects of chlorypyrifos, permethrin, phenol, strychnine and 2,4 dinitrophenol to 30 day old Japanse medaka (*Oryzias latipes*). Environmental

its amelioration through dietary supplementation of ascorbic acid in freshwater

stress biomarkers of exposure to deltamethrin in freshwater sh, *Channa punctatus*

(2000). Diazinon disrupts antipredator and homing behaviors in chinook salmon

C.D. (2005). Carbofuran and malathion inhibit nucleotide hydrolysis in zebrash

activity and lipid peroxidation of *Poecilia reticulate*. Research Journal of

Assessment of persistence and residue of diazinon and malathion in three Rivers (Mond, Shahpour and Dalaky) of Bushehr province in 2004-2005 years. Iranian

as a mechanism for enhancement of goldfish virus-2 replication. Appl. Environ.

sub-chronic exposure to selected current-use pesticides in rainbow trout

endosulfan and its effect on behaviour and some hematological parameters of Asian swamp eel (*Monopterus albus*, Zuiew). Pesticide Biochemistry and

response to Cythion and BHC in the vitellogenic catfish *clarias batrachus*. Environ.


**7** 

**Production of Insecticidal Baculoviruses in** 

Juan D. Claus1, Verónica V. Gioria1,

*Universidad Nacional del Litoral,* 

*1República Argentina;* 

*2New Zealand* 

Gabriela A. Micheloud1 and Gabriel Visnovsky2

*2Chemical and Process Engineering, University of Canterbury* 

*1Laboratory of Virology; Facultad de Bioquímica y Ciencias Biológicas;* 

**Insect Cell Cultures: Potential and Limitations** 

The potential of baculoviruses to be employed as insecticides is known since more than 75 years ago (Benz, 1986). To date, over 30 different baculoviruses are used to control several insect plagues in agriculture, horticulture and forestry (Moscardi, 1999). The use of baculovirus as insecticides is based on a set of useful properties, such as pathogenicity, specificity, narrow host range, environmental persistence, ability to act synergistically with other natural enemies of the pest and ability to induce artificial epizootics. Despite these advantages, very few baculoviruses have become widely used as insecticides, standing out as some successful examples the use of the *Anticarsia gemmatalis multiple nucleopolyhedrovirus* (AgMNPV) to control the velvetbean caterpillar in soybean crops in Latin America, *Cidia pommonella granulovirus* (CpGV) to fight the codling moth attacks in fruit orchards, and *Spodoptera exigua multiple nucleopolyhedrovirus* (SeMNPV) to control the armyworm in vegetable crops under cover in Europe (Moscardi, 1999). The causes of the limited acceptance of baculoviruses as insecticides are diverse, including slow speed of action, problems to register and market these biological insecticides and difficulties to produce

The technologies currently used to produce insecticidal baculoviruses are based on the infection of susceptible insect larvae (Black et al., 1997). However, the implementation of processes to produce baculovirus in insect larvae is hampered by several limitations: high labour requirements, lack of expertise in standardization and validation of such processes, difficulties in scaling production to levels consistent with the profitability of the process and difficulties to properly control both the process production and product quality. While several improvements in production systems in insect larvae have been described in the last years which could help overcome some of the problems described above (van Beek & Davis, 2007), it has been also proposed that the adoption of an alternative technology based on the viral propagation in insect cell cultures could enable the development of well standardized, controlled and scalable production processes for insecticidal baculoviruses (Szewczyk et al.,

**1. Introduction** 

them at an appropriate scale.

2006).


### **Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations**

Juan D. Claus1, Verónica V. Gioria1, Gabriela A. Micheloud1 and Gabriel Visnovsky2 *1Laboratory of Virology; Facultad de Bioquímica y Ciencias Biológicas; Universidad Nacional del Litoral, 2Chemical and Process Engineering, University of Canterbury 1República Argentina; 2New Zealand* 

#### **1. Introduction**

126 Insecticides – Basic and Other Applications

Viran, R. Erkoc, F.U. Polat, H. & Kocak, O. (2003). Investigation of acute toxicity of

Vryzas, Z. Vassiliou, G. Alexoudis, C. & Papadopoulou-Mourkidou, E. (2009). Spatial and

Wang, C. Lu, G. Gui, J. & Wang, P. (2009). Sublethal effects of pesticide mixtures on selected

Wang, X. Xing, H. Li, X. Xu, S. & Wang, X. (2011). Effects of atrazine and chlorpyrifos on the

Werimo, K. Bergwerff, A.A. & Seinen, W. (2009). Residue levels of organochlorines and

Wetzel, J. (1986) Static Acute 96-hour LC50 of Linuron (IN Z-326118) to Bluegill (Lepomis

Woodward, D. & Mauck, W. (1980) Toxicity of five forest insecticides to cutthroat trout and

Xu, C. Tu, W. Lou, C. Hong, Y. & Zhao, M. (2010). Enantioselective separation and zebrash

Xu, W.N. Liu, W.B. & Liu, Z.P. (2009). Trichlorfon-induced apoptosis in hepatocyte primary

Yaji, A.J. Auta, J. Oniye, S.J. Adakole, A.J. & Usman, J.I. (2011). Effects of cypermethrin on

Yi, M.Q. Liu, H.X. Shi, X.Y. Liang, P. & Gao, X.W. (2006). Inhibitory effects of four carbamate

Zaheer Khan, M. & Law, F.C.P. (2005). Adverse effects of pesticides and related chemicals

Zelikoff, J.T. Raymond, A. Carlson, E. Li, Y. Beaman, J.R. & Anderson, M. (2000). Biomarkers

cultures of *Carassius auratus gibelio*. Chemosphere, 77:895-901.

Comparative Biochemistry and Physiology, Part C, 143: 113–116.

portion. Aquatic Ecosystem Health & Management 12 337-341.

Safety, 55: 82–85.

414-419.

248989-O).

331.

Greece. Water Research, 43: 1-10.

Shellsh Immunology, 31: 126-133.

Nemours and Co., Inc. 14 p.

Sciences, 22(5): 738–743.

EJEAFChe, 10(2): 1927-1934.

Pakistan Acad. Sci. 42(4): 315-323.

deltamethrin on guppies (*Poecilia reticulata*). Ecotoxicology and Environmental

temporal distribution of pesticide residues in surface waters in northeastern

biomarkers of *Carassius auratus*. Environmental Toxicology and Pharmacology, 28:

mRNA levels of IL-1β and IFN- γ2b in immune organs of common carp. Fish &

organophosphates in water, fish and sediments from Lake Victoria-Kenyan

macrochirus): Rept. No. 52786. Unpublished study prepared by E.I. du Pont de

two species of aquatic invertebrates. Bull. Environm. Contam. Toxicol. 25:846-854. (Submitter 69597. also in unpublished submission received Dec 9, 1982 under 3125327. submitted by Mobay Chemical Corp., Kansas City, MO. CDL:

embryo toxicity of insecticide beta-cypermethrin. Journal of Environmental

behavior and biochemical indices of fresh water fish *Oreochromis niloticus*.

insecticides on acetylcholinesterase of male and female *Carassius auratus in vitro*.

on enzyme and hormone systems of fish, amphibians and reptiles: a review. Proc.

of immunotoxicity in sh: from the lab to the ocean. Toxicol. Lett. (Amst.), 325–

The potential of baculoviruses to be employed as insecticides is known since more than 75 years ago (Benz, 1986). To date, over 30 different baculoviruses are used to control several insect plagues in agriculture, horticulture and forestry (Moscardi, 1999). The use of baculovirus as insecticides is based on a set of useful properties, such as pathogenicity, specificity, narrow host range, environmental persistence, ability to act synergistically with other natural enemies of the pest and ability to induce artificial epizootics. Despite these advantages, very few baculoviruses have become widely used as insecticides, standing out as some successful examples the use of the *Anticarsia gemmatalis multiple nucleopolyhedrovirus* (AgMNPV) to control the velvetbean caterpillar in soybean crops in Latin America, *Cidia pommonella granulovirus* (CpGV) to fight the codling moth attacks in fruit orchards, and *Spodoptera exigua multiple nucleopolyhedrovirus* (SeMNPV) to control the armyworm in vegetable crops under cover in Europe (Moscardi, 1999). The causes of the limited acceptance of baculoviruses as insecticides are diverse, including slow speed of action, problems to register and market these biological insecticides and difficulties to produce them at an appropriate scale.

The technologies currently used to produce insecticidal baculoviruses are based on the infection of susceptible insect larvae (Black et al., 1997). However, the implementation of processes to produce baculovirus in insect larvae is hampered by several limitations: high labour requirements, lack of expertise in standardization and validation of such processes, difficulties in scaling production to levels consistent with the profitability of the process and difficulties to properly control both the process production and product quality. While several improvements in production systems in insect larvae have been described in the last years which could help overcome some of the problems described above (van Beek & Davis, 2007), it has been also proposed that the adoption of an alternative technology based on the viral propagation in insect cell cultures could enable the development of well standardized, controlled and scalable production processes for insecticidal baculoviruses (Szewczyk et al., 2006).

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 129

can also infect lepidopteran insects, but they produce granulosis. On the other hand, the viruses classified into the genus *γ-baculovirus* can infect hymenopteran insects to produce nuclear polyhedrosis, while the viruses of the genus *δ-baculovirus* are associated to the production of nuclear polyhedrosis in dipteran insects. As most baculoviruses used as insecticides so far belong to the genus *α-baculovirus*, the subject henceforth will be focused on

The main route of infection of lepidopteran larvae with nucleopolyhedrovirus is the ingestion of food contaminated with viral OBs (Granados & Williams, 1986). Once ingested, OBs are transported to the larvae´s midgut, where they are dissolved to release the occluded virions, due to the combined action of the alkaline environment and the presence of alkaline proteases. The released OVs pass through the midgut peritrophic matrix and find the brush border membrane of the columnar midgut epithelial cells, which fuse with the viral envelope to enter the viral nucleocapsids within the cytoplasm. The ability of OVs to infect midgut epithelial cells is dependent on the expression of a set of genes whose products are denominated "*per os* infectivity factors" (PIFs) (Rohrmann, 2011). Most nucleocapsids are then transported to the nucleus through a process that is dependent on actin polymerization. Once the nucleocapsid has been entered into the cell nucleus, the viral DNA is naked and starts the transcriptional cascade that lead ultimately to the assembly of progeny nucleocapsids. A distinctive feature of the primary replication of nucleopolyhedroviruses in the midgut is that the nucleocapsids that were assembled in the nucleus are almost totally exported to the basal cytoplasmatic membrane, from where they finally egress as BVs. The budding of BVs occurs at regions of the plasmatic membrane that have been modified by insertion of the glycoproteins characteristic of the BV progeny, GP64 and/or F. Then, BVs would cross the basal lamina to begin the dissemination process that leads to secondary systemic infections. Alternatively, it has been proposed that baculoviruses can reach the main insect cavity through previous infection of tracheal cells (Pasarelli, 2011). Secondary infections, that affect almost all insect tissues, start when BVs penetrate into cells through a receptor-mediated endocytosis process. After the fusion of the viral envelope with the membrane of acidified endosomes, viral nucleocapsids are released into the cytoplasm and then transported to the nucleus, where the viral genome is naked. Differently from what occur in midgut epithelial cells, the transcriptional cascade in secondary infections drives the replication process to the production of nucleocapsids that, besides of feeding the budding of BVs, are assembled into the nucleus to form OVs. The assembly of OVs implies the retention of nucleocapsids inside the nucleus and the acquisition of an envelope synthesized *de novo* at the expense of the inner nuclear membrane. The OVs are finally occluded inside a crystalline matrix consisting mainly of the viral protein polyhedrin , whose gene is expressed at very high levels during the very late stage of the transcriptional cascade. The products of the occlusion process are the OBs, which are retained inside the nucleus until the death and lysis of the infected cell. At the end of the pathogenic process, the infected insect is full of OBs that, after its death and

liquefaction of its tissues, are released into the environment to restart the cycle again.

The symptoms of the disease associated to baculovirus infection are not usually apparent during the first days post-infection (Granados & Williams, 1986). The change of color and the altered behavior of infected insects are often the earliest signs of infection with αbaculovirus. The lack of appetite, which after several days culminates in the total

lepidopteran nucleopolyhedroviruses.

**2.2 Natural cycle and pathogenicity** 

The purpose of this chapter is to review the current state of the art about insect cell culture technology and its application to the production of viral insecticides belonging to the family *Baculoviridae*. The several restrictions still existing to develop feasible processes as well as the prospects for overcoming these limitations will be also reviewed.

### **2. The baculoviruses**

#### **2.1 Structure and classification**

The baculoviruses (family *Baculoviridae*) are a group of arthropod-specific viral pathogens that have a circular and supercoiled double-stranded DNA genome (Rohrmann, 2011). The size of the genome ranges from 80 to 180 kbp. The genome is contained in a rod-shaped nucleocapsid with helical symmetry. The baculoviruses generate two different progenies, called budded virus (BVs) and occluded virus (OVs) that share the same nucleocapsid. Both viral progenies play different roles in the natural cycle of these viruses. BVs, consisting of a single nucleocapsid surrounded by an envelope derived from the cellular plasmatic membrane, are responsible for the transmission of the infection from cell to cell in an infected animal. In OVs, on the other hand, one or more nucleocapsids are contained by an envelope synthesized *de novo* in the nucleus of infected cells. These virions are then included in a crystalline protein matrix, consisting mainly of a single polypeptide which is product of the hyperexpression of a very late viral gene, resulting in the so-called occlusion bodies (OBs). The OBs, whose polypeptide structure gives protection to the OVs contained therein, are responsible for the transmission of the infection between susceptible animals in nature and, in fact, constitute the viral progeny useful as insecticide. The structures of BVs, OVs and OBs are shown in Figure 1.

Fig. 1. schematic structures of budded virus, occluded virus and occlusion bodies of baculoviruses.

The members of the *Baculoviridae* family are classified into four different genera (Jehle et al., 2006). The viruses classified into the genus *α-baculovirus* are able to infect lepidopteran insects to produce nuclear polyhedrosis. The viruses belonging to the genus *β-baculovirus*

can also infect lepidopteran insects, but they produce granulosis. On the other hand, the viruses classified into the genus *γ-baculovirus* can infect hymenopteran insects to produce nuclear polyhedrosis, while the viruses of the genus *δ-baculovirus* are associated to the production of nuclear polyhedrosis in dipteran insects. As most baculoviruses used as insecticides so far belong to the genus *α-baculovirus*, the subject henceforth will be focused on lepidopteran nucleopolyhedroviruses.

#### **2.2 Natural cycle and pathogenicity**

128 Insecticides – Basic and Other Applications

The purpose of this chapter is to review the current state of the art about insect cell culture technology and its application to the production of viral insecticides belonging to the family *Baculoviridae*. The several restrictions still existing to develop feasible processes as well as

The baculoviruses (family *Baculoviridae*) are a group of arthropod-specific viral pathogens that have a circular and supercoiled double-stranded DNA genome (Rohrmann, 2011). The size of the genome ranges from 80 to 180 kbp. The genome is contained in a rod-shaped nucleocapsid with helical symmetry. The baculoviruses generate two different progenies, called budded virus (BVs) and occluded virus (OVs) that share the same nucleocapsid. Both viral progenies play different roles in the natural cycle of these viruses. BVs, consisting of a single nucleocapsid surrounded by an envelope derived from the cellular plasmatic membrane, are responsible for the transmission of the infection from cell to cell in an infected animal. In OVs, on the other hand, one or more nucleocapsids are contained by an envelope synthesized *de novo* in the nucleus of infected cells. These virions are then included in a crystalline protein matrix, consisting mainly of a single polypeptide which is product of the hyperexpression of a very late viral gene, resulting in the so-called occlusion bodies (OBs). The OBs, whose polypeptide structure gives protection to the OVs contained therein, are responsible for the transmission of the infection between susceptible animals in nature and, in fact, constitute the viral progeny useful as insecticide. The structures of BVs, OVs

Fig. 1. schematic structures of budded virus, occluded virus and occlusion bodies of

The members of the *Baculoviridae* family are classified into four different genera (Jehle et al., 2006). The viruses classified into the genus *α-baculovirus* are able to infect lepidopteran insects to produce nuclear polyhedrosis. The viruses belonging to the genus *β-baculovirus*

the prospects for overcoming these limitations will be also reviewed.

**2. The baculoviruses** 

**2.1 Structure and classification** 

and OBs are shown in Figure 1.

baculoviruses.

The main route of infection of lepidopteran larvae with nucleopolyhedrovirus is the ingestion of food contaminated with viral OBs (Granados & Williams, 1986). Once ingested, OBs are transported to the larvae´s midgut, where they are dissolved to release the occluded virions, due to the combined action of the alkaline environment and the presence of alkaline proteases. The released OVs pass through the midgut peritrophic matrix and find the brush border membrane of the columnar midgut epithelial cells, which fuse with the viral envelope to enter the viral nucleocapsids within the cytoplasm. The ability of OVs to infect midgut epithelial cells is dependent on the expression of a set of genes whose products are denominated "*per os* infectivity factors" (PIFs) (Rohrmann, 2011). Most nucleocapsids are then transported to the nucleus through a process that is dependent on actin polymerization. Once the nucleocapsid has been entered into the cell nucleus, the viral DNA is naked and starts the transcriptional cascade that lead ultimately to the assembly of progeny nucleocapsids. A distinctive feature of the primary replication of nucleopolyhedroviruses in the midgut is that the nucleocapsids that were assembled in the nucleus are almost totally exported to the basal cytoplasmatic membrane, from where they finally egress as BVs. The budding of BVs occurs at regions of the plasmatic membrane that have been modified by insertion of the glycoproteins characteristic of the BV progeny, GP64 and/or F. Then, BVs would cross the basal lamina to begin the dissemination process that leads to secondary systemic infections. Alternatively, it has been proposed that baculoviruses can reach the main insect cavity through previous infection of tracheal cells (Pasarelli, 2011). Secondary infections, that affect almost all insect tissues, start when BVs penetrate into cells through a receptor-mediated endocytosis process. After the fusion of the viral envelope with the membrane of acidified endosomes, viral nucleocapsids are released into the cytoplasm and then transported to the nucleus, where the viral genome is naked. Differently from what occur in midgut epithelial cells, the transcriptional cascade in secondary infections drives the replication process to the production of nucleocapsids that, besides of feeding the budding of BVs, are assembled into the nucleus to form OVs. The assembly of OVs implies the retention of nucleocapsids inside the nucleus and the acquisition of an envelope synthesized *de novo* at the expense of the inner nuclear membrane. The OVs are finally occluded inside a crystalline matrix consisting mainly of the viral protein polyhedrin , whose gene is expressed at very high levels during the very late stage of the transcriptional cascade. The products of the occlusion process are the OBs, which are retained inside the nucleus until the death and lysis of the infected cell. At the end of the pathogenic process, the infected insect is full of OBs that, after its death and liquefaction of its tissues, are released into the environment to restart the cycle again.

The symptoms of the disease associated to baculovirus infection are not usually apparent during the first days post-infection (Granados & Williams, 1986). The change of color and the altered behavior of infected insects are often the earliest signs of infection with αbaculovirus. The lack of appetite, which after several days culminates in the total

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 131

same viral enzyme in charge of the late transcription. Although some BVs are produced during the very late stage, the hallmark of this period is the assembly of occlusion bodies,

Baculoviruses are arthropod-specific pathogens, with a host range that is generally very narrow, and lack the ability to replicate and produce pathogenic effects in other animals and plants, properties that have promoted their use as safe insecticides with reduced environmental impact (Huber, 1986). In addition, the expression of very late genes in the baculovirus genome is under the control of regulatory elements with a very high promoter activity, a property that has allowed the development of one of the most widely used expression systems for the production of recombinant proteins, the baculovirus vector expression system (Luckow & Summers, 1988). Also, baculoviruses are able to penetrate into mammalian cells, although they can not replicate into them. This property permits the use of these viruses as vectors for gene delivery (Kost & Condreay, 2002). Baculoviruses also exhibit a potent immunostimulating activity in mammals, opening the possibility of their use as adjuvants in the formulation of novel vaccines (Abe & Matsuura, 2010). Some of these applications have yet to demonstrate its market potential, but others are a reality and products based on these viruses are used today in agriculture, veterinary medicine and

Baculoviruses presents a number of advantages over traditional synthetic chemical insecticides (Moscardi, 1999). Their high specificity makes them safe for other insects, and thus helps to preserve and even enhance the natural mechanisms of plagues control. In addition, although baculoviruses can infect mammalian cells, including human cells, they can not replicate in them and therefore they lack of pathogenicity for the human being and other animals, making safe their use. The multiplication in their natural hosts, and their capacity to persist in the environment make them suitable for the inoculative control of plagues in forestry. In addition, the same properties coupled with the aforementioned preservation of natural enemies, permit the reduction of the number of the applications needed to keep the insect plague under control in annual crops, thus contributing to reduce the costs of protection. Finally, its use in replacement of synthetic insecticides helps to

The use of baculoviruses as insecticides also has limitations. Their high specificity is also a disadvantage to their widespread use, since they are only useful when the damage to the crop to be protected is produced predominantly by a single insect, and they are not effective in controlling pest complexes. The insecticidal effect of baculoviruses is not evident immediately after application, and the delay usually is accompanied by an increase of the level of crop damage. This defect can be counteracted by an earlier application of the virus, but it requires a close quantitative following of the insect population. Also, since baculovirus production processes are based on viral replication in living hosts, their yields can not match the high yields obtained at relatively low costs in the synthesis of chemical insecticides. Table 2 presents a list of selected baculoviruses belonging to the genus *α-baculovirus* registered for their use as insecticides. As can be

which extends up to the cell death.

human medicine, among others.

**3.1 Baculoviruses as insecticides** 

reduce the overall levels of chemical pollution.

**3. Technological applications of baculoviruses** 

interruption of feeding, is another sign of infection. The growth of infected larvae is delayed with respect to uninfected controls, and the death occurs after several days post-infection. The length of the interval of time between infection and death of the insect varies between 3 days and 3 weeks, and depends on many factors, including larval age, nutritional status, dose of virus and virulence of the viral strain, as well as on environmental factors. Anyway, nor the cessation of ingestion or death occur immediately to infection, facts that constitute strong constraints to the acceptance of baculoviruses as insecticides.

#### **2.3 The transcriptional regulation of the baculovirus replication cycle and the production of viral progenies**

The replication cycle of baculoviruses is mainly regulated at the transcriptional level. The baculovirus transcriptional program occurs in three stages, called early, late and very late, respectively, which are coordinated in a cascade (Table 1) (Blissard & Rohrmann, 1990; Rohrmann, 2011).


Table 1. Cascade of transcriptional events during the replication of baculoviruses and temporal distribution of production of viral progenies. \*AcMNPV replication.

Each transcriptional stage is characterized by the expression of a specific group of genes. Early transcription begins immediately after the parental viral DNA is naked into the nucleus. It is carried out by the cellular RNA polymerase II, and includes a set of genes whose products are trans-activators and enzymes that will then have a role in viral DNA replication. As an exception, in the early stage is also transcribed the *gp64* gene whose expression´s product is the principal glycoprotein of the group I nucleopolyhedrovirus´ envelope. The end of the early stage is determined by the onset of viral DNA synthesis, and involves a change in the pattern of transcription by which some of the genes initially transcribed are no longer expressed, and a new set of genes begins to be transcribed by a viral RNA polymerase, starting the late stage of transcription. The late stage involves the expression of genes whose products are proteins and glycoproteins that are part of the structure of the budded virions, which are assembled and released from the infected cell during this time. Finally, there occurs a further change in the pattern of transcription, whose most notable feature is the hyperexpression of genes whose products are proteins and glycoproteins involved in the assembly of occluded virions and occlusion bodies, such as polyhedrin and P10. The RNA polymerase involved in the very late transcription is the same viral enzyme in charge of the late transcription. Although some BVs are produced during the very late stage, the hallmark of this period is the assembly of occlusion bodies, which extends up to the cell death.

#### **3. Technological applications of baculoviruses**

130 Insecticides – Basic and Other Applications

interruption of feeding, is another sign of infection. The growth of infected larvae is delayed with respect to uninfected controls, and the death occurs after several days post-infection. The length of the interval of time between infection and death of the insect varies between 3 days and 3 weeks, and depends on many factors, including larval age, nutritional status, dose of virus and virulence of the viral strain, as well as on environmental factors. Anyway, nor the cessation of ingestion or death occur immediately to infection, facts that constitute

The replication cycle of baculoviruses is mainly regulated at the transcriptional level. The baculovirus transcriptional program occurs in three stages, called early, late and very late, respectively, which are coordinated in a cascade (Table 1) (Blissard & Rohrmann, 1990;

strong constraints to the acceptance of baculoviruses as insecticides.

**production of viral progenies** 

Rohrmann, 2011).

**2.3 The transcriptional regulation of the baculovirus replication cycle and the** 

Table 1. Cascade of transcriptional events during the replication of baculoviruses and temporal distribution of production of viral progenies. \*AcMNPV replication.

Each transcriptional stage is characterized by the expression of a specific group of genes. Early transcription begins immediately after the parental viral DNA is naked into the nucleus. It is carried out by the cellular RNA polymerase II, and includes a set of genes whose products are trans-activators and enzymes that will then have a role in viral DNA replication. As an exception, in the early stage is also transcribed the *gp64* gene whose expression´s product is the principal glycoprotein of the group I nucleopolyhedrovirus´ envelope. The end of the early stage is determined by the onset of viral DNA synthesis, and involves a change in the pattern of transcription by which some of the genes initially transcribed are no longer expressed, and a new set of genes begins to be transcribed by a viral RNA polymerase, starting the late stage of transcription. The late stage involves the expression of genes whose products are proteins and glycoproteins that are part of the structure of the budded virions, which are assembled and released from the infected cell during this time. Finally, there occurs a further change in the pattern of transcription, whose most notable feature is the hyperexpression of genes whose products are proteins and glycoproteins involved in the assembly of occluded virions and occlusion bodies, such as polyhedrin and P10. The RNA polymerase involved in the very late transcription is the Baculoviruses are arthropod-specific pathogens, with a host range that is generally very narrow, and lack the ability to replicate and produce pathogenic effects in other animals and plants, properties that have promoted their use as safe insecticides with reduced environmental impact (Huber, 1986). In addition, the expression of very late genes in the baculovirus genome is under the control of regulatory elements with a very high promoter activity, a property that has allowed the development of one of the most widely used expression systems for the production of recombinant proteins, the baculovirus vector expression system (Luckow & Summers, 1988). Also, baculoviruses are able to penetrate into mammalian cells, although they can not replicate into them. This property permits the use of these viruses as vectors for gene delivery (Kost & Condreay, 2002). Baculoviruses also exhibit a potent immunostimulating activity in mammals, opening the possibility of their use as adjuvants in the formulation of novel vaccines (Abe & Matsuura, 2010). Some of these applications have yet to demonstrate its market potential, but others are a reality and products based on these viruses are used today in agriculture, veterinary medicine and human medicine, among others.

#### **3.1 Baculoviruses as insecticides**

Baculoviruses presents a number of advantages over traditional synthetic chemical insecticides (Moscardi, 1999). Their high specificity makes them safe for other insects, and thus helps to preserve and even enhance the natural mechanisms of plagues control. In addition, although baculoviruses can infect mammalian cells, including human cells, they can not replicate in them and therefore they lack of pathogenicity for the human being and other animals, making safe their use. The multiplication in their natural hosts, and their capacity to persist in the environment make them suitable for the inoculative control of plagues in forestry. In addition, the same properties coupled with the aforementioned preservation of natural enemies, permit the reduction of the number of the applications needed to keep the insect plague under control in annual crops, thus contributing to reduce the costs of protection. Finally, its use in replacement of synthetic insecticides helps to reduce the overall levels of chemical pollution.

The use of baculoviruses as insecticides also has limitations. Their high specificity is also a disadvantage to their widespread use, since they are only useful when the damage to the crop to be protected is produced predominantly by a single insect, and they are not effective in controlling pest complexes. The insecticidal effect of baculoviruses is not evident immediately after application, and the delay usually is accompanied by an increase of the level of crop damage. This defect can be counteracted by an earlier application of the virus, but it requires a close quantitative following of the insect population. Also, since baculovirus production processes are based on viral replication in living hosts, their yields can not match the high yields obtained at relatively low costs in the synthesis of chemical insecticides. Table 2 presents a list of selected baculoviruses belonging to the genus *α-baculovirus* registered for their use as insecticides. As can be

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 133

The first studies conducted *in vitro* on tissues of invertebrate animals were made by Goldschmidt in 1915 (Day & Grace, 1959). Thereafter, and for about 40 years, attempts to multiply insect cells and tissues *in vitro* have had limited success. After completion of the Second World War, and already having the air filtration technology that permitted the safe handling of animal cell cultures in sterile environments, the work of Wyatt et al. (1956) on the chemical composition of the insect hemolymph allowed the development of the first culture media specifically designed for the cultivation of lepidopteran cells (Grace, 1958). The establishment of the first insect cell lines obtained from tissues of lepidopteran insects was an achievement reached by Grace (1962). Since then until now, at least half thousand cell lines, from different insects and distinct tissues have been established. A milestone in this process was the establishment of the cell line IPLB-Sf21 (Vaughn et al., 1977). This cell line, used to plaque baculovirus for the first time, exhibited relevant technological properties, such as the ability to grow indistinctly in static cultures and in agitated suspension cultures. Also, IPLB-Sf21 was the insect cell line where the clone Sf9 was produced from. This clone was closely linked to the development of the baculovirus – insect cell expression system for recombinant proteins (Summers & Smith, 1983). At the same time, new culture media were developed, such as MM, TC-100, TNM-FH and IPL-41, and the insect haemolymph that was initially used to supplement them, was replaced by fetal calf serum. At the end of the 80's, two important developments opened the possibility of expanding the cultivation of insect cells to an industrial scale: first, the development of microemulsions of lipids and sterols allowed the formulation of serum-free media, and second, the demonstration of the protective effect of surfactant poli-alcohols on the integrity of insect cells in suspension cultures aerated by sparging permitted the scaling-up to large stirred tank reactors and airlift reactors (Maiorella et al, 1988). In recent years, the main contributions to the technology of cultivation of insect cells have come from the development of genetically modified cell lines, capable, for example, to produce proteins

In a process of baculovirus production in cell cultures is crucial to make a proper selection of the cell line to be used as substrate for virus multiplication. The selected cell line must be susceptible and permissive to the virus, which must replicate in abundance to produce high yields of both budded virus and occlusion bodies. Preferably, nutritional requirements and metabolism should be well characterized, and the cell line should show relevant technological properties such as adaptability to suspension cultures, capability to grow in a low-cost serum-free medium and ability to grow in industrial bioreactors. Furthermore, it

Currently there are hundreds of cell lines established from tissues and organs of lepidopteran insects (Lynn, 2007), but very few meet the requirements described above. Table 3 shows a list of the lepidopteran insect cell lines more used for producing wildtype and/or recombinant baculovirus. The IPLB-Sf21 cell line and its clone Sf9 have been used intensively and they are well characterized. They can grow in suspension cultures at high cell concentration in bioreactors and there are several serum-free media available for them. Both wild-type and recombinant AcMNPV replicate very well in Sf cell lines

should be genetically stable, and should not be a source of viral variability.

**4. Insect cell culture technology** 

with humanized molecular structures (Shi & Jarvis, 2007).

**4.1 A brief history** 

**4.2 Insect cell lines** 


observed, most of them are rather specific for one lepidopteran specie, except AcMNPV that has a wider host range.

Table 2. Examples of *α-baculovirus* registered for insecticidal use.

#### **3.2 Genetically modified baculoviruses as improved insecticides**

As explained above, the adoption of baculoviruses as insecticides is limited by some of its pathogenic properties. One of the strategies developed to overcome this limitation is the modification of the viral genome in order to improve the insecticidal capabilities of the modified virus. To this end, two alternative routes have been followed: the insertion of foreign genes whose expression gives the virus an increased virulence, and the deletion of viral genes responsible for the delay in the evolution of the viral pathogenic process (reviewed by Szewczyk et al., 2006). The genes corresponding to several specific insect toxins, hormones or enzymes have been cloned and expressed in different baculoviruses, resulting in most cases in increased virulence, decreased time to insect death, and decreased plant damage. Besides, the deletion of the viral gene codifying for the ecdysteroid glycosil transferase (*egt*) - involved in the metabolism of the hormone ecdysone - also resulted in reduction of food consumption and faster killing of infected larvae.

Although the genetic modification has demonstrated to be a promissory strategy to improve the insecticidal ability of these viruses, the public perception about the risks that would involve the field release of recombinant viruses has limited the interest in developing novel insectides based on genetically modified baculoviruses. In fact, although have elapsed 20 years since the first publications that described the development of genetically modified baculovirus with enhanced insecticidal activity, no one product based on these recombinant viruses has yet come to market, and companies that were involved initially in these developments have canceled the processes for obtaining approvals for its use.

#### **4. Insect cell culture technology**

#### **4.1 A brief history**

132 Insecticides – Basic and Other Applications

observed, most of them are rather specific for one lepidopteran specie, except AcMNPV

**Virus Insect target Crops** AgMNPV *Anticarsia gemmatalis* Soybean

> *californica*, *Trichoplusia ni*, *Pseudoplusia includens*, etc.

*Heliothis virescens*

LdMNPV *Lymantria dispar* Forest MbMNPV *Mamestra brassicae* Cabbage OpMNPV *Orgya pseudotsugata* Forest SeMNPV *Spodoptera exigua* Vegetables SlNPV *Spodoptera littoralis* Cotton SpltMNPV *Spodoptera litura* Vegetables, cotton

As explained above, the adoption of baculoviruses as insecticides is limited by some of its pathogenic properties. One of the strategies developed to overcome this limitation is the modification of the viral genome in order to improve the insecticidal capabilities of the modified virus. To this end, two alternative routes have been followed: the insertion of foreign genes whose expression gives the virus an increased virulence, and the deletion of viral genes responsible for the delay in the evolution of the viral pathogenic process (reviewed by Szewczyk et al., 2006). The genes corresponding to several specific insect toxins, hormones or enzymes have been cloned and expressed in different baculoviruses, resulting in most cases in increased virulence, decreased time to insect death, and decreased plant damage. Besides, the deletion of the viral gene codifying for the ecdysteroid glycosil transferase (*egt*) - involved in the metabolism of the hormone ecdysone - also resulted in

Although the genetic modification has demonstrated to be a promissory strategy to improve the insecticidal ability of these viruses, the public perception about the risks that would involve the field release of recombinant viruses has limited the interest in developing novel insectides based on genetically modified baculoviruses. In fact, although have elapsed 20 years since the first publications that described the development of genetically modified baculovirus with enhanced insecticidal activity, no one product based on these recombinant viruses has yet come to market, and companies that were involved initially in these

HaNPV *Helicoverpa armigera* Cotton,tomato

Cotton, cabbage, tomato, broccoli

Cotton, corn, tomato, vegetables

AcMNPV *Autographa*

HzSNPV *Helicoverpa zea*,

Table 2. Examples of *α-baculovirus* registered for insecticidal use.

**3.2 Genetically modified baculoviruses as improved insecticides** 

reduction of food consumption and faster killing of infected larvae.

developments have canceled the processes for obtaining approvals for its use.

that has a wider host range.

The first studies conducted *in vitro* on tissues of invertebrate animals were made by Goldschmidt in 1915 (Day & Grace, 1959). Thereafter, and for about 40 years, attempts to multiply insect cells and tissues *in vitro* have had limited success. After completion of the Second World War, and already having the air filtration technology that permitted the safe handling of animal cell cultures in sterile environments, the work of Wyatt et al. (1956) on the chemical composition of the insect hemolymph allowed the development of the first culture media specifically designed for the cultivation of lepidopteran cells (Grace, 1958). The establishment of the first insect cell lines obtained from tissues of lepidopteran insects was an achievement reached by Grace (1962). Since then until now, at least half thousand cell lines, from different insects and distinct tissues have been established. A milestone in this process was the establishment of the cell line IPLB-Sf21 (Vaughn et al., 1977). This cell line, used to plaque baculovirus for the first time, exhibited relevant technological properties, such as the ability to grow indistinctly in static cultures and in agitated suspension cultures. Also, IPLB-Sf21 was the insect cell line where the clone Sf9 was produced from. This clone was closely linked to the development of the baculovirus – insect cell expression system for recombinant proteins (Summers & Smith, 1983). At the same time, new culture media were developed, such as MM, TC-100, TNM-FH and IPL-41, and the insect haemolymph that was initially used to supplement them, was replaced by fetal calf serum. At the end of the 80's, two important developments opened the possibility of expanding the cultivation of insect cells to an industrial scale: first, the development of microemulsions of lipids and sterols allowed the formulation of serum-free media, and second, the demonstration of the protective effect of surfactant poli-alcohols on the integrity of insect cells in suspension cultures aerated by sparging permitted the scaling-up to large stirred tank reactors and airlift reactors (Maiorella et al, 1988). In recent years, the main contributions to the technology of cultivation of insect cells have come from the development of genetically modified cell lines, capable, for example, to produce proteins with humanized molecular structures (Shi & Jarvis, 2007).

#### **4.2 Insect cell lines**

In a process of baculovirus production in cell cultures is crucial to make a proper selection of the cell line to be used as substrate for virus multiplication. The selected cell line must be susceptible and permissive to the virus, which must replicate in abundance to produce high yields of both budded virus and occlusion bodies. Preferably, nutritional requirements and metabolism should be well characterized, and the cell line should show relevant technological properties such as adaptability to suspension cultures, capability to grow in a low-cost serum-free medium and ability to grow in industrial bioreactors. Furthermore, it should be genetically stable, and should not be a source of viral variability.

Currently there are hundreds of cell lines established from tissues and organs of lepidopteran insects (Lynn, 2007), but very few meet the requirements described above. Table 3 shows a list of the lepidopteran insect cell lines more used for producing wildtype and/or recombinant baculovirus. The IPLB-Sf21 cell line and its clone Sf9 have been used intensively and they are well characterized. They can grow in suspension cultures at high cell concentration in bioreactors and there are several serum-free media available for them. Both wild-type and recombinant AcMNPV replicate very well in Sf cell lines

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 135

the quantitative requirements of other important nutrients, such as lipids, sterols, vitamins

Carbohydrates are essential components of all culture media for insect cells, due to their role as main sources of carbon and energy. Insect cells are capable to grow in culture media containing glucose as the unique carbohydrate, but insect cells can also consume other monosaccharides and disaccharides (Mitsuhashi, 1989). Sf9 cells consume glucose without production of lactate. This behavior has been attributed to the existence of an active tricarboxylic acid cycle, where 70 – 80% of the consumed glucose would be totally oxidized (Neermann & Wagner, 1996). However, these results are still controversial, because other studies have found that the percentage of glucose consumed that is derived to the tricarboxylic acid cycle is much lower (Benslimane *et al*., 2005). The production of other metabolites such as alanine, glycerol and ethanol, as well as fatty acid synthesis, could explain the fate of a significant fraction of carbon incorporated to Sf9 cells through glucose consumption (Drews et al., 2000; Bernal et al., 2009). BTI-TN-5B1-4 and BM5 cell lines have a different behavior: both cell lines produce lactate, displaying a behavior similar to that of mammalian cells (Rhiel et al., 1997; Stavroulakis et al., 1991). The flux of glucose was studied in BTI-TN-5B1-4 cells, resulting that the proportion oxidized in the tricarboxylic acids cycle was lower than in Sf9 cells (Benslimane et al., 2005). The influence of baculovirus infection on the metabolism of insect cells, and specifically on glucose metabolism, is a topic that has been scarcely addressed, and the information is contradictory (Bernal et al., 2009; Gioria et

According to Mitsuhashi (1989), 15 amino acids are essential to insect cells. Glutamine is the amino acid consumed faster and in a greater extension in cultures of most lepidopteran insect cell lines characterized to date. However, it has been demonstrated that glutamine is dispensable for the growth of Sf9 cells, providing that the cells have ammonium as nitrogen source (Öhman et al., 1996). Other amino acids that can be consumed in significant quantities are asparagine, aspartate, glutamate and serine, and precisely asparagine is consumed faster than glutamine in cultures of the BTI-TN-5B1-4 cell line (Rhiel et al., 1997). The demand of other amino acids is much lower. Besides serving as precursors for the synthesis of proteins and nucleic acids, amino acids that are consumed faster are used as sources of energy, such as glutamine and asparagine. The metabolism of glutamine has been studied in Sf9 cells, where the utilization pathways are depending on the availability of glucose (Drews et al., 2000). It has been proposed that, in glucose excess, the cytoplasmatic enzyme glutamate synthase transfers the amidic nitrogen of glutamine to the amine-position in glutamate, from where it is transaminated to alanine, the main product of glutamine metabolism. But when glucose is exhausted, glutamine is metabolized in mitochondria, where the amide-nitrogen and the amine-nitrogen of glutamine are sequentially released as ammonium ion, which accumulates as the main product of glutamine metabolism under glucose limitation in Sf9 cells. The metabolism of glutamine and other amino acids used as energy sources in other cell lines, such as BTI-TN-5B1-4 and saUFL-AG-286, is probably regulated differently, because they produce ammonia even in the presence of excess glucose (Gioria et al., 2006; Rhiel et al., 1997). The information about the influence of baculovirus infection on glutamine requirement and metabolism is scarce, but most results appear to indicate that insect cells tend to reduce the demand after infection (Gioria et al., 2006; Bernal

and mineral salts, is much scarce.

al., 2006).

et al., 2009).

(O´Reilly et al., 1994). In addition, these cell lines had shown to be susceptible and permissive to the replication of other baculoviruses (Claus et al, 1993). The cell line BTI-TN-5B1-4, known commercially as High Five®, is also being used widely to produce recombinant proteins due to its susceptibility to AcMNPV and elevated specific productivity (Chung & Shuler, 1993).


Table 3. Lepidopteran insect cell lines used frequently to produce wild-type and recombinant baculoviruses.

Besides the widely used cell lines that were mentioned in the preceding paragraph, other cell lines have been used more narrowly in processes involving the multiplication of other baculoviruses. In general, these lines were established from tissues of the natural host of the baculovirus to replicate, because viral yields tend to be higher in infected cultures of these homologous cell lines than in the heterologous ones. For instance, the cell lines saUFL-AG-286 (*Anticarsia gemmatalis*) (Sieburth & Maruniak, 1988), BM5 (*Bombyx mori*) (Grace, 1967), BCIRL-HZ-AM1 (*Heliothis zea*) (McIntosh & Ignoffo, 1981) and IPLB-LdEIta (*Lymantria dispar*) (Lynn et al, 1988) have been used to produce specifically the viruses AgMNPV, BmNPV, HaSNPV and LdMNPV, respectively. However, these cell lines are not as well characterized as the most widely used lines, and their technological properties (adaptation to suspension, ability to grow in serum-free culture media) are less remarkable or yet unknown.

#### **4.3 Nutrition and metabolism of lepidopteran insect cells**

Most existing data on nutrition and metabolism of lepidopteran insect cells in culture has been obtained from studies of a few cell lines, mostly Sf9 and BTI-TN-5B1-4. The comparative analysis of that information permits to conclude that each cell line is considerably flexible for satisfying their nutritional requirements. However, there are marked differences in metabolic behavior between different cell lines. Carbohydrates and amino acids are the most important nutrients, and the knowledge about their quantitative demands and metabolism will be briefly reviewed below. The information available about

(O´Reilly et al., 1994). In addition, these cell lines had shown to be susceptible and permissive to the replication of other baculoviruses (Claus et al, 1993). The cell line BTI-TN-5B1-4, known commercially as High Five®, is also being used widely to produce recombinant proteins due to its susceptibility to AcMNPV and elevated specific

**Cell line Insect Tissue of origin Susceptibility to**

BTI‐TN‐5B1‐4 *Trichoplusia ni* Embryos AcMNPV,AgMNPV,

BCIRL‐HZ‐AM1 *Heliothis zea* Ovarioles AcMNPV,HaNPV,

Besides the widely used cell lines that were mentioned in the preceding paragraph, other cell lines have been used more narrowly in processes involving the multiplication of other baculoviruses. In general, these lines were established from tissues of the natural host of the baculovirus to replicate, because viral yields tend to be higher in infected cultures of these homologous cell lines than in the heterologous ones. For instance, the cell lines saUFL-AG-286 (*Anticarsia gemmatalis*) (Sieburth & Maruniak, 1988), BM5 (*Bombyx mori*) (Grace, 1967), BCIRL-HZ-AM1 (*Heliothis zea*) (McIntosh & Ignoffo, 1981) and IPLB-LdEIta (*Lymantria dispar*) (Lynn et al, 1988) have been used to produce specifically the viruses AgMNPV, BmNPV, HaSNPV and LdMNPV, respectively. However, these cell lines are not as well characterized as the most widely used lines, and their technological properties (adaptation to suspension, ability to grow in serum-free culture media) are less remarkable or yet

Most existing data on nutrition and metabolism of lepidopteran insect cells in culture has been obtained from studies of a few cell lines, mostly Sf9 and BTI-TN-5B1-4. The comparative analysis of that information permits to conclude that each cell line is considerably flexible for satisfying their nutritional requirements. However, there are marked differences in metabolic behavior between different cell lines. Carbohydrates and amino acids are the most important nutrients, and the knowledge about their quantitative demands and metabolism will be briefly reviewed below. The information available about

Bm5 *Bombyx mori* Ovarioles AcMNPV,BmNPV IPLB‐LdEIta *Lymantria dispar* Embryos AcMNPV, LdMNPV

**baculoviruses**

SfMNPV, SlNPV, TnSNPV

TnSNPV

HzSNPV

Ovarioles AcMNPV,AgMNPV,

Embryos AcMNPV,AgMNPV

productivity (Chung & Shuler, 1993).

IPLB‐Sf21 / Sf9 *Spodoptera*

saUFL‐AG‐286 *Anticarsia*

recombinant baculoviruses.

unknown.

*frugiperda*

*gemmatalis*

**4.3 Nutrition and metabolism of lepidopteran insect cells** 

Table 3. Lepidopteran insect cell lines used frequently to produce wild-type and

the quantitative requirements of other important nutrients, such as lipids, sterols, vitamins and mineral salts, is much scarce.

Carbohydrates are essential components of all culture media for insect cells, due to their role as main sources of carbon and energy. Insect cells are capable to grow in culture media containing glucose as the unique carbohydrate, but insect cells can also consume other monosaccharides and disaccharides (Mitsuhashi, 1989). Sf9 cells consume glucose without production of lactate. This behavior has been attributed to the existence of an active tricarboxylic acid cycle, where 70 – 80% of the consumed glucose would be totally oxidized (Neermann & Wagner, 1996). However, these results are still controversial, because other studies have found that the percentage of glucose consumed that is derived to the tricarboxylic acid cycle is much lower (Benslimane *et al*., 2005). The production of other metabolites such as alanine, glycerol and ethanol, as well as fatty acid synthesis, could explain the fate of a significant fraction of carbon incorporated to Sf9 cells through glucose consumption (Drews et al., 2000; Bernal et al., 2009). BTI-TN-5B1-4 and BM5 cell lines have a different behavior: both cell lines produce lactate, displaying a behavior similar to that of mammalian cells (Rhiel et al., 1997; Stavroulakis et al., 1991). The flux of glucose was studied in BTI-TN-5B1-4 cells, resulting that the proportion oxidized in the tricarboxylic acids cycle was lower than in Sf9 cells (Benslimane et al., 2005). The influence of baculovirus infection on the metabolism of insect cells, and specifically on glucose metabolism, is a topic that has been scarcely addressed, and the information is contradictory (Bernal et al., 2009; Gioria et al., 2006).

According to Mitsuhashi (1989), 15 amino acids are essential to insect cells. Glutamine is the amino acid consumed faster and in a greater extension in cultures of most lepidopteran insect cell lines characterized to date. However, it has been demonstrated that glutamine is dispensable for the growth of Sf9 cells, providing that the cells have ammonium as nitrogen source (Öhman et al., 1996). Other amino acids that can be consumed in significant quantities are asparagine, aspartate, glutamate and serine, and precisely asparagine is consumed faster than glutamine in cultures of the BTI-TN-5B1-4 cell line (Rhiel et al., 1997). The demand of other amino acids is much lower. Besides serving as precursors for the synthesis of proteins and nucleic acids, amino acids that are consumed faster are used as sources of energy, such as glutamine and asparagine. The metabolism of glutamine has been studied in Sf9 cells, where the utilization pathways are depending on the availability of glucose (Drews et al., 2000). It has been proposed that, in glucose excess, the cytoplasmatic enzyme glutamate synthase transfers the amidic nitrogen of glutamine to the amine-position in glutamate, from where it is transaminated to alanine, the main product of glutamine metabolism. But when glucose is exhausted, glutamine is metabolized in mitochondria, where the amide-nitrogen and the amine-nitrogen of glutamine are sequentially released as ammonium ion, which accumulates as the main product of glutamine metabolism under glucose limitation in Sf9 cells. The metabolism of glutamine and other amino acids used as energy sources in other cell lines, such as BTI-TN-5B1-4 and saUFL-AG-286, is probably regulated differently, because they produce ammonia even in the presence of excess glucose (Gioria et al., 2006; Rhiel et al., 1997). The information about the influence of baculovirus infection on glutamine requirement and metabolism is scarce, but most results appear to indicate that insect cells tend to reduce the demand after infection (Gioria et al., 2006; Bernal et al., 2009).

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 137

reach 90% of the final cost of complete culture medium, which can be unacceptable for the development of a production process for a viral bioinsecticide. Finally, the regulatory agencies are becoming increasingly restrictive in relation to the use of raw materials of

The problems about the use of serum as a supplement in animal cell cultures, described above, have driven the development of new culture media capable to support the growth of insect cell cultures in a serum-free environment. As the serum is a very complex substance, and its functions are diverse, it is necessary to use a mixture of various components to replace it. The supply of hydrophobic nutrients is replaced by adding microemulsions that contain a source of lipids and cholesterol (Maiorella et al, 1988; Ikonomou et al., 2001). The most used sources of lipids are the methyl esters of fatty acids isolated from the liver of marine fishes, but recently it has been described the use of cooking soybean oil, a cheaper and more abundant source of lipids (Micheloud et al., 2009). In addition, microemulsions also contain the surfactant Pluronic F68, whose presence protects the cells from the

On the other hand, the contribution of growth factors that made the serum is replaced by mixtures of enzymatic hydrolysates of proteins and yeast extract (Schlaeger, 1996). Hydrolysates from milk proteins such as lactalbumin and casein, and meat proteins, are commonly used as cheap replacements of serum, but the peptides responsible for the growth factor activity have not been identified. The effects of the addition of yeast extract to culture media for insect cells are similar to that of fetal calf serum, modifying the specific

There are currently several commercial serum-free media available that were specifically designed to cultivate either Sf9 or BTI-TN-5B1-4 cell lines, but eventually can also support the growth of other insect cell lines. The growth parameters of cultures in these media are remarkable, as well as the yields of baculovirus and recombinant proteins obtained in infected cultures. However, the cost of the commercial serum-free media is, at least, as high as the cost of complete medium supplemented with fetal calf serum, precluding its

New serum-free media were specifically designed in recent years for culturing a few cell lines of interest, due to their potential application to the development of production processes for insecticidal baculoviruses in insect cell cultures. A prototype low cost medium (LCM) was developed to both cultivate the cell line BCIRL-HZ-AM1 and to produce the baculovirus HaSNPV (Lua & Reid, 2003). Comparable maximum cell densities and growth rates were obtained in both the LCM and a commercial serum-free medium, but lower specific virus yields were reached in LCM. The composition of the LCM medium was not disclosed. On the other hand, the low-cost medium UNL-10 was developed to grow the saUFL-AG-286 cell line, useful to produce the baculovirus AgMNPV (Micheloud et al, 2009). The yields of occlusion bodies in suspension cultures, using optimized parameters of infection, were as high as 3 x 1011 OBs L-1, with specific yields higher than 600 OBs/cell. The composition of the UNL-10 medium, that was optimized to improve the yield of OBs of AgMNPV, has glucose as the only source of carbohydrates, a lower concentration of most amino acids, an improved mixture of vitamins and a lipid emulsion made with cooking oil. The growth factor activity is exerted by an optimized mixture of an enzymatic hydrolysate

growth rate and increasing the maximum cell density (Eriksson & Häggström, 2005).

utilization for the economically feasible production of insecticidal baculoviruses.

animal origin.

**4.4.2.1 Serum-free media for insect cell cultures** 

detrimental influence of bubbles in sparged bioreactors.

of casein, tryptose broth and yeast extract.

#### **4.4 Culture media for lepidopteran insect cells**

Almost all media used for cultivation of insect cells have a chemical composition partially defined. They consist of a basal medium composed of chemically defined mixtures of carbohydrates, amino acids, vitamins, salts and, in some cases, organic acids. In addition, the media must be supplemented with compounds of undefined chemical composition that contribute to cell´s proliferation, such as fetal calf serum, microbial extracts and/or protein hydrolysates.

#### **4.4.1 Basal culture media**

TC-100, IPL-41, Grace and TNM-FH are the most commonly used basal media for *in vitro* culture of lepidopteran insect cells (Schlaeger, 1996).TC-100 and TNM-FH have almost the same amino acids composition as the Grace medium, from which they originated. IPL-41 has the same qualitative composition of amino acids, but with higher concentration for the ones consumed faster: glutamine, asparagine, glutamic acid, aspartic acid and cystine. In any case, it has been demonstrated for several different insect cell lines that the concentrations of most amino acids in basal culture media are oversized with respect to cellular requirements (Bédard et al., 1993; Ferrance et al., 1993; Lua & Reid, 2003) . TC-100 and TNM-FH contain also protein hydrolysates, and the last is additionally supplemented with yeast extract. With regard to carbohydrates, all media contain glucose, although at different concentrations, and IPL-41 contains also an additional monosaccharide, fructose. Grace, TNM-FH and IPL-41 media also contain disaccharides, all of them contain sucrose and the last also maltose. IPL-4 is richer in vitamins and also contains organic acids, not present in the original formulations of the media Grace, TNM-FH and TC-100. The composition of mineral salts of IPL-41 differs from the other three, and is also enriched with oligoelements. In spite of the differences in the chemical composition described above, the four basal media can support the growth of Sf9 and BTI-TN-5B1-4 cell cultures, provided they are supplemented with fetal calf serum. This fact highlights the nutritional plasticity of these cell lines. On the other hand, the richer medium IPL-41 has demonstrated to be better suited than the other three to formulate serum-free media (Ikonomou et al., 2001; Maiorella et al., 1988).

#### **4.4.2 Fetal calf serum**

Blood serum obtained from bovine fetuses is the most used undefined supplement for animal cell cultures, including both mammalian and insect cells. It concentrates, in a single component, several essential functions for cultured cells (Barnes & Sato, 1980). Serum provides, in a water-soluble vehicle, lipids and cholesterol, and it is also a rich source of growth factors, vitamins and mineral oligoelements. Its proteins´ transporters allow the supply of poorly soluble ions, such as Fe+++. In addition, serum has detoxifying activity, and their proteins can contribute to the preservation of the structural integrity of cells when they are subjected to mechanical stress.

In spite of the advantages of fetal calf serum, they are accompanied by several disadvantages. Its composition is undefined and variable from batch to batch. It is also a possible vehicle for the introduction of chemical and biological contaminants, such as plaguicides, virus and prions, among others. The high concentration of serum proteins may involve interference with the extraction and purification of products. Besides, serum proteins are a source of foam in culture processes where aeration is made by sparging. The cost of using fetal bovine serum as a supplement of a basic medium is so high that it can reach 90% of the final cost of complete culture medium, which can be unacceptable for the development of a production process for a viral bioinsecticide. Finally, the regulatory agencies are becoming increasingly restrictive in relation to the use of raw materials of animal origin.

#### **4.4.2.1 Serum-free media for insect cell cultures**

136 Insecticides – Basic and Other Applications

Almost all media used for cultivation of insect cells have a chemical composition partially defined. They consist of a basal medium composed of chemically defined mixtures of carbohydrates, amino acids, vitamins, salts and, in some cases, organic acids. In addition, the media must be supplemented with compounds of undefined chemical composition that contribute to cell´s proliferation, such as fetal calf serum, microbial extracts and/or protein

TC-100, IPL-41, Grace and TNM-FH are the most commonly used basal media for *in vitro* culture of lepidopteran insect cells (Schlaeger, 1996).TC-100 and TNM-FH have almost the same amino acids composition as the Grace medium, from which they originated. IPL-41 has the same qualitative composition of amino acids, but with higher concentration for the ones consumed faster: glutamine, asparagine, glutamic acid, aspartic acid and cystine. In any case, it has been demonstrated for several different insect cell lines that the concentrations of most amino acids in basal culture media are oversized with respect to cellular requirements (Bédard et al., 1993; Ferrance et al., 1993; Lua & Reid, 2003) . TC-100 and TNM-FH contain also protein hydrolysates, and the last is additionally supplemented with yeast extract. With regard to carbohydrates, all media contain glucose, although at different concentrations, and IPL-41 contains also an additional monosaccharide, fructose. Grace, TNM-FH and IPL-41 media also contain disaccharides, all of them contain sucrose and the last also maltose. IPL-4 is richer in vitamins and also contains organic acids, not present in the original formulations of the media Grace, TNM-FH and TC-100. The composition of mineral salts of IPL-41 differs from the other three, and is also enriched with oligoelements. In spite of the differences in the chemical composition described above, the four basal media can support the growth of Sf9 and BTI-TN-5B1-4 cell cultures, provided they are supplemented with fetal calf serum. This fact highlights the nutritional plasticity of these cell lines. On the other hand, the richer medium IPL-41 has demonstrated to be better suited than the other three to formulate serum-free media (Ikonomou et al., 2001; Maiorella

Blood serum obtained from bovine fetuses is the most used undefined supplement for animal cell cultures, including both mammalian and insect cells. It concentrates, in a single component, several essential functions for cultured cells (Barnes & Sato, 1980). Serum provides, in a water-soluble vehicle, lipids and cholesterol, and it is also a rich source of growth factors, vitamins and mineral oligoelements. Its proteins´ transporters allow the supply of poorly soluble ions, such as Fe+++. In addition, serum has detoxifying activity, and their proteins can contribute to the preservation of the structural integrity of cells when they

In spite of the advantages of fetal calf serum, they are accompanied by several disadvantages. Its composition is undefined and variable from batch to batch. It is also a possible vehicle for the introduction of chemical and biological contaminants, such as plaguicides, virus and prions, among others. The high concentration of serum proteins may involve interference with the extraction and purification of products. Besides, serum proteins are a source of foam in culture processes where aeration is made by sparging. The cost of using fetal bovine serum as a supplement of a basic medium is so high that it can

**4.4 Culture media for lepidopteran insect cells** 

hydrolysates.

et al., 1988).

**4.4.2 Fetal calf serum** 

are subjected to mechanical stress.

**4.4.1 Basal culture media** 

The problems about the use of serum as a supplement in animal cell cultures, described above, have driven the development of new culture media capable to support the growth of insect cell cultures in a serum-free environment. As the serum is a very complex substance, and its functions are diverse, it is necessary to use a mixture of various components to replace it. The supply of hydrophobic nutrients is replaced by adding microemulsions that contain a source of lipids and cholesterol (Maiorella et al, 1988; Ikonomou et al., 2001). The most used sources of lipids are the methyl esters of fatty acids isolated from the liver of marine fishes, but recently it has been described the use of cooking soybean oil, a cheaper and more abundant source of lipids (Micheloud et al., 2009). In addition, microemulsions also contain the surfactant Pluronic F68, whose presence protects the cells from the detrimental influence of bubbles in sparged bioreactors.

On the other hand, the contribution of growth factors that made the serum is replaced by mixtures of enzymatic hydrolysates of proteins and yeast extract (Schlaeger, 1996). Hydrolysates from milk proteins such as lactalbumin and casein, and meat proteins, are commonly used as cheap replacements of serum, but the peptides responsible for the growth factor activity have not been identified. The effects of the addition of yeast extract to culture media for insect cells are similar to that of fetal calf serum, modifying the specific growth rate and increasing the maximum cell density (Eriksson & Häggström, 2005).

There are currently several commercial serum-free media available that were specifically designed to cultivate either Sf9 or BTI-TN-5B1-4 cell lines, but eventually can also support the growth of other insect cell lines. The growth parameters of cultures in these media are remarkable, as well as the yields of baculovirus and recombinant proteins obtained in infected cultures. However, the cost of the commercial serum-free media is, at least, as high as the cost of complete medium supplemented with fetal calf serum, precluding its utilization for the economically feasible production of insecticidal baculoviruses.

New serum-free media were specifically designed in recent years for culturing a few cell lines of interest, due to their potential application to the development of production processes for insecticidal baculoviruses in insect cell cultures. A prototype low cost medium (LCM) was developed to both cultivate the cell line BCIRL-HZ-AM1 and to produce the baculovirus HaSNPV (Lua & Reid, 2003). Comparable maximum cell densities and growth rates were obtained in both the LCM and a commercial serum-free medium, but lower specific virus yields were reached in LCM. The composition of the LCM medium was not disclosed. On the other hand, the low-cost medium UNL-10 was developed to grow the saUFL-AG-286 cell line, useful to produce the baculovirus AgMNPV (Micheloud et al, 2009). The yields of occlusion bodies in suspension cultures, using optimized parameters of infection, were as high as 3 x 1011 OBs L-1, with specific yields higher than 600 OBs/cell. The composition of the UNL-10 medium, that was optimized to improve the yield of OBs of AgMNPV, has glucose as the only source of carbohydrates, a lower concentration of most amino acids, an improved mixture of vitamins and a lipid emulsion made with cooking oil. The growth factor activity is exerted by an optimized mixture of an enzymatic hydrolysate of casein, tryptose broth and yeast extract.

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 139

cultures of insect cells will be always the result of a compromise between the satisfaction of an adequate mixing and the preservation of the structural and functional cellular integrity. Stirred tank reactors, where agitation is performed mechanically through impellers, have demonstrated to be useful to cultivate insect cells at large scale (Maranga et al., 2004). The agitation rate should be carefully controlled in stirred reactors, especially when cultures are aerated by sparging, due to deleterious effects on cell functionality and viability that occurs when cultures are stirred at speeds over 300 rpm with Rushton turbines (Cruz et al., 1998). This is because the shear in the zone near to the impeller - where the energy of agitation is introduced into the reactor- is very high. Three different ways to aerate insect cell cultures in stirred tank reactors have been used: surface aeration (Kamen et al., 1991), bubble-free aeration (Chico & Jáger, 2000) and sparging (Cruz et al., 1998). Without a doubt, the aeration method that offers a better chance of escalation in stirred reactors is through direct gas bubbling. Two process parameters should be carefully considered in stirred tank reactors aerated through sparging in order to keep a proper cellular viability and functionality: the aeration rate and the bubble size. High aeration rates, as well as low bubble size are main causes of impaired growth and functionality in insect cell cultures (Trinh et al., 1994). Airlift reactors have been much less used than stirred tank reactors, in spite they offer advantageous characteristics to cultivate large and fragile cells, as insect cells. One of the advantages of airlift reactors, when compared to classical stirred tank reactors is that they have the potential to provide high mass transfer rates with low and homogeneously distributed shear. Thus, adequate oxygen transfer rates from the gas to the liquid phase and from this one to the suspended cells can be obtained within a homogeneous environment and with a reduced exposure to sources of mechanical stress (Merchuk, 1991). On the other hand, these reactors improve their performance as it increases in size, provided that the optimal relations between the reactor geometrical parameters are preserved. In addition, due to its simplicity of design and construction, airlift reactors require less capital investment, and its operation and maintenance costs are also lower than in stirred tank reactors. While some articles have been published on the cultivation of insect cell lines in airlift reactor and its application to the production of baculovirus and recombinant proteins, there are few systematic studies for optimization of the geometrical parameters and operation in processes involving these reactors, nor models for their scaling-up (King et al.,

In addition to the classical airlift and stirred tank reactors, other reactor designs were applied successfully to the cultivation of lepidopteran insect cells, such as the rotating-wall vessel (Cowger et al., 1999). However, given the level of production scale that should be achieved for the development of economically feasible processes, only stirred tank and airlift reactors appear to have the potential to be used for the large scale production of insecticidal baculoviruses in insect cell cultures. This is also because of the experience existing in the scale-up of cultures of microorganisms and animal cells in these reactors.

Figure 3 outlines an infection process of infection with baculovirus in an insect cell culture. The preceding sections have addressed the characteristics of the cells, culture media and reactors. This section will deal with those aspects of the process that relate specifically to infection: the viral inoculums, the parameters of infection, the operation strategy and the

1992; Maiorella et al., 1988; Visnovsky et al.; 2003).

product.

**6. Baculovirus infection in lepidopteran insect cell cultures** 

#### **4.5 Physicochemical conditions in insect cell cultures**

The pH of the medium in insect cell cultures is determined by its chemical composition, depending mainly on the buffer activity of salts, although the mixture of amino acids can also play a role in pH regulation. The optimal pH for all lepidopteran insect cell cultures is acid, between 6.2 and 6.4 (Schlaeger, 1996). While the pH can be modified through the evolution of an insect cell culture, the changes tend to be limited and usually do not compromise the cellular physiology.

The optimum osmolarity can differ for distinct lepidopteran insect cell lines, which can react also differently according to the agents utilized to modify it. The osmolarity of most culture media for insect cells varies between 250 and 350 mOsm kg-1, but the initial value is habitually modified through the evolution of the culture, usually without consequences on the cellular physiology (Kurtti & Munderloh, 1984).

Insect cells are cultivated in vitro at temperatures ranging between 25 and 30 ºC. The optimum temperature for most lepidopteran cell lines is 28 ºC (O´Reilly et al., 1994). Cells cultured in serum-free media are less tolerant of temperature changes that cells grown in media supplemented with serum (Mitsuhashi & Goodwin, 1989).

The dissolved oxygen concentration is a critical parameter in insect cell cultures due to the reduced solubility of oxygen in aqueous culture media (Palomares & Ramírez, 1996). Dissolved oxygen levels between 40 and 70% are usually appropriate to keep acceptable growth parameters in insect cell cultures. In addition, most of the available information indicates that it is also critical to keep a proper level of dissolved oxygen in baculovirusinfected cultures, because oxygen deprivation is a cause of low yield of virus or recombinant protein.

#### **5. Bioreactors in insect cell cultures**

Although the most used insect cell lines can grow in suspension or as static cultures indistinctly, the scaling-up of static cultures is not a feasible alternative for the production of insecticidal baculoviruses. Thus, in this section it will only be reviewed the information about insect cell suspension cultures, from agitated erlenmeyers and spinner-flasks for lowscale cultures up to stirred tank and airlift reactors at larger scale.

The suspension cell cultures at low scale in agitated erlenmeyers or spinner-flasks usually do not offer significant technological difficulties, being able to reach cell densities as high as 1 x 107 viable cells per milliliter, with doubling times from 18 to 30 hours during the exponential growth phase (Bédard et al., 1993; Benslimane et al., 2007; Gioria et al., 2006; Lua & Reid, 2003; Rhiel et al., 1997). This is true for most insect cell lines as long as the ratio between the areas of the gas phase –usually air- to the liquid phase –culture medium- is large enough to ensure that the superficial supply of oxygen is adequate. In practical terms this means that the volume of culture should never exceed 25% of the total volume of the container. In addition, the stirring speed should be adjusted to 60 - 80 rpm in spinner-flasks and 100 - 120 rpm in flasks with orbital shaking.

The large scale culture of animal cells in suspension requires an adequate mixing through agitation, either mechanical or pneumatic, in order to keep cells in suspension, as well as to ensure physicochemical homogeneity and adequate mass transference. But these requirements of scaling collide with certain morphological characteristics of the insect cells, as their large size and lack of cell wall, that make them fragile and sensitive to the effects of agitation and gas sparging (Trinh et al., 1994). A successful scaling-up of suspension

The pH of the medium in insect cell cultures is determined by its chemical composition, depending mainly on the buffer activity of salts, although the mixture of amino acids can also play a role in pH regulation. The optimal pH for all lepidopteran insect cell cultures is acid, between 6.2 and 6.4 (Schlaeger, 1996). While the pH can be modified through the evolution of an insect cell culture, the changes tend to be limited and usually do not

The optimum osmolarity can differ for distinct lepidopteran insect cell lines, which can react also differently according to the agents utilized to modify it. The osmolarity of most culture media for insect cells varies between 250 and 350 mOsm kg-1, but the initial value is habitually modified through the evolution of the culture, usually without consequences on

Insect cells are cultivated in vitro at temperatures ranging between 25 and 30 ºC. The optimum temperature for most lepidopteran cell lines is 28 ºC (O´Reilly et al., 1994). Cells cultured in serum-free media are less tolerant of temperature changes that cells grown in

The dissolved oxygen concentration is a critical parameter in insect cell cultures due to the reduced solubility of oxygen in aqueous culture media (Palomares & Ramírez, 1996). Dissolved oxygen levels between 40 and 70% are usually appropriate to keep acceptable growth parameters in insect cell cultures. In addition, most of the available information indicates that it is also critical to keep a proper level of dissolved oxygen in baculovirusinfected cultures, because oxygen deprivation is a cause of low yield of virus or recombinant

Although the most used insect cell lines can grow in suspension or as static cultures indistinctly, the scaling-up of static cultures is not a feasible alternative for the production of insecticidal baculoviruses. Thus, in this section it will only be reviewed the information about insect cell suspension cultures, from agitated erlenmeyers and spinner-flasks for low-

The suspension cell cultures at low scale in agitated erlenmeyers or spinner-flasks usually do not offer significant technological difficulties, being able to reach cell densities as high as 1 x 107 viable cells per milliliter, with doubling times from 18 to 30 hours during the exponential growth phase (Bédard et al., 1993; Benslimane et al., 2007; Gioria et al., 2006; Lua & Reid, 2003; Rhiel et al., 1997). This is true for most insect cell lines as long as the ratio between the areas of the gas phase –usually air- to the liquid phase –culture medium- is large enough to ensure that the superficial supply of oxygen is adequate. In practical terms this means that the volume of culture should never exceed 25% of the total volume of the container. In addition, the stirring speed should be adjusted to 60 - 80 rpm in spinner-flasks

The large scale culture of animal cells in suspension requires an adequate mixing through agitation, either mechanical or pneumatic, in order to keep cells in suspension, as well as to ensure physicochemical homogeneity and adequate mass transference. But these requirements of scaling collide with certain morphological characteristics of the insect cells, as their large size and lack of cell wall, that make them fragile and sensitive to the effects of agitation and gas sparging (Trinh et al., 1994). A successful scaling-up of suspension

**4.5 Physicochemical conditions in insect cell cultures** 

the cellular physiology (Kurtti & Munderloh, 1984).

**5. Bioreactors in insect cell cultures** 

and 100 - 120 rpm in flasks with orbital shaking.

media supplemented with serum (Mitsuhashi & Goodwin, 1989).

scale cultures up to stirred tank and airlift reactors at larger scale.

compromise the cellular physiology.

protein.

cultures of insect cells will be always the result of a compromise between the satisfaction of an adequate mixing and the preservation of the structural and functional cellular integrity. Stirred tank reactors, where agitation is performed mechanically through impellers, have demonstrated to be useful to cultivate insect cells at large scale (Maranga et al., 2004). The agitation rate should be carefully controlled in stirred reactors, especially when cultures are aerated by sparging, due to deleterious effects on cell functionality and viability that occurs when cultures are stirred at speeds over 300 rpm with Rushton turbines (Cruz et al., 1998). This is because the shear in the zone near to the impeller - where the energy of agitation is introduced into the reactor- is very high. Three different ways to aerate insect cell cultures in stirred tank reactors have been used: surface aeration (Kamen et al., 1991), bubble-free aeration (Chico & Jáger, 2000) and sparging (Cruz et al., 1998). Without a doubt, the aeration method that offers a better chance of escalation in stirred reactors is through direct gas bubbling. Two process parameters should be carefully considered in stirred tank reactors aerated through sparging in order to keep a proper cellular viability and functionality: the aeration rate and the bubble size. High aeration rates, as well as low bubble size are main causes of impaired growth and functionality in insect cell cultures (Trinh et al., 1994).

Airlift reactors have been much less used than stirred tank reactors, in spite they offer advantageous characteristics to cultivate large and fragile cells, as insect cells. One of the advantages of airlift reactors, when compared to classical stirred tank reactors is that they have the potential to provide high mass transfer rates with low and homogeneously distributed shear. Thus, adequate oxygen transfer rates from the gas to the liquid phase and from this one to the suspended cells can be obtained within a homogeneous environment and with a reduced exposure to sources of mechanical stress (Merchuk, 1991). On the other hand, these reactors improve their performance as it increases in size, provided that the optimal relations between the reactor geometrical parameters are preserved. In addition, due to its simplicity of design and construction, airlift reactors require less capital investment, and its operation and maintenance costs are also lower than in stirred tank reactors. While some articles have been published on the cultivation of insect cell lines in airlift reactor and its application to the production of baculovirus and recombinant proteins, there are few systematic studies for optimization of the geometrical parameters and operation in processes involving these reactors, nor models for their scaling-up (King et al., 1992; Maiorella et al., 1988; Visnovsky et al.; 2003).

In addition to the classical airlift and stirred tank reactors, other reactor designs were applied successfully to the cultivation of lepidopteran insect cells, such as the rotating-wall vessel (Cowger et al., 1999). However, given the level of production scale that should be achieved for the development of economically feasible processes, only stirred tank and airlift reactors appear to have the potential to be used for the large scale production of insecticidal baculoviruses in insect cell cultures. This is also because of the experience existing in the scale-up of cultures of microorganisms and animal cells in these reactors.

#### **6. Baculovirus infection in lepidopteran insect cell cultures**

Figure 3 outlines an infection process of infection with baculovirus in an insect cell culture. The preceding sections have addressed the characteristics of the cells, culture media and reactors. This section will deal with those aspects of the process that relate specifically to infection: the viral inoculums, the parameters of infection, the operation strategy and the product.

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 141

population of defective virus, at the expense of the population of wild-type virus (van Lier et al., 1990). The generation of DIPs in cell cultures infected with baculovirus is favored by conditions that increase the probability of homologous recombination, such as in infections at high multiplicity of infection. The proportion of DIPs in a viral population, as well as the proportion of FP mutants, increases with the number of passages in cell cultures, a phenomenon known as "passage effect"(Krell, 1996). This effect impairs the amplification of

The viral stock is the most expensive raw material in processes destined to the production of insecticidal OBs in insect cell cultures. Obtaining the stock for infecting the production reactor usually requires the amplification of the seed virus through successive rounds of infection in insect cell cultures at progressively larger scales (Rhodes, 1996). The optimization of the conditions of infection for the production of BVs would have a double beneficial effect on the whole production process: on the one side, it would allow to reduce the consumption of an expensive raw material and, on the other side, it would permit to reduce the steps of the seed virus amplification process, and therefore to reduce the probabilities to generate and propagate deleterious genomic variants. Despite its importance, the information available about the optimization of infection conditions for the

production of the BV progeny of baculovirus is very scarce (Carinhas et al., 2009).

important impact on the feasibility of the scaling-up process.

quality of the culture medium at the time that they are actually infected.

**6.2 Parameters of infection** 

The quality of baculovirus stocks may also be affected by reduced infectivity in relation to the total amount of viral particles (Dee & Shuler, 1997). Although several causes have been proposed to explain this phenomenon, the inactivation of BVs could be an important detrimental factor, especially in stocks prepared in serum-free media (Jorio et al., 2006). An optimized management of the preservation of serum-free baculovirus stocks could have an

The fate of a baculovirus infection in an insect cell culture is strongly dependent on the selection of the multiplicity of the infection (MOI) and the time of infection (TOI) (Carinhas et al., 2009; Licari & Bailey, 1991; Micheloud et al., 2009). MOI is defined as the ratio between the number of infectious viral particles and the number of cells of the infected culture. The selection of the MOI determines the proportion of cells that become initially infected, as well as the distribution of infectious particles per cell. In cultures infected at high MOI (greater than 5 infectious units per cell), all the cells are infected synchronously. This prompts the viral replicative process to follow the same temporal pattern in all cells, with the emergence of a unique peak of viral progeny. On the other hand, when cultures are infected at lower values of MOI, only a proportion of the cellular population is infected initially. Thus, at least two cellular sub-populations will coexist in a culture infected at low MOI: infected and uninfected cells. Viral replication takes place immediately in initially infected cells, producing a viral progeny after one generation time. Uninfected cells, on the other hand, proliferate and will be infected later with the viral progeny of the initially infected cells. An important difference is that in cultures infected synchronously, viral replication occurs in cells that are in similar physiological state and subjected to similar environmental conditions, while in asynchronously infected ones viral replication takes place through successive rounds of infection, in cells under different physiological states and subjected to different environmental conditions. The importance of this difference is that the replicative capacity of insect cells varies, depending on both their physiological condition and the

baculovirus stocks necessary to infect large scale cultures of insect cells.

#### **6.1 BVs: the viral inoculums in insect cell cultures**

The baculovirus inoculum is composed by BVs that are added to the insect cell culture at the time of infection. The quality of the seed virus is a critical factor to determine the quality of the final product, the occlusion bodies, as well as the process productivity. First, the selected strain of virus should be virulent for the insect to be controlled. It should be also capable to replicate in the cell culture, yielding a high productivity of OBs. In addition, OBs produced from that inoculums should have a high biological activity. To meet these requisites, the viral inoculums should be free of genomic variants capable to reduce either the product yield or the biological activity of OBs. Two main types of genomic variants of baculovirus capable to reduce the OBs yield and biological activity have been described: the "few polyhedra" (FP) phenotype and the defective interfering particles (DIPs).

The mutations responsible for the FP phenotype, often associated with the inactivation of the gene 25k, are expressed through the following features: reduced yield of OBs, reduced content of OVs per occlusion body, reduced biological activity of OBs and increased yield of BVs (Beames & Summers, 1989; Harrison & Summers, 1995). The emergence of the FP phenotype is responsible for a sharp drop of the final yields of OBs, as well as its biological activity (Lua et al., 2002). Once emerged, the population of FP mutants tends to enrich through successive passages in insect cell cultures, due to its increased capability to produce BVs.

Fig. 3. Schematic representation of OBs insecticidal baculovirus production process in insect cell cultures. MOI: multiplicity of infection; TOI: time of infection.

The DIPs are generated as consequence of genomic deletions that originate shorter viral genomes (Kool et al., 1990). The DIP genomes can not replicate autonomously, but they can do it with the help of complete genomes. The replication of the DIPs competes and interferes with the replication of complete genomes, and leads to progressive enrichment of the population of defective virus, at the expense of the population of wild-type virus (van Lier et al., 1990). The generation of DIPs in cell cultures infected with baculovirus is favored by conditions that increase the probability of homologous recombination, such as in infections at high multiplicity of infection. The proportion of DIPs in a viral population, as well as the proportion of FP mutants, increases with the number of passages in cell cultures, a phenomenon known as "passage effect"(Krell, 1996). This effect impairs the amplification of baculovirus stocks necessary to infect large scale cultures of insect cells.

The viral stock is the most expensive raw material in processes destined to the production of insecticidal OBs in insect cell cultures. Obtaining the stock for infecting the production reactor usually requires the amplification of the seed virus through successive rounds of infection in insect cell cultures at progressively larger scales (Rhodes, 1996). The optimization of the conditions of infection for the production of BVs would have a double beneficial effect on the whole production process: on the one side, it would allow to reduce the consumption of an expensive raw material and, on the other side, it would permit to reduce the steps of the seed virus amplification process, and therefore to reduce the probabilities to generate and propagate deleterious genomic variants. Despite its importance, the information available about the optimization of infection conditions for the production of the BV progeny of baculovirus is very scarce (Carinhas et al., 2009).

The quality of baculovirus stocks may also be affected by reduced infectivity in relation to the total amount of viral particles (Dee & Shuler, 1997). Although several causes have been proposed to explain this phenomenon, the inactivation of BVs could be an important detrimental factor, especially in stocks prepared in serum-free media (Jorio et al., 2006). An optimized management of the preservation of serum-free baculovirus stocks could have an important impact on the feasibility of the scaling-up process.

#### **6.2 Parameters of infection**

140 Insecticides – Basic and Other Applications

The baculovirus inoculum is composed by BVs that are added to the insect cell culture at the time of infection. The quality of the seed virus is a critical factor to determine the quality of the final product, the occlusion bodies, as well as the process productivity. First, the selected strain of virus should be virulent for the insect to be controlled. It should be also capable to replicate in the cell culture, yielding a high productivity of OBs. In addition, OBs produced from that inoculums should have a high biological activity. To meet these requisites, the viral inoculums should be free of genomic variants capable to reduce either the product yield or the biological activity of OBs. Two main types of genomic variants of baculovirus capable to reduce the OBs yield and biological activity have been described: the "few

The mutations responsible for the FP phenotype, often associated with the inactivation of the gene 25k, are expressed through the following features: reduced yield of OBs, reduced content of OVs per occlusion body, reduced biological activity of OBs and increased yield of BVs (Beames & Summers, 1989; Harrison & Summers, 1995). The emergence of the FP phenotype is responsible for a sharp drop of the final yields of OBs, as well as its biological activity (Lua et al., 2002). Once emerged, the population of FP mutants tends to enrich through successive passages in insect cell cultures, due to its increased capability to produce

Fig. 3. Schematic representation of OBs insecticidal baculovirus production process in insect

The DIPs are generated as consequence of genomic deletions that originate shorter viral genomes (Kool et al., 1990). The DIP genomes can not replicate autonomously, but they can do it with the help of complete genomes. The replication of the DIPs competes and interferes with the replication of complete genomes, and leads to progressive enrichment of the

cell cultures. MOI: multiplicity of infection; TOI: time of infection.

polyhedra" (FP) phenotype and the defective interfering particles (DIPs).

**6.1 BVs: the viral inoculums in insect cell cultures** 

BVs.

The fate of a baculovirus infection in an insect cell culture is strongly dependent on the selection of the multiplicity of the infection (MOI) and the time of infection (TOI) (Carinhas et al., 2009; Licari & Bailey, 1991; Micheloud et al., 2009). MOI is defined as the ratio between the number of infectious viral particles and the number of cells of the infected culture. The selection of the MOI determines the proportion of cells that become initially infected, as well as the distribution of infectious particles per cell. In cultures infected at high MOI (greater than 5 infectious units per cell), all the cells are infected synchronously. This prompts the viral replicative process to follow the same temporal pattern in all cells, with the emergence of a unique peak of viral progeny. On the other hand, when cultures are infected at lower values of MOI, only a proportion of the cellular population is infected initially. Thus, at least two cellular sub-populations will coexist in a culture infected at low MOI: infected and uninfected cells. Viral replication takes place immediately in initially infected cells, producing a viral progeny after one generation time. Uninfected cells, on the other hand, proliferate and will be infected later with the viral progeny of the initially infected cells. An important difference is that in cultures infected synchronously, viral replication occurs in cells that are in similar physiological state and subjected to similar environmental conditions, while in asynchronously infected ones viral replication takes place through successive rounds of infection, in cells under different physiological states and subjected to different environmental conditions. The importance of this difference is that the replicative capacity of insect cells varies, depending on both their physiological condition and the quality of the culture medium at the time that they are actually infected.

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 143

The production in culture systems that operate continuously appears to be an attractive option to reduce the operation costs in fermentation processes. Given its lytic nature, a continuous process for baculovirus production should involve the use of at least two cultivation devices, one for cellular propagation and a second one for virus infection and replication. The first reactor is continuously fed with fresh medium, and the viral product is continuously harvested from the second reactor, which is in turn fed with cell culture from the first reactor, also in a continuous way and at the same rate. Although it has been demonstrated that is possible to operate this kind of continuous process to produce baculovirus OBs by a limited time, the viral yield is affected rapidly by the passage effect,

The feasibility of a production process for an insecticidal baculovirus will finally reside on the yield and the quality of viral occlusion bodies. Rhodes (1996), through a detailed economic analysis of an *in vitro* process for production of an insecticidal baculovirus, has established that the limit yield to reach the economical feasibility should be at least 2 x 1011 OBs.L-1. More recently, Nguyen et al. (2011) have argued that the minimum volumetric yield for an economically feasible process of production of HaSNPV in insect cell cultures should be 40 times higher. The OBs yields effectively reached for several baculoviruses in serum-free cultures of different cell lines, according to the available information, ranged from 1 x 109 to 3 x 1011 OBsL-1, with cell specific yields ranging from 3 to 700 OBs per cell (Chakraborty et al., 1999; Gioria et al., 2006; Lua & Reid, 2003; McKenna et al., 1997;

The insecticidal potency of baculovirus occlusion bodies that are produced in insect cell cultures is a controversial topic. Some papers have shown that occlusion bodies produced in infected insect cell cultures are less potent than polyhedra produced by infection of insect larvae (Chakraborty et al., 1999; McKenna et al., 1997). However, this reduced activity may not be an intrinsic characteristic of the occlusion bodies produced in cell cultures, but related to the extraction method used. Extraction with a solution of sodium dodecyl sulfate (SDS), a widely used procedure for releasing the occlusion bodies accumulated in the nuclei of infected cells, alters the structure of the polyhedron envelope and would reduce the content of occluded virus per polyhedron, and thus its insecticidal ability (Lua et al., 2003). Consequently, the extraction with SDS should be avoided for the purpose of preserving the quality of occlusion bodies. On the other hand, the occlusion bodies produced by infection in larvae contain an alkaline protease that is not codified in the viral genome, and that would be incorporated from the tissues of the infected insect. This protease, which is not present in the occlusion bodies produced in cell cultures, could be an additional factor of virulence, accelerating the dissolution of occlusion bodies in the insect midgut and thus contributing to increase its biological activity (Rohrmann, 2011). Finally, it has been also reported that the composition of the culture medium could affect the activity of occlusion

Although the production of insecticidal baculoviruses in insect cell cultures has been proposed as an alternative to overcome the limitations of the processes *in vivo*, so far no process *in vitro* could be even implemented on an industrial scale, and occlusion bodies are

invalidating this strategy as an effective approach (van Lier et al, 1990).

**6.4 Product yield and quality** 

Micheloud et al., 2009; Rodas et al., 2005).

bodies, but the causes are unknown (Pedrini et al, 2006).

**7. Scaling-up: limitations and possibilities** 

One factor that has contributed strongly to limiting the development of feasible production processes of insecticidal baculoviruses in insect cell cultures is the so called "cell density effect" (Wood et al., 1982). This phenomenon is characterized by the reduction of the intrinsic ability of insect cells to replicate baculovirus as the infection is delayed in time, and the cell density increases. The phenomenon has been observed also for recombinant proteins produced by genetically modified baculovirus. Several causes have been proposed to explain the "cell density effect", including nutrient limitation (Bédard et al., 1994), accumulation of toxic by-products (Taticek & Shuler, 1997), autocrine factors and cell cycle distribution (Braunagel et al., 1998; Calles et al., 2006), and inhibition of the central energy metabolism (Bernal et al., 2009), among others, but the causes remain to be identified. The cell density effect, that could be observed in several insect cell lines infected with different baculoviruses, both wild-type and recombinant, can be overcome, at least partially in Sf9 cell cultures by adopting alternative strategies for cell culture and viral infection, such as fedbatch or perfusion, as will be explained hereinafter.

#### **6.3 Operation strategy**

The typical strategy for the production of viruses in cultured animal cells is the infection of batch cultures. This strategy implies that a cell culture is cultivated in a proper medium up to reach the desired cellular concentration, when it is added the viral inoculums and the infection is allowed to progress without ulterior modification of the system until the harvest of the product is made. The batch production of insecticidal baculoviruses can be easily implemented, and it is possible to obtain high yields of occlusion bodies (Lua & Reid, 2003; Micheloud et al., 2009; Rodas et al., 2005). However, the cell density effect is a strong limitation to reach economically significant yields of virus, so that the MOI and the time of infection should be optimized to avoid an increase in cell density of infected cultures. This limitation could be partially overcome through the medium exchange before infection (Bédard et al., 2004). Although this strategy is feasible for working at low scale in shakenflasks or spinner-flasks, it is difficult to implement at larger scale.

Fed-batch is an alternative operation strategy to overcome the cell density effect in cultures of the *Spodoptera frugiperda* cell lines. Concentrated solutions containing the more demanded nutrients (glucose, glutamine and other amino acids, yeast extract and lipids´ emulsion) were added to high density cell cultures for obtaining high yields of recombinant proteins and occlusion bodies (Bédard et al., 1994; Elias et al., 2000). The main advantage of this strategy resides in its technological simplicity, which makes it proper to be implemented in both low and large scale. However, the use of this kind of operation is limited to insect cell lines that do not accumulate toxic by-products, such as IPLB-Sf21 and Sf9. For other, cell lines, like BTI-TN-5B1-4 and saUFL-AG-286, fed-batch would not be a suitable strategy, since ammonia accumulation could be the cause of inhibition of viral replication.

The perfusion strategy, that implies the continuous removal of spent medium and its replacement by fresh medium with retention of the cell mass inside the cultivation device, has been also used. It has been employed to obtain high yields of recombinant proteins in insect cell cultures of high density, infected with genetically modified baculoviruses (Chico & Jäger, 2000). The advantage of this strategy with regard to fed-batch resides in the possibility to use it with cell lines that accumulate toxic by products, which are continuously removed with the spent medium. However, its implementation requires sophisticated devices to remove spent cell-free medium, making it unlikely use in the development of economically feasible processes for the production of insecticidal baculoviruses in large scale.

The production in culture systems that operate continuously appears to be an attractive option to reduce the operation costs in fermentation processes. Given its lytic nature, a continuous process for baculovirus production should involve the use of at least two cultivation devices, one for cellular propagation and a second one for virus infection and replication. The first reactor is continuously fed with fresh medium, and the viral product is continuously harvested from the second reactor, which is in turn fed with cell culture from the first reactor, also in a continuous way and at the same rate. Although it has been demonstrated that is possible to operate this kind of continuous process to produce baculovirus OBs by a limited time, the viral yield is affected rapidly by the passage effect, invalidating this strategy as an effective approach (van Lier et al, 1990).

#### **6.4 Product yield and quality**

142 Insecticides – Basic and Other Applications

One factor that has contributed strongly to limiting the development of feasible production processes of insecticidal baculoviruses in insect cell cultures is the so called "cell density effect" (Wood et al., 1982). This phenomenon is characterized by the reduction of the intrinsic ability of insect cells to replicate baculovirus as the infection is delayed in time, and the cell density increases. The phenomenon has been observed also for recombinant proteins produced by genetically modified baculovirus. Several causes have been proposed to explain the "cell density effect", including nutrient limitation (Bédard et al., 1994), accumulation of toxic by-products (Taticek & Shuler, 1997), autocrine factors and cell cycle distribution (Braunagel et al., 1998; Calles et al., 2006), and inhibition of the central energy metabolism (Bernal et al., 2009), among others, but the causes remain to be identified. The cell density effect, that could be observed in several insect cell lines infected with different baculoviruses, both wild-type and recombinant, can be overcome, at least partially in Sf9 cell cultures by adopting alternative strategies for cell culture and viral infection, such as fed-

The typical strategy for the production of viruses in cultured animal cells is the infection of batch cultures. This strategy implies that a cell culture is cultivated in a proper medium up to reach the desired cellular concentration, when it is added the viral inoculums and the infection is allowed to progress without ulterior modification of the system until the harvest of the product is made. The batch production of insecticidal baculoviruses can be easily implemented, and it is possible to obtain high yields of occlusion bodies (Lua & Reid, 2003; Micheloud et al., 2009; Rodas et al., 2005). However, the cell density effect is a strong limitation to reach economically significant yields of virus, so that the MOI and the time of infection should be optimized to avoid an increase in cell density of infected cultures. This limitation could be partially overcome through the medium exchange before infection (Bédard et al., 2004). Although this strategy is feasible for working at low scale in shaken-

Fed-batch is an alternative operation strategy to overcome the cell density effect in cultures of the *Spodoptera frugiperda* cell lines. Concentrated solutions containing the more demanded nutrients (glucose, glutamine and other amino acids, yeast extract and lipids´ emulsion) were added to high density cell cultures for obtaining high yields of recombinant proteins and occlusion bodies (Bédard et al., 1994; Elias et al., 2000). The main advantage of this strategy resides in its technological simplicity, which makes it proper to be implemented in both low and large scale. However, the use of this kind of operation is limited to insect cell lines that do not accumulate toxic by-products, such as IPLB-Sf21 and Sf9. For other, cell lines, like BTI-TN-5B1-4 and saUFL-AG-286, fed-batch would not be a suitable strategy,

The perfusion strategy, that implies the continuous removal of spent medium and its replacement by fresh medium with retention of the cell mass inside the cultivation device, has been also used. It has been employed to obtain high yields of recombinant proteins in insect cell cultures of high density, infected with genetically modified baculoviruses (Chico & Jäger, 2000). The advantage of this strategy with regard to fed-batch resides in the possibility to use it with cell lines that accumulate toxic by products, which are continuously removed with the spent medium. However, its implementation requires sophisticated devices to remove spent cell-free medium, making it unlikely use in the development of economically feasible

since ammonia accumulation could be the cause of inhibition of viral replication.

processes for the production of insecticidal baculoviruses in large scale.

batch or perfusion, as will be explained hereinafter.

flasks or spinner-flasks, it is difficult to implement at larger scale.

**6.3 Operation strategy** 

The feasibility of a production process for an insecticidal baculovirus will finally reside on the yield and the quality of viral occlusion bodies. Rhodes (1996), through a detailed economic analysis of an *in vitro* process for production of an insecticidal baculovirus, has established that the limit yield to reach the economical feasibility should be at least 2 x 1011 OBs.L-1. More recently, Nguyen et al. (2011) have argued that the minimum volumetric yield for an economically feasible process of production of HaSNPV in insect cell cultures should be 40 times higher. The OBs yields effectively reached for several baculoviruses in serum-free cultures of different cell lines, according to the available information, ranged from 1 x 109 to 3 x 1011 OBsL-1, with cell specific yields ranging from 3 to 700 OBs per cell (Chakraborty et al., 1999; Gioria et al., 2006; Lua & Reid, 2003; McKenna et al., 1997; Micheloud et al., 2009; Rodas et al., 2005).

The insecticidal potency of baculovirus occlusion bodies that are produced in insect cell cultures is a controversial topic. Some papers have shown that occlusion bodies produced in infected insect cell cultures are less potent than polyhedra produced by infection of insect larvae (Chakraborty et al., 1999; McKenna et al., 1997). However, this reduced activity may not be an intrinsic characteristic of the occlusion bodies produced in cell cultures, but related to the extraction method used. Extraction with a solution of sodium dodecyl sulfate (SDS), a widely used procedure for releasing the occlusion bodies accumulated in the nuclei of infected cells, alters the structure of the polyhedron envelope and would reduce the content of occluded virus per polyhedron, and thus its insecticidal ability (Lua et al., 2003). Consequently, the extraction with SDS should be avoided for the purpose of preserving the quality of occlusion bodies. On the other hand, the occlusion bodies produced by infection in larvae contain an alkaline protease that is not codified in the viral genome, and that would be incorporated from the tissues of the infected insect. This protease, which is not present in the occlusion bodies produced in cell cultures, could be an additional factor of virulence, accelerating the dissolution of occlusion bodies in the insect midgut and thus contributing to increase its biological activity (Rohrmann, 2011). Finally, it has been also reported that the composition of the culture medium could affect the activity of occlusion bodies, but the causes are unknown (Pedrini et al, 2006).

#### **7. Scaling-up: limitations and possibilities**

Although the production of insecticidal baculoviruses in insect cell cultures has been proposed as an alternative to overcome the limitations of the processes *in vivo*, so far no process *in vitro* could be even implemented on an industrial scale, and occlusion bodies are

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 145

cultures at high density, could also be an alternative strategy to increase the yield of occlusion bodies. A deeper understanding of the causes that lead to the manifestations of the cell density effect could help to design more rational feeding schedules than those used to date, and thus increase the viral productivity. However, the usefulness of the fed-batch

A large-scale process to produce insecticidal baculovirus OBs in insect cell cultures requires the completion of successive steps of viral amplification in growing scale (Rhodes, 1996). OBs are the final product of the whole process, but BVs are the product for each of the intermediate steps of scaling. Despite the importance to improve the yield of BVs, few studies have systematically explored the optimization of the production of this viral progeny (Carinhas *et al*, 2009). The optimization of BVs production could help to reduce the number of scaling steps necessary to get the number of virions needed to feed the OBs production reactor, and therefore reduce scaling cost. Furthermore, the reduction in the number of stages of scaling would contribute to limiting the probability of emergence of unproductive viral variants, such as FP mutants and DIPs. The approach patented by Lua and Reid (2005), using occluded virions extracted from occlusion bodies as seed, could alleviate the need for viral inoculum at the beginning of the scaling-up process, but does not prevent the need to improve the yields of BVs in the later stages. Additionally, the improvement of the ratio infectious particles/total particles, through better BVs preservation, could mean significant savings in the demand for seed virus, and therefore a step towards the feasible scaling-up of the viral insecticide production process in insect cell

Baculoviruses are a group of arthropod-specific pathogens which have a significant potential to be used as safe and environmentally friendly insecticides in agriculture, horticulture and forestry. The replication of baculoviruses produces two viral progenies, budded and occluded viruses. The last are included into proteinaceous structures called occlusion bodies, which display insecticidal activity when ingested by susceptible insects. The current technology to produce insecticidal occlusion bodies is based on the viral infection of susceptible insects, but an alternative technology based on the viral replication in insect cell cultures could aid to overcome some of the limitations of the former. The insect cell line, the culture medium, the bioreactor, the virus, the infection parameters and the culture strategy are elements of the insect cell culture technology that must be optimized in order to develop i*n vitro* production processes for insecticidal baculoviruses. While it is now possible to grow insect cells in large-scale industrial reactors using serum-free media to produce high yields of occlusion bodies for several baculoviruses, the current technology is still insufficient to achieve economic feasibility. To do that, in the next future the efforts




strategy is restricted to cell lines that do not accumulate toxic by-products.

cultures.

**8. Concluding remarks and perspectives** 

should be mainly orientated:

insect cell cultures;

occlusion bodies;

responsible of the cell density effect;

still produced in infected insect larvae. Some factors that 25 years ago have hindered the development of large-scale production processes for insecticidal baculoviruses in insect cell cultures, such as the sensitivity of insect cells to the stresses linked to the mechanical agitation in stirred tank reactors and to the bubble rupture in sparged bioreactors, have been resolved and several cell lines can be cultivated today in industrial bioreactors of large volume to produce occlusion bodies or recombinant proteins. However, other factors that still limit the development of feasible processes have not yet been satisfactorily resolved, and will be reviewed below.

Obtaining a cell line with relevant technological properties and with the ability to replicate the virus at a high yield of OBs, is a requirement to develop a feasible process for the production of an insecticidal baculovirus in insect cell cultures. Besides Sf9 and BTI-TN-5B1- 4, there are few cell lines that fulfill these requisites. The cell line BCIRL-HZ-AM1, used to produce HaSNPV, is capable to grow in suspension cultures in a low-cost serum-free medium in stirred tank reactors. BCIRL-HZ-AM1 cells can produce high specific yields of HaSNPV OBs in infected cultures (Lua & Reid, 2003), but its ability to produce high yields of BVs, a property that is important for the scaling-up, is more limited (Pedrini et al., 2011). The cell line saUFL-AG-286, of election to produce AgMNPV, can generate high specific yields of OBs in serum-free suspension cultures, but the production of OBs is strongly inhibited at cell densities higher 8x105 cells mL-1, thus limiting the possibility to reach very high volumetric yields of OBs (Micheloud et al., 2009). As these cell lines are heterogeneous, the isolation of cell clones with improved ability to produce baculovirus OBs appears to be a reliable possibility to enhance the productivity of viral insecticides (Nguyen et al., 2011; Pasumarthy & Murhammer, 1994). For the production of other insecticidal baculoviruses will be necessary to establish new cell lines, obtained preferably from tissues of their respective target insects.

Another requisite that must be resolved before confronting the scaling-up of an insecticide baculovirus production process is the development of a low cost serum-free culture medium for the selected cell line. It has been indicated that the cost of the culture medium for an economically feasible process should not be higher than U\$S 2.5 per liter (Rhodes et al., 1996), or it even should be lower than U\$S 1 (Gong et al., 1997). Commercial serum-free media for Sf9 and BTI-TN-5B1-4 cells are sold at prices that are 30 times greater, and therefore are not useful for producing insecticidal baculoviruses at industrial scale. Besides, the cost of media specifically developed for producing insecticidal baculoviruses are yet above the acceptable limit for an economically feasible process. The rational approach to further reduce the cost of culture media for insect cell cultures is the simplification of the chemical composition, based on the deep knowledge of the nutritional demands and metabolism of insect cells, both uninfected and infected. However, most cell lines have not been sufficiently characterized as to progress towards a simplification of the composition of the culture medium. A more empirical approach to reduce the cost of the culture medium is the replacement of costly ingredients, such as amino acids and lipids, by optimized mixtures of raw materials of lower cost such as protein hydrolysates and cooking oil.

The usual strategy to produce baculovirus occlusion bodies in insect cell cultures has been the infection of batch cultures. However, the possibility to obtain high volumetric yields of viral OBs in batch cultures is impaired by the "cell density effect". Whenever possible, the adoption of alternative strategies of infection could be a way to overcome the cell density effect and thus improve the viral productivity. The fed-batch culture, which has proven to be a feasible alternative to increase the yield of recombinant proteins and BVs in Sf9 cell cultures at high density, could also be an alternative strategy to increase the yield of occlusion bodies. A deeper understanding of the causes that lead to the manifestations of the cell density effect could help to design more rational feeding schedules than those used to date, and thus increase the viral productivity. However, the usefulness of the fed-batch strategy is restricted to cell lines that do not accumulate toxic by-products.

A large-scale process to produce insecticidal baculovirus OBs in insect cell cultures requires the completion of successive steps of viral amplification in growing scale (Rhodes, 1996). OBs are the final product of the whole process, but BVs are the product for each of the intermediate steps of scaling. Despite the importance to improve the yield of BVs, few studies have systematically explored the optimization of the production of this viral progeny (Carinhas *et al*, 2009). The optimization of BVs production could help to reduce the number of scaling steps necessary to get the number of virions needed to feed the OBs production reactor, and therefore reduce scaling cost. Furthermore, the reduction in the number of stages of scaling would contribute to limiting the probability of emergence of unproductive viral variants, such as FP mutants and DIPs. The approach patented by Lua and Reid (2005), using occluded virions extracted from occlusion bodies as seed, could alleviate the need for viral inoculum at the beginning of the scaling-up process, but does not prevent the need to improve the yields of BVs in the later stages. Additionally, the improvement of the ratio infectious particles/total particles, through better BVs preservation, could mean significant savings in the demand for seed virus, and therefore a step towards the feasible scaling-up of the viral insecticide production process in insect cell cultures.

#### **8. Concluding remarks and perspectives**

144 Insecticides – Basic and Other Applications

still produced in infected insect larvae. Some factors that 25 years ago have hindered the development of large-scale production processes for insecticidal baculoviruses in insect cell cultures, such as the sensitivity of insect cells to the stresses linked to the mechanical agitation in stirred tank reactors and to the bubble rupture in sparged bioreactors, have been resolved and several cell lines can be cultivated today in industrial bioreactors of large volume to produce occlusion bodies or recombinant proteins. However, other factors that still limit the development of feasible processes have not yet been satisfactorily resolved,

Obtaining a cell line with relevant technological properties and with the ability to replicate the virus at a high yield of OBs, is a requirement to develop a feasible process for the production of an insecticidal baculovirus in insect cell cultures. Besides Sf9 and BTI-TN-5B1- 4, there are few cell lines that fulfill these requisites. The cell line BCIRL-HZ-AM1, used to produce HaSNPV, is capable to grow in suspension cultures in a low-cost serum-free medium in stirred tank reactors. BCIRL-HZ-AM1 cells can produce high specific yields of HaSNPV OBs in infected cultures (Lua & Reid, 2003), but its ability to produce high yields of BVs, a property that is important for the scaling-up, is more limited (Pedrini et al., 2011). The cell line saUFL-AG-286, of election to produce AgMNPV, can generate high specific yields of OBs in serum-free suspension cultures, but the production of OBs is strongly inhibited at cell densities higher 8x105 cells mL-1, thus limiting the possibility to reach very high volumetric yields of OBs (Micheloud et al., 2009). As these cell lines are heterogeneous, the isolation of cell clones with improved ability to produce baculovirus OBs appears to be a reliable possibility to enhance the productivity of viral insecticides (Nguyen et al., 2011; Pasumarthy & Murhammer, 1994). For the production of other insecticidal baculoviruses will be necessary to establish new cell lines, obtained preferably from tissues of their

Another requisite that must be resolved before confronting the scaling-up of an insecticide baculovirus production process is the development of a low cost serum-free culture medium for the selected cell line. It has been indicated that the cost of the culture medium for an economically feasible process should not be higher than U\$S 2.5 per liter (Rhodes et al., 1996), or it even should be lower than U\$S 1 (Gong et al., 1997). Commercial serum-free media for Sf9 and BTI-TN-5B1-4 cells are sold at prices that are 30 times greater, and therefore are not useful for producing insecticidal baculoviruses at industrial scale. Besides, the cost of media specifically developed for producing insecticidal baculoviruses are yet above the acceptable limit for an economically feasible process. The rational approach to further reduce the cost of culture media for insect cell cultures is the simplification of the chemical composition, based on the deep knowledge of the nutritional demands and metabolism of insect cells, both uninfected and infected. However, most cell lines have not been sufficiently characterized as to progress towards a simplification of the composition of the culture medium. A more empirical approach to reduce the cost of the culture medium is the replacement of costly ingredients, such as amino acids and lipids, by optimized mixtures

The usual strategy to produce baculovirus occlusion bodies in insect cell cultures has been the infection of batch cultures. However, the possibility to obtain high volumetric yields of viral OBs in batch cultures is impaired by the "cell density effect". Whenever possible, the adoption of alternative strategies of infection could be a way to overcome the cell density effect and thus improve the viral productivity. The fed-batch culture, which has proven to be a feasible alternative to increase the yield of recombinant proteins and BVs in Sf9 cell

of raw materials of lower cost such as protein hydrolysates and cooking oil.

and will be reviewed below.

respective target insects.

Baculoviruses are a group of arthropod-specific pathogens which have a significant potential to be used as safe and environmentally friendly insecticides in agriculture, horticulture and forestry. The replication of baculoviruses produces two viral progenies, budded and occluded viruses. The last are included into proteinaceous structures called occlusion bodies, which display insecticidal activity when ingested by susceptible insects. The current technology to produce insecticidal occlusion bodies is based on the viral infection of susceptible insects, but an alternative technology based on the viral replication in insect cell cultures could aid to overcome some of the limitations of the former. The insect cell line, the culture medium, the bioreactor, the virus, the infection parameters and the culture strategy are elements of the insect cell culture technology that must be optimized in order to develop i*n vitro* production processes for insecticidal baculoviruses. While it is now possible to grow insect cells in large-scale industrial reactors using serum-free media to produce high yields of occlusion bodies for several baculoviruses, the current technology is still insufficient to achieve economic feasibility. To do that, in the next future the efforts should be mainly orientated:


Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 147

Carinhas N., Bernal, V., Yokomizo, A.Y., Carrondo, M.T.J., Oliveira, R. & Alves, P.M.

Chakraborty, S., Monsour, C., Teakle, R. & Reid, S. Yield, biological activity, and field

Chico, E. & Jäger, V. Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a

Claus, J.D., Remondetto, G.E., Guerrero, S.A., Demonte, A.M., Murguía, M. & Marcipar, A.J.

Cowger, N.L., O´Connor, K.C., Hammond, D.J., Lacks, D.J. & Navar, G.L. Characterization

*Biotechnology and Bioengineering*. Vol.64, (1999), ISSN 0006-3592, pp. 14-26. Cruz, P.E., Cuhna, A., Peixoto, C.C., Clemente, J., Moreira, J.L. & Carrondo, M.J.T.

Day, M.F & Grace, T.D.C. Culture of insect tissues. *Annual Review of Entomology*. Vol.4,

Dee, K.U. & Shuler, M.L. Optimization of an assay for baculovirus titer and design of

Drews, M., Doverskog, M., Öhman, L., Chapman, B.E., Jacobson, U., Kuchel, P.W. &

Elias, C.B., Zeiser, A., Bédard, C. & Kamen, A. Enhanced growth of Sf-9 cells to a máximum

Eriksson, U. & Häggström, L. Yeast extract from Express Five serum-free medium contains

Ferrance, J.P., Goel, A. & Ataai, M.M. Utilization of glucose and amino acids in insect cell

*Biotechnology Letters*. Vol.27, (2005), ISSN 0141-5492, pp. 1623-1627.

*and Bioengineering*. Vol.60, ISSN 0006-3592, pp. 408–418.

(2009), ISSN 1432-0614, pp. 1041-1049.

199-205.

0141-5492, pp. 1007-1012.

(1959), ISSN 00664170, pp. 17-38.

(1997), ISSN 8756-7938, pp. 14-24.

0168-1656, pp. 23-37.

0006-3592, pp. 381-388.

0168-1656, pp. 1-15.

Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange. *Applied Microbiology and Biotechnology*. Vol.81,

performance of a wild-type *Helicoverpa* nucleopolyhedrovirus produced in *H. zea* cell cultures. *Journal of Invertebrate Pathology*. Vol.73, (1999), ISSN 0022-2011, pp.

method to restore cell-specific beta-trace glycoprotein productivity at high cell density. *Biotechnology and Bioengineering*. Vol.70, ISSN 0006-3592, pp. 574-586. Chung, I.S. & Shuler, M.L. Effect of Trichoplusia ni BTI-Tn-5B1-4 cell density on human

secreted alkaline phosphatase production. *Biotechnology Letters*. Vol.15, (1993), ISSN

*Anticarsia gemmatalis* nuclear polyhedrosis virus replication in serum-free and serum-reduced insect cell cultures. *Journal of Biotechnology*. Vol.31, (1993), ISSN

of bimodal cell death of insect cells in a rotating-wall vessel and shaker-flasks.

Optimization of the production of virus-like particles in insect cells. *Biotechnology* 

regimens for the synchronous infection of insect cells. *Biotechnology Progress*. Vol.13,

Häggström, L. Pathways of glutamine metabolism in *Spodoptera frugiperda* (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR. *Journal of Biotechnology*. Vol.78, (2000), ISSN

density of 5.2 x 107 cells per mL and production of β-galactosidase ast high cell density by fed batch culture. *Biotechnology and Bioengineering*. Vol.68, (2000), ISSN

factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells.

cultures: Quantifying the metabolic flows within the primary pathways and


Only obtaining satisfactory solutions for these remaining problems will make possible to establish economically viable processes for the production of insecticidal baculoviruses in insect cell cultures on an industrial scale.

#### **9. References**



Only obtaining satisfactory solutions for these remaining problems will make possible to establish economically viable processes for the production of insecticidal baculoviruses in

Abe, T., Matsuura, Y. Host innate immune responses induced by baculovirus in mammals.

Barnes, D. & Sato, G. Serum-free cell culture: a unifying approach*. Cell*, Vol.22, (1980), ISSN

Beames, B. & Summers, M.D, .Location and nucleotide sequence of the 25K protein missing

Bédard, C., Kamen, A.A., Tom, R. & Massie, B. Maximization of recombinant protein yield

Benslimane, C., Elias, C.B., Hawari, J., Kamen A. Insights into the central metabolism of

Benz, G.A. (1986) Introduction: Historical Perspectives. In: *The Biology of Baculoviruses.* 

Black, B.C., Brennan, L.S., Dierks, P.M. & Gard, I.E. (1997). Commercialization of Baculoviral

Blissard, G.W. & Rohrmann, G.F. Baculovirus diversity and molecular biology. *Annual* 

Braunagel, S.C., Parr, R., Belyavskyi, M. & Summers, M.D. *Autographa californica* 

Calles, K., Erikson, U. & Häggström, L. Effect of conditioned medium factors on

*Review of Entomology*. Vol.35, (1990), ISSN 00664170, pp. 127–155.

Vol. 244, (1998), ISSN 0042-6822, pp. 195–211.

*Progress*. Vol.22, (2006), ISSN 8756-7938, pp. 653–659.

(Eds.), 1-36, CRC Press, ISBN 0849359872, Boca Raton, Florida, USA. Bernal, V., Carinhas, N., Yokomizo, A.Y., Carrondo, M.T.J. & Alves, P.M. Cell density effect

from baculovirus few polyhedra (FP) mutants. *Virology*. Vol.168, (1989), ISSSN

in the insect cell/baculovirus system by one-time addition of nutrients to high density batch cultures. *Cytotechnology*. Vol.15, (1994), ISSN 0920-9069, pp. 129–138. Bédard, C., Tom, R., Kamen, A. Growth, nutrient consumption and end-product

accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism. *Biotechnology Progress*. Vol. 9, (1993), ISSN 8756-7938,

Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies. *Biotechnology Progress*. Vol.21, (2005), ISSN 8756-7938, pp.

*Volume I: Biological Properties and Molecular Biology*, R. R. Granados & B.A. Federici,

in the baculovirus-insect cells system: a quantitative analysis of energetic metabolism. *Biotechnology and Bioengineering*. Vol.104, ISSN 0006-3592, pp. 162-180.

Insecticides. In: *The Baculoviruses*, L.K. Miller (Ed.), 341-397, Plenum Press, ISBN

*nucleopolyhedrovirus* infection results in Sf9 cell cycle arrest at G2/M phase. *Virology*.

productivity and cell physiology in *Trichoplusia ni* insect cell cultures. *Biotechnology* 

*Current Gene Therapy*. Vol.10, ( 2010), ISSN 15665232, pp. 226-231.

unproductive viral variants.

**9. References** 

insect cell cultures on an industrial scale.

0092-8674 , pp. 649-55.

0042-6822, pp. 344-53.

0306456419, New York.

pp. 615-624.

78-86.


Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 149

Krell, P. J. Passage effect of virus infection in insect cells. *Cytotechnology*. Vol.20, (1996), ISSN

Kurtti, T.J. & Munderloh, U.G. (1984) Mosquito cell culture. In*: Advances in Cell Culture,* 

Licari, P. & Bailey, J.E. Factors influencing recombinant protein yields in an insect cell-

Lua, L.H., Nielsen, L.K. & Reid, S. Sensitivity of Helicoverpa armigera nucleopoly-

Lua, L.H., Pedrini, M.R., Reid. S., Robertson, A. & Tribe, D.E. Phenotypic and genotypic

Luckow, V.L. & Summers, M.D. Trends in the development of baculovirus expression

Lynn, D.E. (2007). Available Lepidopteran Insect Cell Lines. In: *Baculovirus and Cell Expresion* 

Lynn, D.E., Dougherty, E.M., McClintock, J.T. & Loeb, M.(1988). Development of cell lines

Maiorella, B., Inlow, D., Shauger, A. & Harano, D. Large-scale insect cell culture for

Maranga, L., Cunha, A., Clemente, C., Cruz, P. & Carrondo, M.J.T. Scale-up of virus-like

McIntosh, A.H. & Ignoffo, C.M. Replication and infectivity of the single-embedded nuclear

McKenna, K.A., Shuler, M.L. & Granados, R.R. Increased virus production in suspension

Merchuk, J.C. Shear effects on suspended cells. *Advances in Biochemical Engineering and* 

*Invertebrate Pathology*. Vol. 37, (1981), ISSN 0022-2011, pp. 258-264.

*Protocols: Second Edition*. Murhammer, D.W. (Ed.), 117-137, Humana Press, ISBN

from various tissues of Lepidoptera. In: *Invertebrate and Fish Tissue Culture*. Y. Kuroda, E. Kurstak, & K. Maramorosch (Eds.), 239-242, Japan Scientific Societies

recombinant protein production. *Bio/Technology*. Vol.6, (1988), ISSN 0733-222X, pp.

particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity. *Journal of Biotechnology*. Vol.107, (2004),

polyhedrosis virus, baculovirus Heliothis, in homologous cell line. *Journal of* 

culture by a Trichoplusia ni cell line in serum-free media. *Biotechnology Progress*.

vectors. *Bio/Technology*, Vol.6, (1988), ISSN 0733-222X, pp. 47-55.

Press/Springer-Verlag, ISBN 9780387192086, Tokyo/Berlin.

*Vol.3*, K Maramorosch (Ed.), 259-302, Academic Press, ISBN 0120079038, New York,

baculovirus expression system: multiplicity of infection and intracellular protein degradation. *Biotechnology and Bioengineering*. Vol.37, (1991), ISSN 0006-3592, pp.

hedrovirus polyhedra to sodium dodecyl sulfate. *Biological Control*. Vol.26, (2003),

analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. *Journal of General Virology*. Vol.83, (2002), ISSN 0022-1317, pp. 945-955. Lua, L.H. & Reid, S. Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture. *Cytotechnology*, Vol.42, (2003), ISSN 0920-9069, pp: 109-120. Lua, L.H. & Reid, S. Method of producing baculovirus. PCT Nº WO2005/045014. Retrieved

0920-9069, pp. 125-137.

ISSN 1049-9644, pp. 57-67.

1588295370, Totowa, USA.

ISSN 0168-165 , pp. 55-64.

Vol. 13, (1997), ISSN 8756-7938, pp. 605-609.

*Biotechnology*.Vol. 44,.(1991), ISSN 0724-6145, pp. 65-95.

1406-1410.

from http://ip.com/pdf/patent/US7521219.pdf.

USA.

238-246.

medium development. *Biotechnology & Bioengineering*. Vol.42, (1993), ISSN 0006- 3592, pp. 697-707.


Gioria, V.V., Jäger, V. & Claus, J.D. Growth, metabolism and baculovirus production in

Grace, T.C. The prolonged growth and survival of ovarian tissue of the promethea moth

Grace, T.D. Establishment of four strains of cells from insect tissues grown in vitro. *Nature*.

Grace, T.D. Establishment of a line of cells from the silkworm *Bombyx mori*. *Nature*, Vol.216,

Granados, R.R. & Williams, K.A. (1986) In Vivo Replication of Baculoviruses. In: *The Biology* 

Harrison, R.L., Summers, M.D. Mutations in the Autographa californica multinucleocapsid

Huber, J. (1986) Use of Baculovirus in Pest Management Programs. In: *The Biology of* 

*Developmental Biology Animal*.Vol. 37, (2001), ISSN 1071-2690 , pp. 549-559. Jehle, J.A., Blissard, G.W., Bonning, B.C., Cory, J.S., Herniou, E.A., Rohrmann, G.F.,

Jorio, H., Tran, R. & Kamen, A. Stability of serum-free and purified baculovirus stocks under

Kamen, A.A., Tom, R.L., Caron, A.W., Chavarie, C., Massie, B & Archambault, J. Culture of

King, G.A.., Daugulis, A.J., Faulkner, P., Goosen, M.F.A. Recombinant beta-galactosidase

Kool, M., Voncken, J.W., van Lier, F.L.J., Tramper, J., Vlak, J.M. Detection and analysis of

interfering properties. *Virology*. Vol.183, (1990), ISSN 0042-6822, pp. 739-746. Kost, T.A. & Condreay, J.P. Recombinant baculoviruses as mammalian cell gene-delivery vectors. *Trends in Biotechnology*, Vol.20, (2002), ISSN 01677799, pp. 173-180.

3592, pp. 697-707.

USA.

ISSN 0920-9069, pp. 113-124.

0022-1295, pp. 1027-1034.

0304-8608, pp. 1257-1266.

Vol.38, (1991), ISSN 0006-3592, pp. 619-628.

*Progress*. Vol.8, (1992), ISSN 8756-7938, pp. 567-571.

pp. 319-325.

(1967), ISSN 0028-0836, p. 613.

Vol.195, (1962), ISSN 0028-0836, pp. 788-789.

*Virology*.Vol. 76, (1995), ISSN 0022-1317, pp. 1451-1459.

medium development. *Biotechnology & Bioengineering*. Vol.42, (1993), ISSN 0006-

suspension cultures of an *Anticarsia gemmatalis* cell line. *Cytotechnology*.Vol. 52,

(*Callosamia promethea*) *in vitro*. *Journal of General Physiology*. Vol.41, (1958), ISSN

*of Baculoviruses. Volume I: Biological Properties and Molecular Biology*, R. R. Granados & B.A. Federici, (Eds.), 89-108, CRC Press, ISBN 0849359872, Boca Raton, Florida,

nuclear polyhedrosis virus 25kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. *Journal of General* 

*Baculoviruses. Volume II: Practical Application for Insect Control*, R. R. Granados & B.A. Federici, (Eds.), 181-202, CRC Press, ISBN 0849359872, Boca Raton, Florida, USA. Ikonomou, L., Bastin, G., Schneider, Y.J. & Agathos, S.N. Design of an efficient medium for

insect cell growth and recombinant protein production. *In Vitro Cellular &* 

Theilmann, D.A., Thiem, S.M. & Vlak, .J.M. On the classification and nomenclature of baculoviruses: a proposal for revision. *Archives of Virology*, Vol.151, (2006), ISSN

various storage conditions. *Biotechnology Progress*. Vol.22, (1996), ISSN 8756-7938,

insect cells in a helical ribbon impeller bioreactor. *Biotechnology and Bioengineering*.

production in serum-free medium in a 14-liter airlift bioreactor. *Biotechnology* 

Autographa californica nuclear polyhedrosis virus mutants with defective


Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 151

Rodas, V., Marques, F., Honda, M., Soares, D., Jorge, S.; Antoniazzi, M., Medugno, C.,

Palomares, L.A. & Ramírez, O.T. The effect of dissolved oxygen tension and the utility of

Rohrmann, G.F. (2011). *Baculovirus Molecular Biology: Second Edition*. National Library of

Schlaeger, E-J. Medium design for insect cell culture. *Cytotechnology*. Vol.20, (1996), ISSN

Shi, X. & Jarvis, D.L. Protein N-glycosylation in the baculovirus-insect cell system. *Current* 

Sieburth, P., Maruniak, J. Growth characteristics of a continuous cell line from the

Stavroulakis, D.A., Kalogerakis, N. & Behie, L.A. Kinetic data for the BM-5 insect cell line in

Szewczyk, B., Hoyos-Carvajal, L., Paluszek, M., Skrzecz, I. & Lobo de Souza, M..

Taticek, R. & Shuler, M. Effect of elevated oxygen and glutamine levels on foreign protein

Trinh, K., García-Briones, M., Hink, F. & Chalmers, J. Quantification of damage to

van Beek, N. & Davis, D.C. Baculovirus insecticide production in insect larvae. *Methods in* 

van Lier, F.L., van den End, E.J., de Gooijer, C.D., Vlak, J.M. & Tramper, J. Continuous

Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., McCawley, P. The establishment of two cell

Visnovsky, G., Claus, J. & Merchuk, J.C. Airlift reactors as a tool for insect cells and

Wood, H.A., Johnston, L.B., Burand, J.P. Inhibition of Autographa californica nuclear

*Molecular Biology*, Vol.388, (2007), ISSN 1064-3745 , pp. 367-378.

*and Biotechnology*. Vol.33, (1990), ISSN 1432-0614, pp. 43-47.

*Cytotechnology*. Vol. 48, (2005), ISSN 0920-9069, pp. 27-39.

http://www.ncbi.nlm.nih.gov/books/NBK49500/

*Drug Targets*.Vol.8, ( 2007), ISSN 1389-4501, pp. 1116-1125.

9069, pp. 225–237.

0920-9069, pp. 57-70.

ISSN 0006-3592, pp. 116-126.

ISSN 07349750, pp. 143-160.

Vol.43, (1994), ISSN 0006-3592, pp. 37-45.

(1977), ISSN 0073-5655, pp. 213-177.

(1982), ISSN 0042-6822, pp. 245–254.

0327-0793, pp. 117-121.

152.

Castro, M., Ribeiro, B., Souza, M., Tonso, A. & Pereira, C. Cell culture derived AgMNPV bioinsecticide: biological constraints and bioprocess issues.

oxygen uptake rate in insect cell culture. *Cytotechnology*. Vol.22, (1996), ISSN 0920-

Medicine (US), National Center for Biotechnology Information, retrieved from

velvetbean caterpillar, *Anticarsia gemmatalis* Hübner (Lepidoptera: Noctuidae*). In Vitro Cellular and Developmental Biology*.Vol.24, (1988), ISSN 1071-2690, pp. 195-198.

repeated-batch suspension cultures. *Biotechnology and Bioengineering*. Vol.38, (1991),

Baculoviruses: re-emerging biopesticides. *Biotechnology Advances*, Vol. 24, (2006),

production at high cell densities using the insect cell-baculovirus expression system. *Biotechnology and Bioengineering*. Vol.54, (1997), ISSN 0006-3592, pp. 142–

suspended insect cells as a result of buble rupture. *Biotechnology and Bioengineering*.

production of baculovirus in a cascade of insect-cell reactors. *Applied Microbiology* 

lines from the insect *Spodoptera frugiperda* (Lepidoptera; Noctuidae). *In Vitro*. Vol.13,

baculovirus mass production*. Latin-America Applied Research*. Vol 33, (2003), ISSN

polyhedrosis virus in high-density Trichoplusia ni cell cultures*. Virology*. Vol.119,


Micheloud, G.A., Gioria, V.V., Pérez, G. & Claus, J.D. Production of occlusion bodies of

Mitsuhashi, J. (1989). Nutritional Requirements of Insect Cells *In Vitro*. In: *Invertebrate Cell* 

Mitsuhashi, J. & Goodwin, R.H. (1989) Serum-free culture of insect cells in vitro. In:

Moscardi, F. Assessment of the application of baculoviruses for control of Lepidoptera. *Annual Review of Entomology*, Vol. 44, (1999), ISSN 0066-4170, pp. 257-289. Neermann J, Wagner R. Comparative analysis of glucose and glutamine metabolism in

Öhman, L., Alarcón, M., Ljunggren, J., Ramqvist, A.K. & Häggström, L. (1996) Glutamine is

O´Reilly, D.R., Miller, L.K., Luckow, V.A. (1994). *Baculovirus Expression Vectors. A Laboratory Manual*. Oxford University Press, ISBN 0795091310, New York, USA. Pasarelli, A.L. Barriers to success: how baculoviruses establish efficient systemic infections.

Pasumarthy, M.K. & Murhammer, D.W. Clonal variation in the *Spodoptera frugiperda* IPLB-

Pedrini, M.R., Christian, P., Nielsen, L.K., Reid, S. & Chan, L.C. Importance of virus-medium

Pedrini, M.R., Reid, S., Nielsen, L.K. & Chan, L.C. Kinetic characterization of the group II

Nguyen, Q., Qi, Y.M, Wu, Y., Chan, L.C.L., Nielsen, L.K. & Reid, S. In vitro production of

Rhiel, M., Mitchell-Logean, C.M. & Murhammer, D.W. Comparison of *Trichoplusia ni* BTI-

Rhodes, D.J. Economics of baculovirus – insect cell production systems. *Cytotechnology*.

*Virological Methods*. Vol.136, (2006), ISSN 0166-0934, pp. 267-72.

266.

Raton, Florida, USA.

0141-5492, pp. 765-770.

7938, pp. 314-319.

3592, pp. 909-920.

0849343739, Boca Raton, Florida, USA.

*Physiology*. Vol.166, (1996), ISSN 0021-9541, pp. 152-169.

*Virology*, Vol. 411, (2011), ISSN 0042-6822, pp. 383-392.

*Progress*. Vol.27, (2011), ISSN 8756-7938, pp. 614-624.

Vol.175, (2011), ISSN 0166-0934, pp. 197-205.

Vol.20, (1996), ISSN 0920-9069, pp. 291-297.

*Anticarsia gemmatalis multiple nucleopolyhedrovirus* in serum-free suspension cultures of the saUFL-AG-286 cell line: influence of infection conditions and statistical optimization. *Journal of Virological Methods*. Vol.162, (2009), ISSN 0166-0934, pp 258-

*System Applications*. Mitsuhashi, J. (Ed.), 3-21, CRC Press, ISBN 0849343739, Boca

*Invertebrate Cell System Applications*. Mitsuhashi, J. (Ed.), 31-44, CRC Press, ISBN

transformed mammalian cell lines, insect and primary liver cells*. Journal of Cellular* 

not an essential amino acid for Sf-9 insect cells. *Biotechnology Letters*. Vol.18, ISSN

SF21-AE insect cell population. *Biotechnology Progress*. Vol. 10, (1994), ISSN 8756-

interactions on the biological activity of wild-type Heliothine nucleopolyhedroviruses propagated via suspension insect cell cultures. *Journal of* 

*Helicoverpa armigera nucleopolyhedrovirus* propagated in suspension cell cultures: Implications for development of a biopesticides production process. *Biotechnology* 

Helicoverpa baculovirus biopesticides—Automated selection of insect cell clones for manufacturing and systems biology studies*. Journal of Virological Methods*.

Tn-5B1-4 (High FiveTM) and *Spodoptera frugiperda* Sf-9 insect cell line metabolism in suspension cultures. *Biotechnology and Bioengineering*. Vol.55, (1997), ISSN 0006-


**8** 

**Factors Affecting Performance** 

Although baits have increased in popularity in recent years, the application of liquid termiticide to soil remains the most widely used method for protecting structures against subterranean termites (Anonymous, 2008). In addition to fast acting, repellent toxicants such as bifenthrin and other pyrethroids that act as barriers to termite movement, non-repellent, slower acting compounds including fipronil, imidacloprid and thiamethoxam are now among the preferred soil treatments. Delayed toxicity can provide opportunity for horizontal transfer of the active ingredient, potentially reducing termite activity (Remmen & Su, 2005; Shelton & Grace, 2003). While there is some evidence of colony suppression or elimination following perimeter treatments with imidacloprid (Parman & Vargo, 2010), other studies have shown that a reduction in activity occurs over only a small portion of a colony's foraging range, making it unlikely that soil treatments affect the overall termite population (Osbrink et al., 2005; Rust & Saran, 2006; Saran & Rust, 2007; Su, 2005). This limited potential for transfer emphasizes the importance of bioavailability of termiticides in

Failure of soil termiticide treatments is often related to factors other than the active ingredient (Su & Scheffrahn, 1990b). Efficacy and longevity of soil treatments varies greatly with application rate, soil properties (Gold et al., 1996; Su & Scheffrahn, 1990b), termite pressure (Jones, 1990), and application technique (Forschler, 1994; Su et al., 1995). Factors influencing the performance of soil termiticides can be grouped into those that determine toxicity, bioavailability, or persistence. Each of these factors is affected by properties of the termiticide and soil (Gold et al., 1996; Spomer et al., 2009; Wiltz, 2010). Although some generalizations can be made about relative toxicity of different termiticides, susceptibility differences occur among species and colonies (Beal & Smith, 1971; Osbrink & Lax, 2002). Termiticide rate and application technique influence both bioavailability and long-term persistence (Peterson, 2010). Termite population pressure and satellite nests can reduce availability of the toxicant. Finally, other environmental factors such as moisture, temperature, and microbial communities affect

Long-term studies evaluating chemicals as potential termiticides were initiated in the 1920's and 1930's (Randall & Doody, 1934), but it was not until after World War II that the

termiticide degradation (Baskaran et al. 1999, Saran & Kamble 2008).

**1. Introduction** 

soil over an extended period of time.

**2. Soil termiticides** 

**of Soil Termiticides** 

Beverly A. Wiltz

*USDA-ARS New Orleans, LA* 

*USA* 

Wyatt, G.R., Lougheed, T.C., Wyatt, S.S. The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm, Bombyx mori, and two other species. *Journal of General Physiology*, Vol.39, (1956), ISSN 0022-1295, pp. 853-868.

### **Factors Affecting Performance of Soil Termiticides**

Beverly A. Wiltz *USDA-ARS New Orleans, LA USA* 

#### **1. Introduction**

152 Insecticides – Basic and Other Applications

Wyatt, G.R., Lougheed, T.C., Wyatt, S.S. The chemistry of insect hemolymph; organic

components of the hemolymph of the silkworm, Bombyx mori, and two other species. *Journal of General Physiology*, Vol.39, (1956), ISSN 0022-1295, pp. 853-868.

> Although baits have increased in popularity in recent years, the application of liquid termiticide to soil remains the most widely used method for protecting structures against subterranean termites (Anonymous, 2008). In addition to fast acting, repellent toxicants such as bifenthrin and other pyrethroids that act as barriers to termite movement, non-repellent, slower acting compounds including fipronil, imidacloprid and thiamethoxam are now among the preferred soil treatments. Delayed toxicity can provide opportunity for horizontal transfer of the active ingredient, potentially reducing termite activity (Remmen & Su, 2005; Shelton & Grace, 2003). While there is some evidence of colony suppression or elimination following perimeter treatments with imidacloprid (Parman & Vargo, 2010), other studies have shown that a reduction in activity occurs over only a small portion of a colony's foraging range, making it unlikely that soil treatments affect the overall termite population (Osbrink et al., 2005; Rust & Saran, 2006; Saran & Rust, 2007; Su, 2005). This limited potential for transfer emphasizes the importance of bioavailability of termiticides in soil over an extended period of time.

> Failure of soil termiticide treatments is often related to factors other than the active ingredient (Su & Scheffrahn, 1990b). Efficacy and longevity of soil treatments varies greatly with application rate, soil properties (Gold et al., 1996; Su & Scheffrahn, 1990b), termite pressure (Jones, 1990), and application technique (Forschler, 1994; Su et al., 1995). Factors influencing the performance of soil termiticides can be grouped into those that determine toxicity, bioavailability, or persistence. Each of these factors is affected by properties of the termiticide and soil (Gold et al., 1996; Spomer et al., 2009; Wiltz, 2010). Although some generalizations can be made about relative toxicity of different termiticides, susceptibility differences occur among species and colonies (Beal & Smith, 1971; Osbrink & Lax, 2002). Termiticide rate and application technique influence both bioavailability and long-term persistence (Peterson, 2010). Termite population pressure and satellite nests can reduce availability of the toxicant. Finally, other environmental factors such as moisture, temperature, and microbial communities affect termiticide degradation (Baskaran et al. 1999, Saran & Kamble 2008).

#### **2. Soil termiticides**

Long-term studies evaluating chemicals as potential termiticides were initiated in the 1920's and 1930's (Randall & Doody, 1934), but it was not until after World War II that the

Factors Affecting Performance of Soil Termiticides 155

Saran and Rust (2007) found that *R. hesperus* tunneled through untreated sand and stopped near the interface of fipronil treated sand. There was little tunneling in the treated sand, but termites tunneled close enough to obtain a lethal dose of fipronil. To some extent, *C. formosanus* and *Reticulitermes flavipes* penetrated sand treated with 0 - 64ppm fipronil, indicating non-repellency, but complete penetration of the treated sand was prevented by high mortality (≥88% for *C. formosanus* and ≥89% for *R. flavipes* after 7 d) (Remmen & Su, 2005). While several studies conducted in small laboratory arenas have found high mortality in fipronil treatments, extended foraging arena assays demonstrated that fipronil barriers can split termite populations, with high mortality occurring close to the treatment site, but

Although imidacloprid is slow to induce mortality, mobility impairment occurs within hours of exposure (Thorne & Breisch, 2001). Imidacloprid is non-repellent (Remmen & Su, 2005), but this combination of delayed mortality and rapid mobility impairment results in limited movement of termites into treated barriers and limited mortality after 7d in close

Several studies have demonstrated differences in degradation rates among insecticides. Baker and Bellamy (2006) found that of the termiticides tested, the organophosphate, chlorpyrifos, degraded the quickest, while chloronicotinyls and pyrethroids degraded at slower rates. Horwood (2007) measured termiticide residues in a weathered sand: loam mixture, finding that bifenthrin and chlorfenapyr were more persistent than chlorpyrifos, fipronil, and imidacloprid. Horwood (2007) found that after 15 months, chlorpyrifos and fipronil concentrations at lower depths were little changed from the time of treatment, but

Because soil consists of a heterogeneous mixture of mineral and organic particles, it is difficult to predict the influence of soil type on termiticides. When soil conditions fall outside an optimum range, termiticides can be immobilized or adsorbed by the soil or

Laboratory studies have found interactions between soil and termiticide properties. Effects of clay (Henderson et al. 1998; Smith & Rust 1993) and organic carbon (Felsot & Lew 1989; Forschler & Townsend, 1996; Gold et al., 1996; Spomer et al., 2009) content on bioavailability to termites differ with termiticide. Termiticide effectiveness diminishes over time, especially on soils that pose bioavailability problems (Gold et al., 1996; Su et al., 1993; Tamashiro et al.,

Variation in soil properties, such as pH, clay and organic matter content, soil moisture, and electrolyte concentration, influence the adsorption and desorption characteristics of termiticides to soils. Of equal importance are the physical and chemical properties of the

Mobility is one of the most important factors in determining bioavailability and efficacy of a soil treatment. If a pesticide is too mobile, it fails to protect the structure, while increasing risk of groundwater contamination. However, if the chemical is too tightly bound to soil particles, bioavailability is limited. Mobility is affected by the pesticide's sorption, water solubility, and vapor pressure and by external influences that include soil properties,

there was a major reduction in imidacloprid concentration at all depths.

little mortality at distances >5 m (Su, 2005).

proximity to imidacloprid-treated sand.

**3. Soil-termiticide interactions** 

altered chemically to an inactive form.

toxicant, including concentration, pH, and solubility.

1987).

**3.1 Mobility** 

cyclodienes, a class of chemical compounds identified as highly effective termiticides, became commercially available (Ware, 2000). Pre-construction soil treatments with cyclodienes became the standard method of subterranean termite prevention from the late 1940s until 1988 (Lewis, 1980; Su & Scheffrahn, 1990b). The cyclodienes, particularly chlordane, were extremely efficacious and stable in soil, often protecting structures from subterranean termite infestation for several decades (Grace et al., 1993; Lenz et al., 1990; Su & Scheffrahn, 1990b).

Because of their residual longevity, questions were raised about the environmental impact of these chemicals (Lewis, 1980; Su & Scheffrahn, 1990a; Wood & Pierce, 1991). Chlordane and related chemicals were banned in most of the world in the 1970's and 1980's (Ware, 2000). However, they constitute a major environmental problem because of their high toxicity, persistence in the environment, and ability to bioaccumulate in the food chain and because they are still being used in certain countries for agricultural and public health purposes (Itawa et al., 1993; Ntow, 2005; Xue et al., 2006).

Following the loss of chlordane as a soil termiticide, the only termiticides available for use as soil barrier treatments were chlorpyrifos (an organophosphate) and several pyrethroids. The residual activity of chlorpyrifos was significantly shorter than that of the cyclodienes (Grace et al., 1993; Lenz et al., 1990). As a result of the Food Quality Protection Act of 1996, the U. S. Environmental Protection Agency (EPA) revised its risk assessment of chlorpyrifos and, in 2000, the use of chlorpyrifos as a soil termiticide was canceled (EPA, 2000).

With the loss of chlorpyrifos, pyrethroids were the primary weapon available for subterranean termite prevention. The pyrethroids are more persistent than chlorpyrifos, but less stable in the soil than the cyclodienes (Lenz et al., 1990; Pawson & Gold, 1996; Su & Scheffrahn, 1990b). Soil barriers composed of pyrethroids are more likely to fail than barriers composed of cyclodienes or chlorpyrifos (Forschler, 1994; Kard, 1999; Lenz et al., 1990; Su & Scheffrahn, 1990b; Su et al., 1993) because pyrethroids are repellant to subterranean termites (Rust & Smith, 1993; Su & Scheffrahn, 1990b; Su et al., 1993).

Beginning in 2000, several new nonrepellant soil termiticides appeared on the market: fipronil, a phenyl pyrazole (Aventis Corp., 2001), imidacloprid, a chloronicotinyl (Bayer Corp., 2000), and chlorfenapyr, a pyrrole (BASF Corp., 2001). Nonrepellant termiticides are an improvement over the pyrethroids because subterranean termites cannot detect gaps in the treatment and use them to gain access to structures (Potter & Hillery, 2001). Subterranean termites are unable to detect the termiticide and do not avoid soil that has been treated with them (Kuriachan & Gold, 1998). Chlorantraniliprole is a new termiticide belonging to the anthranilic diamide class of insecticides. It targets a unique receptor site, the ryanodine receptor, causing the release of stored calcium, resulting in loss of muscle control, cessation of feeding, and eventually death of the termite (Cordova et al., 2006). Unlike other soil termiticides, chlorantraniliprole has no known health effects to humans and no personal protective equipment is required for application (Dupont, 2010). Also being developed for subterranean termite control is indoxacarb, an oxadiazine proinsecticide that is metabolically activated after entering the insect (Spomer et al. 2009; Wing et al., 2000).

A large amount of the variability in effectiveness of different soil treatments can be attributed to the termiticide itself. In a study evaluating *Coptotermes formosanus* mortality on treated soils, bifenthrin performed better than fipronil or chlorfenapyr (Wiltz 2010). Bifenthrin was also found to have the highest activity against *Reticulitermes hesperus* when compared with other pyrethroids (Smith & Rust, 1990).

cyclodienes, a class of chemical compounds identified as highly effective termiticides, became commercially available (Ware, 2000). Pre-construction soil treatments with cyclodienes became the standard method of subterranean termite prevention from the late 1940s until 1988 (Lewis, 1980; Su & Scheffrahn, 1990b). The cyclodienes, particularly chlordane, were extremely efficacious and stable in soil, often protecting structures from subterranean termite infestation for several decades (Grace et al., 1993; Lenz et al., 1990; Su

Because of their residual longevity, questions were raised about the environmental impact of these chemicals (Lewis, 1980; Su & Scheffrahn, 1990a; Wood & Pierce, 1991). Chlordane and related chemicals were banned in most of the world in the 1970's and 1980's (Ware, 2000). However, they constitute a major environmental problem because of their high toxicity, persistence in the environment, and ability to bioaccumulate in the food chain and because they are still being used in certain countries for agricultural and public health

Following the loss of chlordane as a soil termiticide, the only termiticides available for use as soil barrier treatments were chlorpyrifos (an organophosphate) and several pyrethroids. The residual activity of chlorpyrifos was significantly shorter than that of the cyclodienes (Grace et al., 1993; Lenz et al., 1990). As a result of the Food Quality Protection Act of 1996, the U. S. Environmental Protection Agency (EPA) revised its risk assessment of chlorpyrifos and, in

With the loss of chlorpyrifos, pyrethroids were the primary weapon available for subterranean termite prevention. The pyrethroids are more persistent than chlorpyrifos, but less stable in the soil than the cyclodienes (Lenz et al., 1990; Pawson & Gold, 1996; Su & Scheffrahn, 1990b). Soil barriers composed of pyrethroids are more likely to fail than barriers composed of cyclodienes or chlorpyrifos (Forschler, 1994; Kard, 1999; Lenz et al., 1990; Su & Scheffrahn, 1990b; Su et al., 1993) because pyrethroids are repellant to subterranean termites

Beginning in 2000, several new nonrepellant soil termiticides appeared on the market: fipronil, a phenyl pyrazole (Aventis Corp., 2001), imidacloprid, a chloronicotinyl (Bayer Corp., 2000), and chlorfenapyr, a pyrrole (BASF Corp., 2001). Nonrepellant termiticides are an improvement over the pyrethroids because subterranean termites cannot detect gaps in the treatment and use them to gain access to structures (Potter & Hillery, 2001). Subterranean termites are unable to detect the termiticide and do not avoid soil that has been treated with them (Kuriachan & Gold, 1998). Chlorantraniliprole is a new termiticide belonging to the anthranilic diamide class of insecticides. It targets a unique receptor site, the ryanodine receptor, causing the release of stored calcium, resulting in loss of muscle control, cessation of feeding, and eventually death of the termite (Cordova et al., 2006). Unlike other soil termiticides, chlorantraniliprole has no known health effects to humans and no personal protective equipment is required for application (Dupont, 2010). Also being developed for subterranean termite control is indoxacarb, an oxadiazine proinsecticide that is metabolically activated after entering the insect (Spomer et al. 2009;

A large amount of the variability in effectiveness of different soil treatments can be attributed to the termiticide itself. In a study evaluating *Coptotermes formosanus* mortality on treated soils, bifenthrin performed better than fipronil or chlorfenapyr (Wiltz 2010). Bifenthrin was also found to have the highest activity against *Reticulitermes hesperus* when

2000, the use of chlorpyrifos as a soil termiticide was canceled (EPA, 2000).

purposes (Itawa et al., 1993; Ntow, 2005; Xue et al., 2006).

(Rust & Smith, 1993; Su & Scheffrahn, 1990b; Su et al., 1993).

compared with other pyrethroids (Smith & Rust, 1990).

& Scheffrahn, 1990b).

Wing et al., 2000).

Saran and Rust (2007) found that *R. hesperus* tunneled through untreated sand and stopped near the interface of fipronil treated sand. There was little tunneling in the treated sand, but termites tunneled close enough to obtain a lethal dose of fipronil. To some extent, *C. formosanus* and *Reticulitermes flavipes* penetrated sand treated with 0 - 64ppm fipronil, indicating non-repellency, but complete penetration of the treated sand was prevented by high mortality (≥88% for *C. formosanus* and ≥89% for *R. flavipes* after 7 d) (Remmen & Su, 2005). While several studies conducted in small laboratory arenas have found high mortality in fipronil treatments, extended foraging arena assays demonstrated that fipronil barriers can split termite populations, with high mortality occurring close to the treatment site, but little mortality at distances >5 m (Su, 2005).

Although imidacloprid is slow to induce mortality, mobility impairment occurs within hours of exposure (Thorne & Breisch, 2001). Imidacloprid is non-repellent (Remmen & Su, 2005), but this combination of delayed mortality and rapid mobility impairment results in limited movement of termites into treated barriers and limited mortality after 7d in close proximity to imidacloprid-treated sand.

Several studies have demonstrated differences in degradation rates among insecticides. Baker and Bellamy (2006) found that of the termiticides tested, the organophosphate, chlorpyrifos, degraded the quickest, while chloronicotinyls and pyrethroids degraded at slower rates. Horwood (2007) measured termiticide residues in a weathered sand: loam mixture, finding that bifenthrin and chlorfenapyr were more persistent than chlorpyrifos, fipronil, and imidacloprid. Horwood (2007) found that after 15 months, chlorpyrifos and fipronil concentrations at lower depths were little changed from the time of treatment, but there was a major reduction in imidacloprid concentration at all depths.

#### **3. Soil-termiticide interactions**

Because soil consists of a heterogeneous mixture of mineral and organic particles, it is difficult to predict the influence of soil type on termiticides. When soil conditions fall outside an optimum range, termiticides can be immobilized or adsorbed by the soil or altered chemically to an inactive form.

Laboratory studies have found interactions between soil and termiticide properties. Effects of clay (Henderson et al. 1998; Smith & Rust 1993) and organic carbon (Felsot & Lew 1989; Forschler & Townsend, 1996; Gold et al., 1996; Spomer et al., 2009) content on bioavailability to termites differ with termiticide. Termiticide effectiveness diminishes over time, especially on soils that pose bioavailability problems (Gold et al., 1996; Su et al., 1993; Tamashiro et al., 1987).

Variation in soil properties, such as pH, clay and organic matter content, soil moisture, and electrolyte concentration, influence the adsorption and desorption characteristics of termiticides to soils. Of equal importance are the physical and chemical properties of the toxicant, including concentration, pH, and solubility.

#### **3.1 Mobility**

Mobility is one of the most important factors in determining bioavailability and efficacy of a soil treatment. If a pesticide is too mobile, it fails to protect the structure, while increasing risk of groundwater contamination. However, if the chemical is too tightly bound to soil particles, bioavailability is limited. Mobility is affected by the pesticide's sorption, water solubility, and vapor pressure and by external influences that include soil properties,

Factors Affecting Performance of Soil Termiticides 157

Because sorption coefficient values for the same pesticide vary widely with soil properties, reported values are not included in Table 1. However, Kd values are useful for comparing sorption of different chemicals to the same soil. The organic carbon sorption coefficient is a property of the pesticide and is independent of soil organic matter. The sorption coefficient and organic carbon sorption coefficient are related by

 Kd = Koc (%O.C.) (1) Where O.C. is the percentage organic carbon the soil contains. This relationship shows that as the organic fraction of soil increases, the distribution coefficient, Kd, increases. For this relationship to hold true, the chemical must be non-ionic because soil pH affects sorption of

As Table 1 illustrates, KOC values for a pesticide are not constant. Pesticide concentration affects adsorption (Kamble & Saran, 2005), but not to an extent that prevents comparison of relative mobility of different pesticides. For polar solutes, surfaces other than organic carbon can become important sorbents particularly when soils are low in organic matter (Cheung et

Another useful measure of potential pesticide mobility is the octanol - water partition coefficient (Kow). Kow is a measure of the hydrophobicity of an organic compound. The more hydrophobic a compound, the less soluble it is, therefore the more likely it will adsorb to soil particles (Bedient et al, 1994). To evaluate hydrophobicity, the organic solvent octanol is used as a surrogate for organic matter. The octanol-water partition coefficient is the ratio of the concentration of a chemical in octanol and in water at equilibrium and at a specified temperature. Kow is determined by adding a known amount of the pesticide to equal volumes of octanol and water. The coefficient is determined by calculating the concentration in the octanol phase compared to the concentration in the water phase. Kow values vary by several orders of magnitude and may be reported as either Kow or log Kow values. The octanol-water partition coefficient is correlated with water solubility; therefore, the water solubility of a substance can be used to estimate its

Water solubility describes the amount of pesticide that will dissolve in a known amount of water. Highly soluble pesticides are more likely to be moved by runoff or leaching. As with sorption parameters, solubility values are useful as a means of comparison, but actual values will vary with field conditions. Solubility is affected by temperature, water pH, and the presence of other chemicals. The solubility of a compound tends to be inversely

In addition to being adsorbed to soil or transported by water, pesticides can be volatilized. Pesticide volatilization from moist soil is described by the Henry's law constant (Kh). Kh is defined as the concentration of pesticide in air divided by the concentration in water. Kh can be measured experimentally or estimated by dividing the saturation vapor pressure of the compound by its solubility (Suntio et al., 1988). Like other sorption parameters, Kh is temperature dependent, but values are useful for comparing volatility of different compounds. Pesticides with higher Kh are more likely to volatilize from moist soil. Because sorption affects the amount of pesticide in the soil water, the tendency to volatilize from

moist soil depends on both the Henry's law constant and sorption coefficients.

the equation:

ionic sorbates.

al., 1979; Cox et al., 1998; Means et al., 1982).

octanol-water partition coefficient.

proportional to the amount of sorption that it can undergo.

weather, topography, and vegetation. Sorption describes the attraction between a chemical and soil, vegetation, or other surfaces. However, the term most often refers to the binding of a chemical to soil particles. Sorption is defined as the attraction of an aqueous species to the surface of a solid (Alley, 1993). The sorbing species, usually an organic compound, is called the sorbate, and the solid, usually soil, to which the sorbate is attracted is known as the sorbent. This attraction results from some form of bonding between the chemical and adsorption receptor sites on the solid. Several mechanisms may operate in a particular situation, including ionic attraction, hydrophobic attraction, and hydrogen bonding. For pesticides that are weak acids or bases, sorption is influenced by soil pH.

Sorption is also influenced by soil moisture, organic matter content, and texture. Pesticides are more readily sorbed onto dry soil because water competes with pesticides for binding sites in moist soil. More sorption occurs in soils made largely of clay and organic matter. Organic matter and clay particles have small particle size, large surface area, and high surface charge. Sand particles provide less surface area for sorption, making pesticides more likely to move away from the point of application.

Several parameters are used to describe a pesticide's sorption behavior in soils. Table 1 contains sorption parameters for selected chemicals currently and previously used as soil termiticides.

Two related measures of a pesticide's sorption are the sorption coefficient (Kd) and the soil organic carbon coefficient (KOC). These coefficients are ratios of adsorbed to dissolved pesticide for a specific soil (Kd) or for the organic carbon fraction of a soil (KOC). These values are useful for broadly discriminating between leaching classes of pesticides, but actual adsorption depends on many factors, including soil moisture, temperature, soil pH, and type of organic matter (Wauchope et al., 2002).


Table 1. Soil sorption parameters of selected soil termiticides.

weather, topography, and vegetation. Sorption describes the attraction between a chemical and soil, vegetation, or other surfaces. However, the term most often refers to the binding of a chemical to soil particles. Sorption is defined as the attraction of an aqueous species to the surface of a solid (Alley, 1993). The sorbing species, usually an organic compound, is called the sorbate, and the solid, usually soil, to which the sorbate is attracted is known as the sorbent. This attraction results from some form of bonding between the chemical and adsorption receptor sites on the solid. Several mechanisms may operate in a particular situation, including ionic attraction, hydrophobic attraction, and hydrogen bonding. For

Sorption is also influenced by soil moisture, organic matter content, and texture. Pesticides are more readily sorbed onto dry soil because water competes with pesticides for binding sites in moist soil. More sorption occurs in soils made largely of clay and organic matter. Organic matter and clay particles have small particle size, large surface area, and high surface charge. Sand particles provide less surface area for sorption, making pesticides more

Several parameters are used to describe a pesticide's sorption behavior in soils. Table 1 contains sorption parameters for selected chemicals currently and previously used as soil

Two related measures of a pesticide's sorption are the sorption coefficient (Kd) and the soil organic carbon coefficient (KOC). These coefficients are ratios of adsorbed to dissolved pesticide for a specific soil (Kd) or for the organic carbon fraction of a soil (KOC). These values are useful for broadly discriminating between leaching classes of pesticides, but actual adsorption depends on many factors, including soil moisture, temperature, soil pH,

> H2O Solubility (mg/L)


(average) 2.8 1.023 (20C) 3.1 x 10-15 USEPA

(average) 6.6 4 x 10-3 (20C) 2.5 x 10-7 Jones (1999)



Henry's law constant (atmm3 /mol)

2.2 (pH 9) 3.7 x 10-5 Connelly

Reference

(1986)

(2008)

(2001)

(2000)

pesticides that are weak acids or bases, sorption is influenced by soil pH.

likely to move away from the point of application.

and type of organic matter (Wauchope et al., 2002).

(L/kg)

Table 1. Soil sorption parameters of selected soil termiticides.

Log Kow

– 1.2 x 104 4.01 2.4 (pH 5)

Chlordane 4.19 – 4.39 2.78 1.0 x 10-4 1.3 x 10-3 USEPA

Termiticide KOC

Bifenthrin 1.31 x 105

Chlorantraniliprole 3.3 x 102

Cypermethrin 6.1 x 104

Fipronil 3.8 x 103

Imidacloprid 1.3 x 102

Indoxacarb 2.2 x 103

termiticides.

Because sorption coefficient values for the same pesticide vary widely with soil properties, reported values are not included in Table 1. However, Kd values are useful for comparing sorption of different chemicals to the same soil. The organic carbon sorption coefficient is a property of the pesticide and is independent of soil organic matter. The sorption coefficient and organic carbon sorption coefficient are related by the equation:

$$\mathbf{K}\_d = \text{Koc}\left(\% \text{O.C.}\right) \tag{1}$$

Where O.C. is the percentage organic carbon the soil contains. This relationship shows that as the organic fraction of soil increases, the distribution coefficient, Kd, increases. For this relationship to hold true, the chemical must be non-ionic because soil pH affects sorption of ionic sorbates.

As Table 1 illustrates, KOC values for a pesticide are not constant. Pesticide concentration affects adsorption (Kamble & Saran, 2005), but not to an extent that prevents comparison of relative mobility of different pesticides. For polar solutes, surfaces other than organic carbon can become important sorbents particularly when soils are low in organic matter (Cheung et al., 1979; Cox et al., 1998; Means et al., 1982).

Another useful measure of potential pesticide mobility is the octanol - water partition coefficient (Kow). Kow is a measure of the hydrophobicity of an organic compound. The more hydrophobic a compound, the less soluble it is, therefore the more likely it will adsorb to soil particles (Bedient et al, 1994). To evaluate hydrophobicity, the organic solvent octanol is used as a surrogate for organic matter. The octanol-water partition coefficient is the ratio of the concentration of a chemical in octanol and in water at equilibrium and at a specified temperature. Kow is determined by adding a known amount of the pesticide to equal volumes of octanol and water. The coefficient is determined by calculating the concentration in the octanol phase compared to the concentration in the water phase. Kow values vary by several orders of magnitude and may be reported as either Kow or log Kow values. The octanol-water partition coefficient is correlated with water solubility; therefore, the water solubility of a substance can be used to estimate its octanol-water partition coefficient.

Water solubility describes the amount of pesticide that will dissolve in a known amount of water. Highly soluble pesticides are more likely to be moved by runoff or leaching. As with sorption parameters, solubility values are useful as a means of comparison, but actual values will vary with field conditions. Solubility is affected by temperature, water pH, and the presence of other chemicals. The solubility of a compound tends to be inversely proportional to the amount of sorption that it can undergo.

In addition to being adsorbed to soil or transported by water, pesticides can be volatilized. Pesticide volatilization from moist soil is described by the Henry's law constant (Kh). Kh is defined as the concentration of pesticide in air divided by the concentration in water. Kh can be measured experimentally or estimated by dividing the saturation vapor pressure of the compound by its solubility (Suntio et al., 1988). Like other sorption parameters, Kh is temperature dependent, but values are useful for comparing volatility of different compounds. Pesticides with higher Kh are more likely to volatilize from moist soil. Because sorption affects the amount of pesticide in the soil water, the tendency to volatilize from moist soil depends on both the Henry's law constant and sorption coefficients.

Factors Affecting Performance of Soil Termiticides 159

Initial concentration of termiticides in soil varies from several hundred to over one thousand micrograms per gram. Kard and McDaniel (1993) reported initial concentrations of 858± 990mg/g after application to a Mississippi soil, and Davis and Kamble (1992) reported initial concentrations of chlorpyrifos as high as 1500mg/g in a Nebraska loamy sand soil. Application rate affects both initial availability and degradation rate. Saran and Kamble (2008) reported an inverse relationship between the initial concentrations of bifenthrin, fipronil, and imidacloprid and their LT50 and LT90 values against *R. flavipes*. Greater bioavailability at higher concentrations may explain similar trends reported by Smith and Rust (1992), Forschler and Townsend (1996), and Ramakrishnan et al. (2000). At low rates, fipronil has low soil affinity, but adsorption increases with concentration (Bobé et al., 1997). Kamble and Saran (2005) found that at termiticide application rates of 0.06–0.125%, there is a reversal in the fipronil adsorption process, whereby there is a decrease in adsorption coefficient with an increase in concentration, resulting in an increase in bioavailability. Chlorpyrifos exhibited a lower degradation rate when applied at ≈1,000 μg/g soil than when applied at typical agricultural levels of 0.3–32 μg/g (Racke et al., 1994). When fipronil was applied at the labeled rate for locust control (8g AI per ha), 75% degraded within 3 d (Bobé et al., 1998). However, when applied at termiticidal rates (60-125 μg AI per g), fipronil did not show much degradation, and no metabolites were detected in residue analysis after 180 d (Saran and Kamble 2008). Gahlhoff and Koehler (2001) found that concentration and treatment thickness significantly affected both mortality and penetration by *R. flavipes* into imidacloprid-treated soil, with mortality remaining low after 7 d exposure to low concentrations, as well as affecting bioavailability, high termiticide concentrations may indirectly affect degradation by negatively impacting bacterial and fungal populations, resulting in prolonged inhibition of soil dehydrogenase and esterase activities (Felsot & Dzantor, 1995). Racke et al. (1996) examined hydrolysis of chlorpyrifos in 37 soils at agricultural application rates (10mg/g) and observed that in some alkaline soils hydrolysis constituted the major degradation pathway. However, they also noted that in several soils, with pH values in the range of 7.1 to 8.5, the hydrolytic reaction was inhibited at higher

Termites can circumvent soil treatments by using untreated gaps, building materials, or debris as bridges between the surrounding soil and structure (Forschler, 1994; Smith & Zungoli, 1995; Su & Scheffrahn, 1998). Subterranean termite foragers are able to detect and avoid repellant termiticides so areas treated with pyrethroids are rarely contacted. The subterranean termites' ability to detect chemical barriers allows termite foragers to follow the edge of the pyrethroid treated area until they find a gap in the treatment (Forschler, 1994; Rust & Smith, 1993; Su & Scheffrahn, 1990b; Su et al., 1982). Thus, gaps in pyrethroid applications may actually funnel foragers toward the structures they are intended to protect (Forschler, 1994; Kuriachan & Gold, 1998). The inevitability of gaps in soil termiticide barriers is a major limitation to the efficacy of repellant liquid termiticides (Forschler, 1994; Kuriachan & Gold, 1998). Gaps may exist in a soil termiticide treatment for a number of reasons. Pre-construction treatments often contain gaps due to imperfect initial application or physical disturbance of the soil after application (Koehler et al., 2000; Su & Scheffrahn, 1990a, 1998). When an existing structure becomes infested and requires a remedial termiticide application, it is difficult to create a continuous horizontal barrier of liquid termiticide beneath the structure (Su & Scheffrahn, 1990a, 1998; Koehler et al., 2000). Finally, all termiticides degrade over time. An ageing soil treatment, applied below the foundation

concentrations (1000mg/g).

#### **3.2 Clay**

Soil texture has a strong impact on termiticide performance, but effects differ with insecticide. In assays conducted with bifenthrin, chlorfenapyr, and fipronil, *C. formosanus* mortality was generally highest when clay content was low (Wiltz, 2010). Clay content of soil was significantly related to termite mortality across all termiticides, rates, and exposure times (Wiltz, 2010). Likewise, Osbrink and Lax (2002) found that *C. formosanus* workers experienced greater mortality in fipronil-treated sand than in treated potting soil or a mixture of soil and clay. Bobé et al. (1997) reported that for fipronil there was a significant decrease in adsorption coefficient as the soil clay content decreased, thereby increasing bioavailability. However, the opposite result occurs with some insecticides. Smith and Rust (1993) found that increased clay content increased the toxicity of certain pyrethroids, such as cypermethrin. The authors concluded that cypermethrin and clay apparently interacted creating a formulation similar to a wettable powder, which may have an increased affinity for the nonpolar termite integument. Gao et al. (1998) investigated the adsorption of seven pesticides and metabolites with different physiochemical properties, finding that adsorption was generally more effective on smaller and larger soil particles than on intermediate-sized particles.

#### **3.3 pH**

Effects of pH on adsorption and desorption vary with insecticide chemistry and interact with other soil properties. Low pH soils increase the adsorption of weakly acidic pesticides (Boivin et al., 2005; Carrizosa et al., 2000; Halfon et al., 1996,). Desorption of endosulfan was higher at both acidic and alkaline pH ranges compared to neutral pH (Kumar & Philip, 2006). The authors found that in clay soil, adsorption decreased drastically when the pH was reduced. In soil column experiments, deltamethrin was essentially immobile in three different soils. Kaufman et al. (1981) suggested that for nonacidic soils, the pH may be a primary factor affecting mobility of deltamethrin. In bioassays of treated soils against *C. formosanus*, there was an interaction between effects of soil pH and clay content on effectiveness of chlorfenapyr and fipronil (Wiltz, 2010).

#### **3.4 Organic carbon**

Partitioning of insecticides between soil organic matter and soil solution affects bioavailability (Felsot & Lew, 1989). Like clay, organic matter decreases adsorption of fipronil (Bobé et al., 1997). Mulrooney and Gerard (2007) applied fipronil to 3 soils and found that *R. flavipes* mortality decreased with increasing organic carbon. Although no soil effects were found when soils were treated at label rates, pyrethroids applied at low rates were less available in soils with high OC (Henderson et al., 1998). Soil OC has been shown to affect adsorption of several non-acidic pesticides, but have little or no effect on weakly acidic chemicals (Barriuso & Calvet, 1992; Boivin et al., 2005; Worrall et al., 2001).

#### **4. Application technique**

In addition to properties of the soil and chemicals, variations in application technique can influence availability, persistence, and impenetrability of toxins. Such variables include gaps in soil treatment, thickness of treated layer, and watering method. Within certain ranges of application rates, availability increases with rate; however, the opposite is true at other rates.

Soil texture has a strong impact on termiticide performance, but effects differ with insecticide. In assays conducted with bifenthrin, chlorfenapyr, and fipronil, *C. formosanus* mortality was generally highest when clay content was low (Wiltz, 2010). Clay content of soil was significantly related to termite mortality across all termiticides, rates, and exposure times (Wiltz, 2010). Likewise, Osbrink and Lax (2002) found that *C. formosanus* workers experienced greater mortality in fipronil-treated sand than in treated potting soil or a mixture of soil and clay. Bobé et al. (1997) reported that for fipronil there was a significant decrease in adsorption coefficient as the soil clay content decreased, thereby increasing bioavailability. However, the opposite result occurs with some insecticides. Smith and Rust (1993) found that increased clay content increased the toxicity of certain pyrethroids, such as cypermethrin. The authors concluded that cypermethrin and clay apparently interacted creating a formulation similar to a wettable powder, which may have an increased affinity for the nonpolar termite integument. Gao et al. (1998) investigated the adsorption of seven pesticides and metabolites with different physiochemical properties, finding that adsorption was generally more effective on smaller and larger soil particles than on intermediate-sized

Effects of pH on adsorption and desorption vary with insecticide chemistry and interact with other soil properties. Low pH soils increase the adsorption of weakly acidic pesticides (Boivin et al., 2005; Carrizosa et al., 2000; Halfon et al., 1996,). Desorption of endosulfan was higher at both acidic and alkaline pH ranges compared to neutral pH (Kumar & Philip, 2006). The authors found that in clay soil, adsorption decreased drastically when the pH was reduced. In soil column experiments, deltamethrin was essentially immobile in three different soils. Kaufman et al. (1981) suggested that for nonacidic soils, the pH may be a primary factor affecting mobility of deltamethrin. In bioassays of treated soils against *C. formosanus*, there was an interaction between effects of soil pH and clay content on

Partitioning of insecticides between soil organic matter and soil solution affects bioavailability (Felsot & Lew, 1989). Like clay, organic matter decreases adsorption of fipronil (Bobé et al., 1997). Mulrooney and Gerard (2007) applied fipronil to 3 soils and found that *R. flavipes* mortality decreased with increasing organic carbon. Although no soil effects were found when soils were treated at label rates, pyrethroids applied at low rates were less available in soils with high OC (Henderson et al., 1998). Soil OC has been shown to affect adsorption of several non-acidic pesticides, but have little or no effect on weakly

In addition to properties of the soil and chemicals, variations in application technique can influence availability, persistence, and impenetrability of toxins. Such variables include gaps in soil treatment, thickness of treated layer, and watering method. Within certain ranges of application rates, availability increases with rate; however, the opposite is true at other rates.

acidic chemicals (Barriuso & Calvet, 1992; Boivin et al., 2005; Worrall et al., 2001).

effectiveness of chlorfenapyr and fipronil (Wiltz, 2010).

**3.2 Clay** 

particles.

**3.3 pH** 

**3.4 Organic carbon** 

**4. Application technique** 

Initial concentration of termiticides in soil varies from several hundred to over one thousand micrograms per gram. Kard and McDaniel (1993) reported initial concentrations of 858± 990mg/g after application to a Mississippi soil, and Davis and Kamble (1992) reported initial concentrations of chlorpyrifos as high as 1500mg/g in a Nebraska loamy sand soil. Application rate affects both initial availability and degradation rate. Saran and Kamble (2008) reported an inverse relationship between the initial concentrations of bifenthrin, fipronil, and imidacloprid and their LT50 and LT90 values against *R. flavipes*. Greater bioavailability at higher concentrations may explain similar trends reported by Smith and Rust (1992), Forschler and Townsend (1996), and Ramakrishnan et al. (2000). At low rates, fipronil has low soil affinity, but adsorption increases with concentration (Bobé et al., 1997). Kamble and Saran (2005) found that at termiticide application rates of 0.06–0.125%, there is a reversal in the fipronil adsorption process, whereby there is a decrease in adsorption coefficient with an increase in concentration, resulting in an increase in bioavailability. Chlorpyrifos exhibited a lower degradation rate when applied at ≈1,000 μg/g soil than when applied at typical agricultural levels of 0.3–32 μg/g (Racke et al., 1994). When fipronil was applied at the labeled rate for locust control (8g AI per ha), 75% degraded within 3 d (Bobé et al., 1998). However, when applied at termiticidal rates (60-125 μg AI per g), fipronil did not show much degradation, and no metabolites were detected in residue analysis after 180 d (Saran and Kamble 2008). Gahlhoff and Koehler (2001) found that concentration and treatment thickness significantly affected both mortality and penetration by *R. flavipes* into imidacloprid-treated soil, with mortality remaining low after 7 d exposure to low concentrations, as well as affecting bioavailability, high termiticide concentrations may indirectly affect degradation by negatively impacting bacterial and fungal populations, resulting in prolonged inhibition of soil dehydrogenase and esterase activities (Felsot & Dzantor, 1995). Racke et al. (1996) examined hydrolysis of chlorpyrifos in 37 soils at agricultural application rates (10mg/g) and observed that in some alkaline soils hydrolysis constituted the major degradation pathway. However, they also noted that in several soils, with pH values in the range of 7.1 to 8.5, the hydrolytic reaction was inhibited at higher concentrations (1000mg/g).

Termites can circumvent soil treatments by using untreated gaps, building materials, or debris as bridges between the surrounding soil and structure (Forschler, 1994; Smith & Zungoli, 1995; Su & Scheffrahn, 1998). Subterranean termite foragers are able to detect and avoid repellant termiticides so areas treated with pyrethroids are rarely contacted. The subterranean termites' ability to detect chemical barriers allows termite foragers to follow the edge of the pyrethroid treated area until they find a gap in the treatment (Forschler, 1994; Rust & Smith, 1993; Su & Scheffrahn, 1990b; Su et al., 1982). Thus, gaps in pyrethroid applications may actually funnel foragers toward the structures they are intended to protect (Forschler, 1994; Kuriachan & Gold, 1998). The inevitability of gaps in soil termiticide barriers is a major limitation to the efficacy of repellant liquid termiticides (Forschler, 1994; Kuriachan & Gold, 1998). Gaps may exist in a soil termiticide treatment for a number of reasons. Pre-construction treatments often contain gaps due to imperfect initial application or physical disturbance of the soil after application (Koehler et al., 2000; Su & Scheffrahn, 1990a, 1998). When an existing structure becomes infested and requires a remedial termiticide application, it is difficult to create a continuous horizontal barrier of liquid termiticide beneath the structure (Su & Scheffrahn, 1990a, 1998; Koehler et al., 2000). Finally, all termiticides degrade over time. An ageing soil treatment, applied below the foundation

Factors Affecting Performance of Soil Termiticides 161

adsorption at higher temperatures. Dios-Cancela et al. (1990) found that sorption of the

Microbial degradation occurs when fungi, bacteria, and other soil microorganisms use pesticides as food or consume pesticides along with other substances. Activity of microbes is affected by soil organic matter and texture and is usually highest in warm, moist, wellaerated soils with a neutral pH. Because microbial degradation is mediated by enzymes, temperature is important in determining the rate degradation: the rate of most reactions catalyzed by enzymes tends to double for each 10C increase in temperature between 10

Naturally-occurring pesticide-degrading microorganisms may be relatively rare in pristine environments and non-exposed agricultural soils (Bartha, 1990). Some of the pesticidedegrading microbes that have been identified include *Arthrobacter, Brevibacterium, Clavibacter, Corynebacterium, Micromonospora, Mycobacterium, Nocardia, Nocardioides,* 

A review of earlier work on organophosphate and carbamate insecticide degradation was prepared by Laveglia and Dahm (1977). Although organophosphates are no longer used in many parts of the world, there have been several recent studies on their degradation by microbes. Li et al. (2007) reported the isolation of a bacterium, *Sphingomonas* sp., that degrades chlorpyrifos, parathion, parathion-methyl, fenitrothion and profenofos. However, several other studies have found little microbial degradation of chlorpyrifos. Goda et al. (2010) showed that the intact cells of *Pseudomonas putida* IS168 were able to degrade fenitrothion, diazinon and profenofos when present as sole carbon sources, but failed to grow on chlorpyrifos. Trichloropyridinol (TCP), one of the main chlorpyrifos metabolites, has antimicrobial properties (Cáceres et al., 2007; Feng et al., 1997; Racke et al., 1990), possibly accounting for the scarcity of chlorpyrifos-degrading microorganisms. Degradation of pyrethroids in soil has also been extensively studied (Gan et al., 2005; Jorhan & Kaufman, 1986; Kaufman et al., 1981; Lee et al., 2004; Lord et al., 1982). Most of these studies show that microorganisms play an important role in the degradation of pyrethroid compounds in soils

In studies evaluating termite tunneling through chlordane, chlorpyrifos, or permethrin treated soil, large groups of termites were able to tunnel farther than small groups (Beal & Smith, 1971; Jones, 1990). At low population density, different colonies of *C. formosanus* either totally avoided permethrin-treated soil or tunneled slightly (Jones, 1989, 1990). Jones (1990) found that while large groups of termites tunneled more than small groups in soils treated with chlordane, chlorpyrifos, or permethrin, group size had different effects on mortality in different soil treatments. Several experiments have demonstrated correlations between termite survival rates and population density (Lenz et al., 1984; Lenz, 2009; Santos et al., 2004). At population densities below 0.1 g termites / ml, Lenz et al. (1984) found that, in the absence of termiticide treatment, survival of *Coptotermes lacteus* (Froggatt) and

Susceptibility differences occur among termite species and colonies. Most soil termiticide evaluations have included only one target species. However, in studies comparing

herbicide cyanazine to peats decreased with increasing temperature.

and 45C and is greatly reduced above and below these temperatures.

*Rhodococcus* and *Streptomyces* genera (De Schrijver and De Mot, 1999).

**5.3 Micromial degradation** 

and sediments.

**6. Termite pressure and susceptibility** 

*Nasutitermes exitiosus* (Hill) increased with population density.

before a structure was built, is inaccessible after construction and cannot be reapplied (Su & Scheffrahn, 1990a; Su, 1997; Koehler et al., 2000).

The total volume of pesticide suspension applied to soil affects penetration depth and concentration in the soil. In tests using imidacloprid and fipronil in five different soils, when equal amounts of pesticide were diluted in different volumes of water, the higher volume treatments penetrated further into the soil, but the more concentrated treatments deposited more pesticide in the top 1cm of soil (Peterson 2010). It is likely that the thicker barrier of lower active ingredient concentration would provide better protection, at least in the short term because it might be better able to withstand disturbances to the top 1 cm of soil. Additionally, termites are less able to tunnel through thicker barriers of lower active ingredient concentration than through thinner barriers of higher concentration (Smith et al. 2008). However, pesticide treatments with low initial concentrations degrade faster than those with higher initial concentrations (Bobé et al., 1998; Felsot & Dzantor, 1995; Saran & Kamble, 2008). In addition to total volume of liquid applied, initial thickness of the treated zone depends on soil and termiticide properties. Smith and Rust (1992) found that termiticidal amounts of chlordane and cypermethrin moved to soil depths of at least 7 cm, while chlorpyrifos moved to a depth of at least 30 cm.

#### **5. Environmental factors**

Both biotic and abiotic pathways have been found to be important for insecticide degradation and transformation in soils (Racke et al., 1996).

#### **5.1 Moisture**

Water can compete with pesticides for sorption sites on soil particles. Dry soils become more sorptive for both polar and non-polar chemicals (Chen et al., 2000). However, chemicals with low polarity are released when soil becomes wet (Harper et al., 1976). Repeated cycles of wetting and drying affect pesticide availability and degradation, but depend on properties of the chemical, soil, number of wetting and drying cycles, time since pesticide application, and time since wetting (Garcia-Valcarcel & Tadeo, 1999; Xia & Brandenburg 2000; Ying & Kookana, 2006; Peterson, 2007).

#### **5.2 Temperature**

Soil temperature affects termiticide bioavailability through its influence on solubility and adsorption. In addition to its effect on the physical and chemical properties of the pesticide, extreme temperatures affect the rate of microbial degradation, as described in the following section. Several studies have demonstrated that temperature affects adsorption of pesticides to soil, but that the nature of this effect varies among pesticides. Although most of the work on pesticide availability and degradation has been conducted in the temperate climates of North America and Europe, soil temperature is likely to play an important role in termiticide degradation in tropical regions. Khan et al. (1996) found that lindane adsorption to silty loam and silty clay loam soils increased with temperature. Likewise, Valverde-Garcia et al. (1988) found that higher temperatures increased the adsorption of the fungicide thiram and the organophosphate insecticide dimethoate to organic soils. Temperature may interact with pH, particularly in saturated soils. In aqueous solutions, fenamiphos, fipronil, and trifluralin degradation increased with temperature and pH (Ramesh & Balasubramanian, 1999). Other studies have demonstrated a reduction in pesticide adsorption at higher temperatures. Dios-Cancela et al. (1990) found that sorption of the herbicide cyanazine to peats decreased with increasing temperature.

#### **5.3 Micromial degradation**

160 Insecticides – Basic and Other Applications

before a structure was built, is inaccessible after construction and cannot be reapplied (Su &

The total volume of pesticide suspension applied to soil affects penetration depth and concentration in the soil. In tests using imidacloprid and fipronil in five different soils, when equal amounts of pesticide were diluted in different volumes of water, the higher volume treatments penetrated further into the soil, but the more concentrated treatments deposited more pesticide in the top 1cm of soil (Peterson 2010). It is likely that the thicker barrier of lower active ingredient concentration would provide better protection, at least in the short term because it might be better able to withstand disturbances to the top 1 cm of soil. Additionally, termites are less able to tunnel through thicker barriers of lower active ingredient concentration than through thinner barriers of higher concentration (Smith et al. 2008). However, pesticide treatments with low initial concentrations degrade faster than those with higher initial concentrations (Bobé et al., 1998; Felsot & Dzantor, 1995; Saran & Kamble, 2008). In addition to total volume of liquid applied, initial thickness of the treated zone depends on soil and termiticide properties. Smith and Rust (1992) found that termiticidal amounts of chlordane and cypermethrin moved to soil depths of at least 7 cm,

Both biotic and abiotic pathways have been found to be important for insecticide

Water can compete with pesticides for sorption sites on soil particles. Dry soils become more sorptive for both polar and non-polar chemicals (Chen et al., 2000). However, chemicals with low polarity are released when soil becomes wet (Harper et al., 1976). Repeated cycles of wetting and drying affect pesticide availability and degradation, but depend on properties of the chemical, soil, number of wetting and drying cycles, time since pesticide application, and time since wetting (Garcia-Valcarcel & Tadeo, 1999; Xia & Brandenburg

Soil temperature affects termiticide bioavailability through its influence on solubility and adsorption. In addition to its effect on the physical and chemical properties of the pesticide, extreme temperatures affect the rate of microbial degradation, as described in the following section. Several studies have demonstrated that temperature affects adsorption of pesticides to soil, but that the nature of this effect varies among pesticides. Although most of the work on pesticide availability and degradation has been conducted in the temperate climates of North America and Europe, soil temperature is likely to play an important role in termiticide degradation in tropical regions. Khan et al. (1996) found that lindane adsorption to silty loam and silty clay loam soils increased with temperature. Likewise, Valverde-Garcia et al. (1988) found that higher temperatures increased the adsorption of the fungicide thiram and the organophosphate insecticide dimethoate to organic soils. Temperature may interact with pH, particularly in saturated soils. In aqueous solutions, fenamiphos, fipronil, and trifluralin degradation increased with temperature and pH (Ramesh & Balasubramanian, 1999). Other studies have demonstrated a reduction in pesticide

Scheffrahn, 1990a; Su, 1997; Koehler et al., 2000).

while chlorpyrifos moved to a depth of at least 30 cm.

2000; Ying & Kookana, 2006; Peterson, 2007).

degradation and transformation in soils (Racke et al., 1996).

**5. Environmental factors** 

**5.1 Moisture** 

**5.2 Temperature** 

Microbial degradation occurs when fungi, bacteria, and other soil microorganisms use pesticides as food or consume pesticides along with other substances. Activity of microbes is affected by soil organic matter and texture and is usually highest in warm, moist, wellaerated soils with a neutral pH. Because microbial degradation is mediated by enzymes, temperature is important in determining the rate degradation: the rate of most reactions catalyzed by enzymes tends to double for each 10C increase in temperature between 10 and 45C and is greatly reduced above and below these temperatures.

Naturally-occurring pesticide-degrading microorganisms may be relatively rare in pristine environments and non-exposed agricultural soils (Bartha, 1990). Some of the pesticidedegrading microbes that have been identified include *Arthrobacter, Brevibacterium, Clavibacter, Corynebacterium, Micromonospora, Mycobacterium, Nocardia, Nocardioides, Rhodococcus* and *Streptomyces* genera (De Schrijver and De Mot, 1999).

A review of earlier work on organophosphate and carbamate insecticide degradation was prepared by Laveglia and Dahm (1977). Although organophosphates are no longer used in many parts of the world, there have been several recent studies on their degradation by microbes. Li et al. (2007) reported the isolation of a bacterium, *Sphingomonas* sp., that degrades chlorpyrifos, parathion, parathion-methyl, fenitrothion and profenofos. However, several other studies have found little microbial degradation of chlorpyrifos. Goda et al. (2010) showed that the intact cells of *Pseudomonas putida* IS168 were able to degrade fenitrothion, diazinon and profenofos when present as sole carbon sources, but failed to grow on chlorpyrifos. Trichloropyridinol (TCP), one of the main chlorpyrifos metabolites, has antimicrobial properties (Cáceres et al., 2007; Feng et al., 1997; Racke et al., 1990), possibly accounting for the scarcity of chlorpyrifos-degrading microorganisms. Degradation of pyrethroids in soil has also been extensively studied (Gan et al., 2005; Jorhan & Kaufman, 1986; Kaufman et al., 1981; Lee et al., 2004; Lord et al., 1982). Most of these studies show that microorganisms play an important role in the degradation of pyrethroid compounds in soils and sediments.

#### **6. Termite pressure and susceptibility**

In studies evaluating termite tunneling through chlordane, chlorpyrifos, or permethrin treated soil, large groups of termites were able to tunnel farther than small groups (Beal & Smith, 1971; Jones, 1990). At low population density, different colonies of *C. formosanus* either totally avoided permethrin-treated soil or tunneled slightly (Jones, 1989, 1990). Jones (1990) found that while large groups of termites tunneled more than small groups in soils treated with chlordane, chlorpyrifos, or permethrin, group size had different effects on mortality in different soil treatments. Several experiments have demonstrated correlations between termite survival rates and population density (Lenz et al., 1984; Lenz, 2009; Santos et al., 2004). At population densities below 0.1 g termites / ml, Lenz et al. (1984) found that, in the absence of termiticide treatment, survival of *Coptotermes lacteus* (Froggatt) and *Nasutitermes exitiosus* (Hill) increased with population density.

Susceptibility differences occur among termite species and colonies. Most soil termiticide evaluations have included only one target species. However, in studies comparing

Factors Affecting Performance of Soil Termiticides 163

Anonymous. (2008). State of the industry. Termites: Kicked to the "B" list? PCT October 10,

Baker, P. B., and D. E. Bellamy. (2006). Field and laboratory evaluation of persistence and

(Isoptera: Rhinotermitidae). *Journal of Economic Entomology,* 99: 1345-1353. Barriuso, E. & Calvet, R. (1992). Soil type and herbicide adsorption. *International Journal of* 

Bartha, R., (1990). Isolation of microorganisms that metabolize xenobiotic compounds. In:

Baskaran, S., Kookana, R. S. & Naidu, R. (1999). Degradation of bifenthrin, chlopyrifos, and

Beal, R. H., & Smith, V. K. (1971). Relative susceptibilities of *Coptotermes formosanus*,

Bedient, P.H., Rifai, H. S. & Newell C. J. (1994). Ground Water Contamination: Transport

Bobé, A., Coste, C. M. & Copper, J –F. (1997). Factors influencing the adsorption of fipronil

Bobé, A., Cooper, J. -F., Coste, C. M. & Muller, M. -A. (1998). Behaviour of fipronil in soil

Boivin, A., Cherrier, R. & Schiavon, M. (2005). A comparison of five pesticides adsorption

Cáceres T, He, W., Naidu, R. & Megharaj, M. (2007). Toxicity of chlorpyrifos and TCP alone

Carrizosa, M. J., Calderon, M. J, Hermosin, M. C. & Cornejo, J. (2000). Organosmectites as

Chen D., Rolston, D. E., and Yumaguchi, T. (2000). Calculating partition coefficients of organic vapors in unsaturated soils and clays. *Soil Science*, 165: 217-225. Cheung, M. W., Mingelgrin, V. & Biggar, J. W. (1979). Equilibrium and kinetics of

Connelly, P. (2001). Environmental fate of fipronil. Environmental Monitoring Branch. Department of Pesticide Regulation. California Environmental Protection Agency. Cox, L. Koskinen W. C., Celis, R., Yeen, P. Y., Hermosin, M. C. & Cornejo, J. (1998). Sorption

and desorption processes in thirteen contrasting field soils. *Chemosphere,* 61: 668-

and in combination to *Daphnia carinata*: the influence of microbial degradation in

sorbent and carrier of the herbicide bentazone. *Science of the Total Environment,* 247:

desorption of picloram and parathion in soils. *Journal of Agricultural and Food* 

of imidacloprid on soil clay mineral and organic components. *Soil Science of America* 

bioavailability of soil termiticides to desert subterranean termite *Heterotermes aureus*

*Isolation of Biotechnological Organisms from Nature*, Labeda, D. P. (Ed.), pp. 283-307,

imidacloprid in soil and bedding materials at termiticidal application rates.

*Reticulitermes flavipes*, and *R. virginicus* to soil insecticides. *Journal of Economic* 

2008. http://www.pctonline.com/Article.aspx?article\_id=37550. Aventis Environmental Science. (2001). Termidor SC termiticide label. Montvale, NJ.

BASF Corporation. (2001). Phantom termiticide label. Triangle Park, NC.

*Environmental Analytical Chemistry,* 46: 117–128.

*Pesticide Science,* 55:1222–1228.

*Entomology*, 64: 472-475.

676.

285–293.

*Chemistry*, 27: 1201-1206.

*Journal,* 69: 911-915.

McGraw-Hill Publishing Company, New York, USA.

Bayer Corporation. (2000). Premise 75 WSP termiticide label. Kansas City, MO.

and Remediation, Prentice Hall, Englewood Cliffs, NJ, 1994.

natural water. *Water Research,* 41: 4497–4503

on soils. *Journal of Agricultural and Food Chemistry,* 45: 4861–4865.

under Sahelian plain field conditions. *Pesticide Science,* 52: 275–281.

responses of two or more species, there are frequently differences in susceptibility. *C. formosanus* penetrated soil treated with aldrin, chlordane, dieldrin, or heptachlor, while *R. virginicus* and *R. flavipes* failed to penetrate lower rates of the same chemicals and were killed more quickly than *C. formosanus* (Beal & Smith 1971). In a laboratory assay, chlorpyrifos, permethrin, cypermethrin, bifenthrin, isofenphos, lambda-cyhalothirn, and fenitrothion all provided equal barrier protection against *R. flavipes* (Su et al. 1993). However, in the same assay, *C. formosanus* generally tunneled deeper into sand treated with organophosphates than with pyrethroids. Penetration of sand treated with thiamethoxam or fipronil was similar for *C. formosanus* and *R. flavipes*, but thiamethoxam was more toxic to *C. formosanus* than to *R. flavipes* (Remmen & Su 2005). Osbrink and Lax (2002) evaluated seven insecticides against termites from colonies that had been previously been identified as either insecticide susceptible or tolerant, finding differences in substrate penetration and mortality among colonies and insecticides.

Termite traits other than population size or susceptibility to toxicants can increase the likelihood that soil treatments will fail to protect a structure. Aerial infestations account for a large percentage of structural infestations by *C. formosanus* (Su & Scheffrahn, 1990), making soil treatments ineffective. Additionally, *C. formosanus* colonies may seal off or avoid treated areas (Su et al. 1982) when repellent toxicants are used, but use gaps in the soil barrier to access the structure (Forschler, 1994).

#### **7. Conclusion**

Several long-term studies of termiticide persistence have been conducted. In USDA Forest Service trials, which have been conducted for the past 40 years, tests consist of treated soil plots covered by concrete slabs. Treatments are considered failures when termites penetrate >50% of field replicates to reach a wood block placed in a pipe running through the slab (Kard, 2003; Wagner, 2003). In these tests, longevity differed with geographic location and termiticide class (Mulrooney et al., 2007). Such studies have the advantage of being performed under natural soil weathering conditions for an extended period of time and provide a standard method of comparing termiticides. However, products are evaluated on a limited number of soils and it is impossible to tell if a lack of penetration into plots should be attributed to effectiveness of the termiticide or to the absence of termite pressure. To overcome this problem, other studies have included laboratory bioassays coupled with field termiticide persistence studies (Gold et al., 1996; Grace, 1991; Su et al., 1993). Unfortunately, most of these studies have evaluated relatively few soil types. Because performance is so dependent on a combination of temiticide and soil properties and weathering, more research is needed to evaluate new and existing products under a larger range of conditions. Soil termiticides have been extensively evaluated for toxicity, bioavailability, and degradation. However, reasons for termiticide failure are complex and often local in nature, indicating the need for more research and localized treatment recommendations regarding choice of toxicant, application technique, and treatment frequency.

#### **8. References**

Alley, W., (1993). *Regional Groundwater Quality*, Van Nostrand Reinhold, New York, New York, 1994.

responses of two or more species, there are frequently differences in susceptibility. *C. formosanus* penetrated soil treated with aldrin, chlordane, dieldrin, or heptachlor, while *R. virginicus* and *R. flavipes* failed to penetrate lower rates of the same chemicals and were killed more quickly than *C. formosanus* (Beal & Smith 1971). In a laboratory assay, chlorpyrifos, permethrin, cypermethrin, bifenthrin, isofenphos, lambda-cyhalothirn, and fenitrothion all provided equal barrier protection against *R. flavipes* (Su et al. 1993). However, in the same assay, *C. formosanus* generally tunneled deeper into sand treated with organophosphates than with pyrethroids. Penetration of sand treated with thiamethoxam or fipronil was similar for *C. formosanus* and *R. flavipes*, but thiamethoxam was more toxic to *C. formosanus* than to *R. flavipes* (Remmen & Su 2005). Osbrink and Lax (2002) evaluated seven insecticides against termites from colonies that had been previously been identified as either insecticide susceptible or tolerant, finding differences in substrate penetration and mortality

Termite traits other than population size or susceptibility to toxicants can increase the likelihood that soil treatments will fail to protect a structure. Aerial infestations account for a large percentage of structural infestations by *C. formosanus* (Su & Scheffrahn, 1990), making soil treatments ineffective. Additionally, *C. formosanus* colonies may seal off or avoid treated areas (Su et al. 1982) when repellent toxicants are used, but use gaps in the soil barrier to

Several long-term studies of termiticide persistence have been conducted. In USDA Forest Service trials, which have been conducted for the past 40 years, tests consist of treated soil plots covered by concrete slabs. Treatments are considered failures when termites penetrate >50% of field replicates to reach a wood block placed in a pipe running through the slab (Kard, 2003; Wagner, 2003). In these tests, longevity differed with geographic location and termiticide class (Mulrooney et al., 2007). Such studies have the advantage of being performed under natural soil weathering conditions for an extended period of time and provide a standard method of comparing termiticides. However, products are evaluated on a limited number of soils and it is impossible to tell if a lack of penetration into plots should be attributed to effectiveness of the termiticide or to the absence of termite pressure. To overcome this problem, other studies have included laboratory bioassays coupled with field termiticide persistence studies (Gold et al., 1996; Grace, 1991; Su et al., 1993). Unfortunately, most of these studies have evaluated relatively few soil types. Because performance is so dependent on a combination of temiticide and soil properties and weathering, more research is needed to evaluate new and existing products under a larger range of conditions. Soil termiticides have been extensively evaluated for toxicity, bioavailability, and degradation. However, reasons for termiticide failure are complex and often local in nature, indicating the need for more research and localized treatment recommendations regarding

Alley, W., (1993). *Regional Groundwater Quality*, Van Nostrand Reinhold, New York, New

choice of toxicant, application technique, and treatment frequency.

among colonies and insecticides.

access the structure (Forschler, 1994).

**7. Conclusion** 

**8. References** 

York, 1994.


BASF Corporation. (2001). Phantom termiticide label. Triangle Park, NC.


Factors Affecting Performance of Soil Termiticides 165

Gold, R. E., Howell, H. N., Pawson, B. M., Wright, M. S. & J. C. Lutz. (1996). Persistence and

Grace, J. K. (1991). Response of eastern and Formosan subterranean termites (Isoptera,

Grace, J. K., Yates, J. R., Tamishiro, M. & Yamamoto, R. T. (1993). Persistence of

Henderson, G., Walthall, P. M., Wiltz, B. A. Rivera-Monroy, V. H., Ganaway, D. R. & H. M.

Horwood, M. A. (2007). Rapid degradation of termiticides under field conditions. *Australian* 

Itawa, H., Tanabe, S., Sakai, N., Tatsukawa, R., (1993). Distribution of persistent

Jones, S. C. (1990). Effects of population density on tunneling by Formosan subterranean

Jorhan, E. G. & Kaufman, D. D. (1986). Degradation of cis- and transpermethrin in flooded

Kamble, S. T. & Saran, R. K. (2005). Effect of concentration on the adsorption of three

Kard, B. M. (2003). Integrated pest management of subterranean termites (Isoptera*). Journal* 

Kard B. M. & McDaniel, C. A. (1993). Field evaluation of the persistence and efficacy of

soil. *Journal of Agricultural and Food Chemistry,* 34: 880-884.

Kard, B. (1999). Termiticides: the Gulfport report. *Pest Control,* 67: 42-46.

from five soil types in Texas. *Sociobiology,* 28: 337-363.

84: 1753–1757.

*Quality*, 5: 236-242.

Diego, CA, April 26-28, 1998.

*Journal of Entomology,* 46: 75-78.

control. *Sociobiology,* 15: 33-41.

*of Entomological Science,* 38: 200–224.

Chemical Society: Washington DC. pp 42-62.

*Entomology*, 83: 875-878.

1085.

bioavailability of termiticides to subterranean termites (Isoptera: Rhinotermitidae)

Rhinotermitidae) to borate dust and soil treatments. *Journal of Economic Entomology,*

organochlorine insecticides for Formosan subterranean termite (Isoptera: Rhinotermitidae) control in Hawaii. *Journal of Economic Entomology*, 86: 761-766. Halfon, E., Galassi, S., Bruggemann, R. &. Provini, A. (1996). Selection of priority properties to assess environmental hazard of pesticides, *Chemosphere,* 33: 1543–1562. Harper, L. A., White, A. W. Jr., Bruce, R. R., Thomas, A. W., and Leonard, R, A. (1976) Soil

and microclimate effects on trifluralin volatilization. *Journal of Environmental* 

Selim. (1998). Analysis of soil properties in relation to termiticide performance in Louisiana. In Proceedings of the National Conference on Urban Entomology, San

organochlorines in the oceanic air and surface seawater and the role of ocean on their global transport and fate. *Environmental Science & Technology,* 27: 1080-1098. Jones, D. (1999). Environmental fate of cypermethrin. Environmental Monitoring and Pest Management Branch. California Department of Pesticide Regulation. Jones S. C. (1989). Field evaluation of fenoxycarb as a bait toxicant for subterranean termite

termite (Isoptera: Rhinotermitidae) through treated soil. *Journal of Economic* 

termiticides in soil. *Bulletin of Environmental Contamination and Toxicology,* 75: 1077-

pesticides used for termite control, in Pesticides in Urban Environments - Fate and Significance, ed by Racke KD and Leslie AR, ACS Symposium Series 522, American


http://www.epa.gov/pesticides/op/chlorpyrifos/agreement.pdf.


Davis, R. W. & Kamble, S. T. (1992). Distribution of sub-slab injected Dursban TC

De Schrijver, A. & De Mot, R., (1999). Degradation of pesticides by actinomycetes. *Critical* 

Dios-Cancela, G., Romero, T. E. & Sanchez-Rasero, F. (1990). Adsorption of cyanazine on

Environmental Protection Agency. (2000). Chlorpyrifos revised risk assessment and

Fecko, A. (1999). Environmental fate of bifenthrin. Environmental Monitoring and Pest Management Branch. California Department of Pesticide Regulation. Felsot, A. S., & Lew, A. (1989). Factors affecting bioactivity of soil insecticides: relationships

Felsot, A. S., & Dzantor, E. K. (1995). Effect of alachlor concentration and an organic

Feng Y, Racke, K. D. & Bollag, J. M. (1997). Isolation and characterization of a chlorinatedpyridinol-degrading bacterium. *Applied Environmental Microbiology,* 63: 4096–4409 Forschler, B. T. (1994). Survivorship and tunneling activity of *Reticulitermes flavipes* (Kollar)

Forschler, B. T. & Townsend, M. L. (1996). Mortality of Eastern subterranean termites

Gahlhoff, J. E. & Koehler, P. G. (2001). Penetration of the eastern subterranean termite into

Gan, J., Lee, S. J., Liu, W. P., Haver, D. L., Kabashima, J. N. (2005). Distribution and

Gao, J. P., Maguhn, J., Spitzauer, P. & Kettrup, A. (1998). Sorption of pesticides in the

Garcia-Valcarcel, A. I. & Tadeo, J. L. (1999). Influence of soil moisture on sorption and

Goda, S. K., Elsayed, I. E., Khodair, T. A., El-Sayed, W. & Mohamed, M. E. (2010). Screening

carboxylestrase in soil bacteria. *Biodegradation,* 21: 903–913.

gaps of untreated soil. *Journal of Entomological Science,* 29: 43–54.

among uptake, desorption, and toxicity of carbofuran and terbufos. *Journal of* 

amendment on soil dehydrogenase activity and pesticide degradation rate*.* 

(Isoptera: Rhinotermitidae) in response to termiticide soil barriers with and without

(Isoptera: Rhinotermitidae) exposed to four soils treated with termiticides. *Journal* 

soil treated at various thicknesses and concentrations of Dursban TC and Premise

persistence of pyrethroids in runoff sediment*. Journal of Environmental Quality*, 36:

sediment of the Teufelsweiher pond (Southern Germany). I: Equilibrium assessments, effect of organic carbon content and pH. *Water Research,* 32: 1662-1672.

degradation of hexazinone and simazine in soil. *Journal of Agricultural and Food* 

for and isolation and identification of malathion-degrading bacteria: cloning and sequencing a gene that potentially encodes the malathion-degrading enzyme,

*Bulletin of Environmental Contamination and Toxicology,* 48: 585-591.

peat and montmorillonite clay surfaces. *Soil Science*, 150: 836-843

http://www.epa.gov/pesticides/op/chlorpyrifos/agreement.pdf.

*Environmental Toxicology and Chemistry,* 14: 23-28.

*Reviews in Microbiology*, 25: 85-119.

Dupont. (2010). Dupont Altriset MSDS.

agreement with registrants. -

*Economic Entomology,* 82: 389-395.

*of Economic Entomology,* 89: 678-681.

834-841.

*Chemistry,* 47: 3895–3900.

75. *Journal of Economic Entomology,* 94: 486-491.

(chlorpyrifos) in a loamy sand soil when used for subterranean termite control,


Factors Affecting Performance of Soil Termiticides 167

Osbrink, W. L A., Cornelius, M. L. & Lax, A. R. (2005). Effect of imidacloprid soil treatments

Peterson, C. J. (2007). Imidacloprid mobility and longevity in soil columns at a termiticidal

Peterson, C. J. (2010). Varying termiticide application rate and volume affect initial soil

Potter, M. H. & Hillery, A. E. (2001). Exterior-targeted liquid termiticides an alternative

Racke, K. D., Fontaine, D. D., Yoder, R. N. & Miller, J. R. (1994). Chlorpyrifos degradation in

Racke K. D., Laskowski D. A. & Schultz M. R. (1990). Resistance of chlorpyrifos to enhanced biodegradation in soil. *Journal of Agricultural and Food Chemistry,* 38: 1430–1436. Racke K. D., Steele, K. P., Yoder, R. N., Dick, W.A. & Avidov E. (1996). Factors affecting the

Ramakrishnan, R., Suiter, D. R., Nakatsu C. H. & Bennett, G. W. (2000). Feeding inhibition

Remmen, L. N. & Su, N.-Y. (2005). Tunneling and mortality of eastern and Formosan

Ramesh, A. & Balasubramanian, M. (1999). Kinetics and Hydrolysis of Fenamiphos,

Rust, M. K. & Saran, R. K. (2006). The toxicity, repellency, and transfer of chlorfenapyr

Rust, M. K. & Smith, J. L. (1993). Toxicity and repellency of components in formulated

imidacloprid-treated soils. *Journal of Economic Entomology,* 93: 422-428. Randall, M. and Doody, T. C. (1934). Wood preservatives and protective treatments, pp. 463-

thiamethoxam or fipronil. *Journal of Economic Entomology,* 98: 906–910. Rice, R. C., Jaynes, D. B. & Bowman, R. S. (1991). Preferential flow of solutes and herbicide

approach to managing subterranean termites (Isoptera: Rhinotermitidae) in

hydrolytic degradation of chlorpyrifos in soil, *Journal of Agricultural and Food* 

and mortality in *Reticulitermes flavipes* (Isoptera: Rhinotermitidae) after exposure to

476. In C.A. Kofoid [ed.] Termites and Termite Control. University of California

subterranean termites (Isoptera: Rhinotermitidae) in sand treated with

under irrigated fields. *Transactions of the American Society of Agricultural Engineers,*

Fipronil, and Trifluralin in Aqueous Buffer Solutions. *Journal of Agricultural and* 

against western subterranean termites (Isoptera: Rhinotermitidae). *Journal of* 

termiticides against western subterranean termites (Isoptera: Rhinotermitidae).

independent monitors. *Journal of Economic Entomology,* 98: 2160–2168. Parman, V. & Vargo, E. L. (2010). Colony-Level Effects of Imidacloprid in Subterranean Termites (Isoptera: Rhinotermitidae). *Journal of Economic Entomology,* 103: 791-798. Pawson, B. M. & R. E. Gold. (1996). Evaluation of baits for termites (Isoptera:

Rhinotermitidae) in Texas. *Sociobiology,* 28: 485-491.

buildings. *Sociobiology* 39: 373–405.

*Chemistry,* 44: 1582-1592.

Press, Berkeley, CA.

*Food Chemistry*, 47: 3367-3371.

*Economic Entomology,* 99: 864–872.

*Journal of Economic Entomology,* 86: 1131-1135.

34: 914-918.

application rate. *Pest Management Science*, 63: 1124-1132.

penetration. *Journal of Economic Entomology*. 103: 433-436.

soil at termiticidal application rates. *Pesticide Science* 42: 43-51.

on occurrence of Formosan subterranean termites (Isoptera: Rhinotermitidae) in


Kaufman, D. D., B. A. Russell, C. S. Helling, and A. Kayser. (1981). Movement of

Koehler, P.G., Su, N. –Y., Scheffrahn, R. H. & Oi, F.M. (2000). Baits to control subterranean

Kumar, M., & Philip, L. (2006). Adsorption and desorption characteristics of hydrophobic pesticide endosulfan in four Indian soils. *Chemosphere,* 62: 1064-1077. Kuriachan, I. & Gold, R. E. (1998). Evaluation of the ability of *Reticulitermes flavipes* Kollar, a

Lenz, M. (2009). Laboratory bioassays with subterranean termites (Isoptera) – the

Lenz, M., Barrett, R. A. & Williams, E. R.. (1984). Implications for comparability of

Li, X., He, J., & Li, S. (2007). Isolation of a chlorpyrifos-degrading bacterium, *Sphingomonas*

Lord, K. A., Mckinley, M. & Walker, N. (1982). Degradation of permethrin in soils.

Masters, G. M., (1991). Introduction to Environmental Engineering and Science, Prentice

Means, J. C., Wood, S. G. Hassett, J. J., & Banwart W. L. (1982). Sorption of amino-and

Mulrooney, J. E., & Gerard, P. D. (2007). Toxicity of fipronil in Mississippi soil types against *Reticulitermes flavipes* (Isoptera: Rhinotermitidae). *Sociobiology* 50: 63-70. Mulrooney, J. E., Wagner, T. L., Shelton, T. G., Peterson, C. J. & Gerard, P. D. (2007).

Ntow, W. J. (2005). Pesticide residues in Volta Lake, Ghana, Lakes Reservoirs. *Research and* 

Osbrink, W. L. A., & Lax, A. R. (2002). Effect of tolerance to insecticides on substrate

sp. strain Dsp-2, and cloning of the mpd gene. *Research in Microbiology* 158(2):14–

carboxy-substituted polynuclead aromatic hydrocarbons by sediments and soils.

Historical review of termite activity at Forest Service termiticide test sites from 1971

penetration by Formosan subterranean termites (Isoptera: Rhinotermitidae). *Journal* 

*Journal of Agricultural and Food Chemistry,* 29: 239-345.

Institute of Food and Agricultural Services.

*Environmental Toxicology & Chemistry* ,23: 1-6.

Sociobiology. 5: 197-203.

Hall, Englewood Cliffs, NJ.

*Management,* 10: 243-248.

*of Economic Entomology,* 95: 989-1000.

*Environmental Pollution,* 29 (2): 81-90.

*Environmental Science Technology,* 16: 93-98.

to 2004. *Journal of Economic Entomology,* 100: 488-494.

143.

importance of termite biology. *Sociobiology*, 53: 573-595.

cypermethrin, decamethrin, permethrin and their degradation productions in soil.

termites: the Sentricon system, ENY 2000, Florida Cooperative Extension Service,

subterranean termite (Isoptera: Rhinotermitidae), to differentiate between termiticide treated and untreated soils in laboratory tests. *Sociobiology,* 32: 151-166. Lee, S, Gan, J. Y., Kim, J. S., Kabashima, J. N. & Crowley, D. E. (2004). Microbial

transformation of pyrethroid insecticides in aqueous and sediment phases.

laboratory experiments revealed in studies on the effects of population density on vigor in *Coptotermes lacteus* (Froggatt) and *Nasutitermes exitious* (Hill) (Isoptera: Rhinotermitidae & Termitidae). *Bulletin of Entomological Research*. 74: 477-485. Lenz, M, Watson, J.A L., Barrett, R.A. & Runko, S. (1990). The effectiveness of insecticidal soil barriers against subterranean termites in Australia*. Sociobiology*, 17: 9-35. Lewis, D. L. (1980). Environmental and health aspects of termite control chemicals.


Factors Affecting Performance of Soil Termiticides 169

Su, N. -Y., Tamashiro, M., Yates, J. R. & Haverty, M. I. (1982). Effect of behavior on the

Su, N. -Y., Wheeler, G. S. & Scheffrahn, R. H. (1995). Subterranean termite (Isoptera:

Sunti, L. R., Shiu, W. Y., Mackay, D., Sieber, J. N. & Glotfelty, D. (1988). Critical review of

Tamashiro, M., Yates, J. R. & Ebesu, R. H. (1987). The Formosan termite in Hawaii: problems

Thorne, B. L. & Breisch, N. L. (2001). Effects of sublethal exposure to imidacloprid on

USEPA. (1986). U.S. Environmental Protection Agency. Pesticide fact sheet: chlordane. U.S.

USEPA. (2001). U.S. Environmental Protection Agency. Pesticide fact sheet: chlorfenapyr.

USEPA. (2000). U.S. Environmental Protection Agency. Pesticide fact sheet. Indoxacarb. U.S.

USEPA. (2008). U.S. Environmental Protection Agency. (2008). Pesticide fact sheet: chlorantraniliprole. U.S. Environmental Protection Agency. Washington, DC.

Ware, G.W. (2000). The Pesticide Book 5th Ed. Thompson Publications. Fresno, CA. 386

Wauchope, R. D., Yeh, S., Linders, J., Kloskowski, R., Tanaka, K., Rubin, B., Katayama, A.,

Wiltz, B. A. (2010). Laboratory Evaluation of Effects of Soil Properties on Termiticide

Wing, K. D., Sacher, M., Kagaya, Y., Tsurubuchi, Y., Mulderig, L., Connair, M. & Schnee, M.

Wood, T. G. & Pierce, M. J. (1991). Termites in Africa: The environmental impact of control

Worrall, F., Fernandez-Perez, M., Johnson, A. C., Flores-Cesperedes, F. & Gonzalez-Pradas,

pesticide leaching, *Journal of Contaminant Hydrology,* 49: 241–262.

Kördel, W., Gerstl, Z., Lane, M. & Unsworth, J. B. (2002). Review: Pesticide soil sorption parameters: theory, measurement, uses, limitations, and reliability. *Pest* 

Performance against Formosan Subterranean Termites (Isoptera: Rhinotermitidae*).* 

(2000). Bioactivation and mode of action of the oxadiazine indoxacarb in insects.

measures and damage to crops, trees, rangeland and rural buildings. *Sociobiology,*

E. (2001). Limitations on the role of incorporated organic matter in reducing

Rhinotermitidae). *Journal of Economic Entomology*, 94: 492–498.

Environmental Protection Agency. Washington, DC.

Environmental Protection Agency. Washington, DC.

U.S. Environmental Protection Agency. Washington, DC.

Wagner, T. L. (2003). U.S. Forest Service termiticide tests. *Sociobiology,* 41: 131–141.

subterranean termite. *Journal of Economic Entomology,* 75: 188-193.

termiticides. *Journal of Economic Entomology*, 88: 1690–1694.

Toxicology. 103: 1−59.

Hawaii, Honolulu.

pages.

*Management Science,* 58: 419–445.

*Sociobiology,* 56 (3): 755-773.

*Crop Protection*, 19: 537-545.

19: 221-234.

evaluation of insecticides for prevention of or remedial control of the Formosan

Rhinotermitidae) penetration into sand treated at various thicknesses with

Henry's law constants for pesticides. Reviews of Environmental Contamination &

and control, pp. 15-22. In Biology and control of the Formosan subterranean termite: M. Tamashiro and N. –Y. Su, eds., 67th Meeting of the Pacific Branch of the Entomological Society of America, Research Extension Series 083, University of

subsequent behavior of subterranean termite *Reticulitermes virginicus* (Isoptera:


Santos, C. A., DeSouza, O. & Guedes, R. N. C. (2004). Social facilitation attenuating

Saran, R. K., & Kamble, S. T. (2008). Concentration-dependent degradation of three

Saran, R. K. & Rust, M. K. (2007). The toxicity, uptake, and transfer efficiency of fipronil in

Shelton, T. G. & Grace, J. K. (2003). Effects of exposure duration on transfer of nonrepellent

Smith, B. C. & Zungoli, P. A. (1995). Rigid board insulation in South Carolina: its impact on

Smith, J. L. & Rust, M. K. (1990). Tunneling response and mortality of the western

Smith, J. L., & Rust, M. K. (1992). Activity of water-induced movement of termiticides in soil.

Smith, J. L. & Rust, M. K. (1993). Cellulose and clay in sand affects termiticide treatments.

Spomer, N. A., Kamble, S. T. & Siegfried, B. D. (2009). Bioavailability of chlorantraniliprole

Su, N.-Y. (1997). Protecting Historic Buildings and structures from termites. Cultural

Su, N. -Y. (2005). Response of the Formosan subterranean termites (Isoptera:

Su, N.-Y. & Scheffrahn, R. H. (1990a). Economically important termites in the United States

Su, N.-Y. & Scheffrahn, R. H. (1990b). Comparison of eleven soil termiticides against the

Su, N.-Y. & Scheffrahn, R. H. (1998). A review of subterranean termite control practices and

Su, N.-Y., Scheffrahn, R. H. & Ban P. M. (1993). Barrier efficacy of pyrethroid and

Rhinotermitidae). *Journal of Economic Entomology,* 83: 1918-1924.

Rhinotermitidae). *Journal of Economic Entomology,* 86: 772-776.

Rhinotermitidae*). Journal of Economic Entomology,* 96: 456–460.

various soils. *Journal of Economic Entomology,* 102: 1922-1927.

*Journal of Economic Entomology,* 83: 1395–1401.

*Journal of Economic Entomology,* 85: 430-434.

*Journal of Economic Entomology,* 86: 53-60.

http://crm.cr.nps.gov/issueindex.cfm.

and their control. *Sociobiology*, 17: 77-92.

*Reviews,* 3: 1 -13.

*Journal of Economic Entomology,* 98: 2143–2152.

539-546.

101: 1373-1383.

*Entomology,* 100: 495–508.

*Entomologist,* 78: 507-515.

insecticide-driven stress in termites (Isoptera: Nasutitermitinae). *Sociobiology*, 44:

termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (Isoptera: Rhinotermitidae). *Journal of Economic Entomology,*

western subterranean termites (Isoptera: Rhinotermitidae). *Journal of Economic* 

termiticides among workers of *Coptotermes formosanus* Shiraki (Isoptera:

damage, inspection and control of termites (Isoptera: Rhinotermitidae). *Florida* 

subterranean termite (Isoptera: Rhinotermitidae) to soil treated with termiticides.

and indoxacarb to eastern subterranean termites (Isoptera: Rhinotermitidae) in

Resource Management 20(7). National Park Service.

Rhinotermitidae) to baits or nonrepellent termiticides in extended foraging arenas.

Formosan subterranean termite and the eastern subterranean termite (Isoptera:

prospects for integrated pest management programs*. Integrated Pest Management* 

organophosphate formulations against subterranean termites (Isoptera:


**9** 

Eric J. Rebek1, Steven D. Frank2,

*1Oklahoma State University 2North Carolina State University* 

> *3Texas A&M University United States of America*

Tom A. Royer1 and Carlos E. Bográn3

**Alternatives to Chemical Control of Insect Pests** 

In 2011, practitioners and advocates of Integrated Pest Management (IPM) find themselves addressing agricultural, societal, and political pressures worldwide resulting from human population growth. This growth brings simultaneous burdens of sustaining a steady food supply; these include preventing losses from pests, dealing with increased human global travel, which in turn intensifies opportunities for the establishment of non-endemic pests into new ecosystems, and addressing global climate change that potentially will shift pest distributions into new areas. Concurrently, societal concerns about pesticide presence in our food and environment have resulted in political and economic pressures to reduce chemical pesticide use, or at a minimum, emphasize the development and use of products that are less toxic and more environmentally safe. These concerns drive the discovery and development of alternatives to chemical control of plant pathogens, weeds, and insect pests. The term Integrated Pest Management has, more often than not, been identified with entomologists. Stern et al. (1959) first used the term "integrated control" to describe the potential for integration of chemical and biological control tactics. Yet from a historical view, the concept of integrating chemical control with other tactics was proposed much earlier (Hoskins et al., 1939). Furthermore, integrating multiple non-chemical tactics to control a pest has been a cornerstone of the discipline of plant pathology throughout much of its early history (Jacobsen, 1997). In fact, because plant pathologists did not have an array of corrective pesticides available to them, the development and integration of control methods that emphasized non-pesticide controls (e.g., genetic host resistance, crop rotations, tillage, and plant sanitation) for plant diseases was a necessity, not simply an option for plant disease management. In contrast, entomologists and weed scientists were more insulated from that necessity due to the availability of relatively inexpensive pesticides to correct a problem. Several events stimulated the necessity for developing IPM programs in entomology, including those that emphasized development of non-chemical methods of insect control (e.g., cultural, biological, and physical control described herein). The chlorinated hydrocarbon, DDT, had been used for control of various insects since the 1950's. Soon after its use began, some pests began to develop resistance to DDT, including house flies, mosquitos, bed bugs, and body lice (Metcalf, 1989). The publication of Rachel Carson's book, "Silent Spring", in 1962 also generated public concern. Carson highlighted the negative

**1. Introduction** 


### **Alternatives to Chemical Control of Insect Pests**

Eric J. Rebek1, Steven D. Frank2,

Tom A. Royer1 and Carlos E. Bográn3 *1Oklahoma State University 2North Carolina State University 3Texas A&M University United States of America* 

#### **1. Introduction**

170 Insecticides – Basic and Other Applications

Xia, Y. & Brandenburg, R. L. (2000). Effect of irrigation on the efficacy of insecticides for

Xue, N., Zhang, D. & Xu, X., (2006). Organochlorinated pesticide multiresidues in surface sediments from Beijing Guanting reservoir. *Water Research,* 40: 183-194. Ying, G. G. & Kookana, R. S. (2006). Persistence and movement of fipronil termiticide with

courses. *Journal of Economic Entomology,* 93: 852–857.

2045–2050.

controlling two species of mole crickets (Orthoptera: Gryllotalpidae) on golf

under slab and trenching treatments. *Environmental Toxicology and Chemistry,* 25:

In 2011, practitioners and advocates of Integrated Pest Management (IPM) find themselves addressing agricultural, societal, and political pressures worldwide resulting from human population growth. This growth brings simultaneous burdens of sustaining a steady food supply; these include preventing losses from pests, dealing with increased human global travel, which in turn intensifies opportunities for the establishment of non-endemic pests into new ecosystems, and addressing global climate change that potentially will shift pest distributions into new areas. Concurrently, societal concerns about pesticide presence in our food and environment have resulted in political and economic pressures to reduce chemical pesticide use, or at a minimum, emphasize the development and use of products that are less toxic and more environmentally safe. These concerns drive the discovery and development of alternatives to chemical control of plant pathogens, weeds, and insect pests. The term Integrated Pest Management has, more often than not, been identified with entomologists. Stern et al. (1959) first used the term "integrated control" to describe the potential for integration of chemical and biological control tactics. Yet from a historical view, the concept of integrating chemical control with other tactics was proposed much earlier (Hoskins et al., 1939). Furthermore, integrating multiple non-chemical tactics to control a pest has been a cornerstone of the discipline of plant pathology throughout much of its early history (Jacobsen, 1997). In fact, because plant pathologists did not have an array of corrective pesticides available to them, the development and integration of control methods that emphasized non-pesticide controls (e.g., genetic host resistance, crop rotations, tillage, and plant sanitation) for plant diseases was a necessity, not simply an option for plant disease management. In contrast, entomologists and weed scientists were more insulated from that necessity due to the availability of relatively inexpensive pesticides to correct a problem.

Several events stimulated the necessity for developing IPM programs in entomology, including those that emphasized development of non-chemical methods of insect control (e.g., cultural, biological, and physical control described herein). The chlorinated hydrocarbon, DDT, had been used for control of various insects since the 1950's. Soon after its use began, some pests began to develop resistance to DDT, including house flies, mosquitos, bed bugs, and body lice (Metcalf, 1989). The publication of Rachel Carson's book, "Silent Spring", in 1962 also generated public concern. Carson highlighted the negative

Alternatives to Chemical Control of Insect Pests 173

Plant resistance to herbivores is a cultural control strategy having the most direct influence on herbivore behavior, fitness, and damage. Plant resistance is achieved through three general mechanisms: antibiosis, antixenosis, and tolerance. Antibiosis is the adverse effect of plant physical or chemical traits on arthropod biology (Painter, 1951). This may include reduced size, survival, fecundity, or longevity and increased development time or mortality. Antixenosis is the effect of plant traits on herbivore behavior that reduces herbivore interactions with the plant (Painter, 1951). These effects can include reduced feeding, preference, residence time, or oviposition on plants having particular traits such as trichomes or defensive compounds. Tolerance is a plant trait that reduces the impact of herbivory on plant growth, allowing tolerant plants to sustain herbivore damage but

Physical plant traits such as leaf pubescence, trichomes, and epicuticular wax, and chemical traits such as alkaloids and terpenoids have antibiotic and antixenotic effects on herbivores (Kennedy & Barbour, 1992; Painter, 1951). In the well-studied tomato production system, effects of leaf trichomes as a plant resistance trait are well documented (Kennedy, 2003; Simmons & Gurr, 2005). Trichomes and associated chemicals confer resistance to some tomato varieties against mites, aphids, whiteflies, beetles, and caterpillars (Gentile & Stoner, 1968; Heinz & Zalom, 1995; Kennedy, 2003; Kennedy & Sorenson, 1985; Simmons & Gurr, 2005). Trichomes are stiff hairs that sometimes contain chemical glands. Glandular trichomes have chemical exudates that confer resistance through antibiosis and kill or reduce longevity of pests feeding on them and entrap pests that forage on the leaves (Simmons & Gurr, 2005). Trichomes also have antixenotic effects on herbivore pests. Increasing trichome density can reduce oviposition by many species of beetles, caterpillars, true bugs, and mites. Of particular relevance is the effect of trichome density on whitefly and mites pests (Simmons & Gurr, 2005). The antibiotic and antixenotic effects of leaf pubescence on whitefly behavior and fitness have been studied in depth in a number of systems such as tomato, tobacco, cucurbits, and ornamental plants (Hoddle et al., 1998;

The soybean aphid offers a current example of how identifying pest resistance in crop plants can benefit IPM. Soybean aphid arrived in the U.S. from Asia in 2000 (Ragsdale et al., 2011). Since that time plant resistance conferred through antibiosis and antixenosis mechanisms has played an important role in mediating the economic impact of this pest on soybean yield (Ragsdale et al., 2011). Aphid fitness is negatively affected in resistant lines because it takes twice as long for aphids to probe into the phloem and initiate feeding (Diaz-Montano et al., 2007). Further, feeding bouts are reduced by more than 90% from less than 7 minutes per bout in resistant lines compared to greater than 60 minutes in susceptible lines (Diaz-Montano et al., 2007). Likewise, production of nymphs was reduced by 50-90% in resistant versus susceptible varieties, confirming antibiosis in resistant lines (Diaz-Montano et al., 2006; Hill et al., 2004). Antixenosis was also demonstrated in resistant varieties wherein adult aphids preferred to colonize susceptible over some resistant lines (Diaz-Montano et

In contrast to conventional breeding programs, plants can now be genetically modified to include lethal traits from other organisms, such as the bacterium, *Bacillus thuringiensis* (Bt). Bt genes are now used in many crop species to confer antibiosis in otherwise susceptible crops. Although we do not focus on this mode of plant resistance here, transgenic traits have had a tremendous effect on modern crop production and yield. However, like any

**2.1 Cultural control via plant resistance** 

Inbar & Gerling, 2008).

al., 2006; Hill et al., 2004).

maintain yields similar to undamaged plants (Painter, 1951).

impacts that widespread use of insecticides could have on the environment and ultimately, human health. What followed was a passionate global reaction that generated intense economic and political pressure to regulate pesticide use and monitor their relative impacts on biological systems. In the United States, the Environmental Protection Agency was created and charged with regulating the registration of all pesticides through the Federal Insecticide, Fungicide and Rodenticide Act (as amended in 1972). Concerns over pesticide use also stimulated the political thrust necessary for support of IPM programs. In the United States and worldwide, IPM flourished in the following three decades and was adopted as policy by various governments (Kogan, 1998).

Today, IPM has attained many successes but fallen short on some issues. Due to the awareness and biological understanding of how insecticide resistance develops, and because insecticides are so expensive to develop, in 1984 the manufacturers of insecticides created the Insecticide Resistance Action Committee (IRAC) to encourage the responsible use of their products in a manner that minimizes the risk of insecticide in target pest populations (IRAC, 2010). New calls have been made for changing the direction of IPM in response to waning political support for funding IPM programs. Frisbie & Smith (1989) coined the term "biologically intensive" IPM, which involves reliance on ecological methods of control based on knowledge of a pest's biology. Benbrook et al. (1996) promoted the idea of moving IPM along a continuum from simple to complex, or 'biointensive". The National Research Council officially introduced the term, "Ecologically Based Pest Management", calling for a new paradigm for IPM in the 21st Century (National Research Council, 1996); eight years earlier, however, Horn (1988) outlined how principles of insect ecology could be incorporated into insect pest management strategies. More recently, Koul & Cuperus (2007) published "Ecologically Based Integrated Pest Management", essentially capturing the breadth and depth of the evolution that IPM has undergone over the past 60 years. While the scope of the "New Solutions" aspect of the NRC's charge has been challenged (Kogan, 1998; Royer et al., 1999), the term "ecologically based" has become infused into the IPM lexicon.

#### **2. Cultural control methods to reduce insecticide applications**

Cultural controls are management tools and activities that make the crop habitat less favorable for pests to survive and cause damage (Horne & Page, 2008). Cultural management practices may make the crop or habitat inhospitable to pests directly, for example, by planting cultivars resistant to pest feeding or rotating crops to deny overwintering pests their preferred food source. Cultural management practices can also make the habitat less hospitable to pests in an indirect manner by encouraging natural enemies (predators and parasitoids) to enhance biological control (see Section 3).

Cultural control is a key pest management tool available to growers because the crop variety, habitat, and selected inputs set the stage for future pest fitness and abundance. Thus, implementing preventive cultural control tactics that slow pest population growth can delay or negate the need for insecticide applications and significant plant damage. In this section we outline the major types of cultural control tactics available to growers and other pest management personnel. Our objective is to demonstrate the breadth of tactics that are used, although we do not have the space to consider them in depth. We draw examples from a diversity of well-studied plant systems from field crops to ornamental landscapes to provide examples of how they affect plant-herbivore-natural enemy interactions to reduce pest abundance and damage.

impacts that widespread use of insecticides could have on the environment and ultimately, human health. What followed was a passionate global reaction that generated intense economic and political pressure to regulate pesticide use and monitor their relative impacts on biological systems. In the United States, the Environmental Protection Agency was created and charged with regulating the registration of all pesticides through the Federal Insecticide, Fungicide and Rodenticide Act (as amended in 1972). Concerns over pesticide use also stimulated the political thrust necessary for support of IPM programs. In the United States and worldwide, IPM flourished in the following three decades and was adopted as

Today, IPM has attained many successes but fallen short on some issues. Due to the awareness and biological understanding of how insecticide resistance develops, and because insecticides are so expensive to develop, in 1984 the manufacturers of insecticides created the Insecticide Resistance Action Committee (IRAC) to encourage the responsible use of their products in a manner that minimizes the risk of insecticide in target pest populations (IRAC, 2010). New calls have been made for changing the direction of IPM in response to waning political support for funding IPM programs. Frisbie & Smith (1989) coined the term "biologically intensive" IPM, which involves reliance on ecological methods of control based on knowledge of a pest's biology. Benbrook et al. (1996) promoted the idea of moving IPM along a continuum from simple to complex, or 'biointensive". The National Research Council officially introduced the term, "Ecologically Based Pest Management", calling for a new paradigm for IPM in the 21st Century (National Research Council, 1996); eight years earlier, however, Horn (1988) outlined how principles of insect ecology could be incorporated into insect pest management strategies. More recently, Koul & Cuperus (2007) published "Ecologically Based Integrated Pest Management", essentially capturing the breadth and depth of the evolution that IPM has undergone over the past 60 years. While the scope of the "New Solutions" aspect of the NRC's charge has been challenged (Kogan, 1998; Royer et al., 1999), the term "ecologically based" has become infused into the IPM

**2. Cultural control methods to reduce insecticide applications** 

enemies (predators and parasitoids) to enhance biological control (see Section 3).

Cultural controls are management tools and activities that make the crop habitat less favorable for pests to survive and cause damage (Horne & Page, 2008). Cultural management practices may make the crop or habitat inhospitable to pests directly, for example, by planting cultivars resistant to pest feeding or rotating crops to deny overwintering pests their preferred food source. Cultural management practices can also make the habitat less hospitable to pests in an indirect manner by encouraging natural

Cultural control is a key pest management tool available to growers because the crop variety, habitat, and selected inputs set the stage for future pest fitness and abundance. Thus, implementing preventive cultural control tactics that slow pest population growth can delay or negate the need for insecticide applications and significant plant damage. In this section we outline the major types of cultural control tactics available to growers and other pest management personnel. Our objective is to demonstrate the breadth of tactics that are used, although we do not have the space to consider them in depth. We draw examples from a diversity of well-studied plant systems from field crops to ornamental landscapes to provide examples of how they affect plant-herbivore-natural enemy interactions to reduce

policy by various governments (Kogan, 1998).

lexicon.

pest abundance and damage.

#### **2.1 Cultural control via plant resistance**

Plant resistance to herbivores is a cultural control strategy having the most direct influence on herbivore behavior, fitness, and damage. Plant resistance is achieved through three general mechanisms: antibiosis, antixenosis, and tolerance. Antibiosis is the adverse effect of plant physical or chemical traits on arthropod biology (Painter, 1951). This may include reduced size, survival, fecundity, or longevity and increased development time or mortality. Antixenosis is the effect of plant traits on herbivore behavior that reduces herbivore interactions with the plant (Painter, 1951). These effects can include reduced feeding, preference, residence time, or oviposition on plants having particular traits such as trichomes or defensive compounds. Tolerance is a plant trait that reduces the impact of herbivory on plant growth, allowing tolerant plants to sustain herbivore damage but maintain yields similar to undamaged plants (Painter, 1951).

Physical plant traits such as leaf pubescence, trichomes, and epicuticular wax, and chemical traits such as alkaloids and terpenoids have antibiotic and antixenotic effects on herbivores (Kennedy & Barbour, 1992; Painter, 1951). In the well-studied tomato production system, effects of leaf trichomes as a plant resistance trait are well documented (Kennedy, 2003; Simmons & Gurr, 2005). Trichomes and associated chemicals confer resistance to some tomato varieties against mites, aphids, whiteflies, beetles, and caterpillars (Gentile & Stoner, 1968; Heinz & Zalom, 1995; Kennedy, 2003; Kennedy & Sorenson, 1985; Simmons & Gurr, 2005). Trichomes are stiff hairs that sometimes contain chemical glands. Glandular trichomes have chemical exudates that confer resistance through antibiosis and kill or reduce longevity of pests feeding on them and entrap pests that forage on the leaves (Simmons & Gurr, 2005). Trichomes also have antixenotic effects on herbivore pests. Increasing trichome density can reduce oviposition by many species of beetles, caterpillars, true bugs, and mites. Of particular relevance is the effect of trichome density on whitefly and mites pests (Simmons & Gurr, 2005). The antibiotic and antixenotic effects of leaf pubescence on whitefly behavior and fitness have been studied in depth in a number of systems such as tomato, tobacco, cucurbits, and ornamental plants (Hoddle et al., 1998; Inbar & Gerling, 2008).

The soybean aphid offers a current example of how identifying pest resistance in crop plants can benefit IPM. Soybean aphid arrived in the U.S. from Asia in 2000 (Ragsdale et al., 2011). Since that time plant resistance conferred through antibiosis and antixenosis mechanisms has played an important role in mediating the economic impact of this pest on soybean yield (Ragsdale et al., 2011). Aphid fitness is negatively affected in resistant lines because it takes twice as long for aphids to probe into the phloem and initiate feeding (Diaz-Montano et al., 2007). Further, feeding bouts are reduced by more than 90% from less than 7 minutes per bout in resistant lines compared to greater than 60 minutes in susceptible lines (Diaz-Montano et al., 2007). Likewise, production of nymphs was reduced by 50-90% in resistant versus susceptible varieties, confirming antibiosis in resistant lines (Diaz-Montano et al., 2006; Hill et al., 2004). Antixenosis was also demonstrated in resistant varieties wherein adult aphids preferred to colonize susceptible over some resistant lines (Diaz-Montano et al., 2006; Hill et al., 2004).

In contrast to conventional breeding programs, plants can now be genetically modified to include lethal traits from other organisms, such as the bacterium, *Bacillus thuringiensis* (Bt). Bt genes are now used in many crop species to confer antibiosis in otherwise susceptible crops. Although we do not focus on this mode of plant resistance here, transgenic traits have had a tremendous effect on modern crop production and yield. However, like any

Alternatives to Chemical Control of Insect Pests 175

theoretical and empirical research suggests that multiple resistance traits or genes and a combination of different modes of action such as antibiosis and antixenosis should confer more stable resistance to crops. In addition, mixing resistant and susceptible varieties in the same field can reduce evolution of resistance by insect pest populations (Gould, 1986, 1998).

Plant fertility and water stress play a major role in plant susceptibility to herbivore feeding, tolerance to herbivore damage, and herbivore population growth. Nitrogen can be a limiting nutrient for herbivorous insects due to the nitrogen-poor quality of their host plants (Mattson, 1980). Therefore, increasing nitrogen concentration within plants by applying fertilizer has a tendency to increase plant quality for herbivores (Mattson, 1980). Increasing foliar nitrogen can reduce pest development time and increase survival and fecundity, leading to more rapid population growth (Mattson, 1980). Research in potato crops has found that increasing nitrogen fertilization increases leaf consumption, reduces development time, and increases abundance of Colorado potato beetles (Boiteau et al., 2008). Likewise, in greenhouse ornamental production, increasing fertilization increases the fecundity, body size, and development rate of citrus mealybug (Hogendorp et al., 2006), and through similar mechanisms increases population growth rates of whiteflies, thrips, aphids, and spider mites (Bentz et al., 1995; Chau et al., 2005; Chau & Heinz, 2006; Chow et al.,

In ornamental landscapes, fertilizer is often applied to improve the growth of trees and other plants and increase their resistance to abiotic and biotic stress, including herbivore feeding. However, nitrogen fertilization of trees has been shown to reduce plant resistance to many arthropod pests (Herms, 2002; Kytö et al., 1996). This reduced resistance occurs through a combination of fertilizer effects on plant nutrition for herbivores and defense against herbivores (Herms & Mattson, 1992). Herms & Mattson (1992) hypothesized that as nitrogen fertilization stimulates rapid plant growth, carbon available for production of defensive compounds is limited. Thus, over-fertilization of trees, shrubs, and other plants provides a dual benefit to many herbivores via increased nitrogen availability and decreased

Pesticide applications are often an essential aspect of plant culture. Managing the type and frequency of applications is a cultural control tactic with well-documented implications. Insecticides can disrupt natural enemy communities and biological control via several mechanisms. Direct toxicity of pyrethroids and organophosphates to natural enemies has been documented frequently (Desneux et al., 2004b; see Galvan et al., 2005). Direct toxicity of insecticides to natural enemies results in smaller natural enemy populations on crop and landscape plants (Frank & Sadof, *in press*; Raupp et al., 2001). Insecticides also cause sublethal effects in parasitoids and predators. For example, the pyrethroid, lambdacyhalothrin, disrupts the host location and oviposition behavior of *Aphidius ervi*, resulting in

Non-target impacts on natural enemy communities are not limited to contact insecticides. Systemic neonicotinoids such as imidacloprid and thiamethoxam have lethal and sublethal effects on natural enemy development, fitness, and efficacy (Cloyd & Bethke, 2009; Desneux et al., 2007). These compounds can reduce survival of developing parasitoids and intoxicate

**2.2 Cultural control via fertility management** 

defensive compounds (Raupp et al., 2010).

**2.3 Cultural control via pesticide selection and management** 

lower parasitism rates of aphids (Desneux et al., 2004a).

2009).

management tactic, Bt crops do not function in a vacuum and effects on natural enemies and other non-targets, secondary pest outbreaks, and evolution of pest resistance have been intensely studied (Gould, 1998; O'Callaghan et al., 2005; Shelton et al., 2002).

#### **2.1.1 Interaction of plant resistance traits and biological control**

Effects of plant resistance and biological control can be contradictory, complementary, or synergistic (Cai et al., 2009; Farid et al., 1998; Johnson & Gould, 1992). Plant resistance can work in conjunction with natural enemies to maintain low pest abundance and damage. In general, natural enemies have slower population growth rates than pests. Thus, by reducing pest population growth rates, plant resistance may help natural enemies better regulate pest populations. For example, research in wheat systems has shown that aphid-resistant wheat varieties do not have negative effects on parasitoid life history parameters such as size and development time (Farid et al., 1998). Parasitism rates may be equal or greater on resistant varieties, which when combined with reduced aphid population growth due to host plant resistance, can improve pest management dramatically (Cai et al., 2009).

Just as trichome exudates reduce herbivore survival they can also have negative effects on natural enemies. Survival and development of natural enemies may be reduced by poisoning or entrapping them, and natural enemy foraging efficiency, predation, or parasitism rate may be inhibited (Kaufman & Kennedy, 1989a, b; Obrycki & Tauber, 1984; Simmons & Gurr, 2005). For example, increasing trichome density and related changes in chemical composition of tomato leaves reduced the walking speed, parasitism rate, and survival of the egg parasitoid, *Trichogramma pretiosum* (Kashyap et al., 1991a, b). Tiny whitefly parasitoids in the genera, *Eretmocerus* and *Encarsia*, are highly affected by plant pubescence and trichome density (Hoddle et al., 1998; van Lenteren et al., 1995). Biological control may be disrupted because these parasitoids avoid highly pubescent plants. Once on the plants, pubescence reduces walking speed, foraging efficiency, oviposition, and parasitism rate (De Barro et al., 2000; Headrick et al., 1996; Hoddle et al., 1998; Inbar & Gerling, 2008).

Trichomes and other plant resistance traits also affect predator behavior and efficacy. Predatory mites used in biological control of spider mite, *Tetranychus urticae*, are readily trapped by trichomes and forage less efficiently due to reduced mobility (Nihoul, 1993a; van Haren et al., 1987). The consequence of mortality and reduced foraging efficiency is reduced biological control on cultivars with high trichome density, although the effect is also dependent on environmental factors such as temperature (Nihoul 1993a, b). Likewise, foraging efficiency of the spotted lady beetle, *Coleomegilla maculata*, and the bigeyed bug, *Geocoris punctipes*, was reduced by high trichome density, resulting in less predation of *Heliothis zea* eggs (Barbour et al., 1993, 1997). Increasing pubescence on poinsettia leaves by just 15% reduced oviposition and whitefly predation by *Delphastus catalinae* and other predators (Heinz & Parrella, 1994).

#### **2.1.2 Herbivore resistance to plant resistance traits**

Herbivores are in a constant evolutionary arms race with plants to overcome resistance traits (Ehrlich & Raven, 1964). It is not surprising then that pests have developed physiological resistance to genetically modified and conventional plant resistance traits (Gould, 1998). For example, certain soybean aphid biotypes are resistant to Rag1 or Rag2 genes that confer resistance to soybean plants (Hill et al., 2009, 2010). Evidence from

management tactic, Bt crops do not function in a vacuum and effects on natural enemies and other non-targets, secondary pest outbreaks, and evolution of pest resistance have been

Effects of plant resistance and biological control can be contradictory, complementary, or synergistic (Cai et al., 2009; Farid et al., 1998; Johnson & Gould, 1992). Plant resistance can work in conjunction with natural enemies to maintain low pest abundance and damage. In general, natural enemies have slower population growth rates than pests. Thus, by reducing pest population growth rates, plant resistance may help natural enemies better regulate pest populations. For example, research in wheat systems has shown that aphid-resistant wheat varieties do not have negative effects on parasitoid life history parameters such as size and development time (Farid et al., 1998). Parasitism rates may be equal or greater on resistant varieties, which when combined with reduced aphid population growth due to host plant

Just as trichome exudates reduce herbivore survival they can also have negative effects on natural enemies. Survival and development of natural enemies may be reduced by poisoning or entrapping them, and natural enemy foraging efficiency, predation, or parasitism rate may be inhibited (Kaufman & Kennedy, 1989a, b; Obrycki & Tauber, 1984; Simmons & Gurr, 2005). For example, increasing trichome density and related changes in chemical composition of tomato leaves reduced the walking speed, parasitism rate, and survival of the egg parasitoid, *Trichogramma pretiosum* (Kashyap et al., 1991a, b). Tiny whitefly parasitoids in the genera, *Eretmocerus* and *Encarsia*, are highly affected by plant pubescence and trichome density (Hoddle et al., 1998; van Lenteren et al., 1995). Biological control may be disrupted because these parasitoids avoid highly pubescent plants. Once on the plants, pubescence reduces walking speed, foraging efficiency, oviposition, and parasitism rate (De Barro et al., 2000; Headrick et al., 1996; Hoddle et al., 1998; Inbar &

Trichomes and other plant resistance traits also affect predator behavior and efficacy. Predatory mites used in biological control of spider mite, *Tetranychus urticae*, are readily trapped by trichomes and forage less efficiently due to reduced mobility (Nihoul, 1993a; van Haren et al., 1987). The consequence of mortality and reduced foraging efficiency is reduced biological control on cultivars with high trichome density, although the effect is also dependent on environmental factors such as temperature (Nihoul 1993a, b). Likewise, foraging efficiency of the spotted lady beetle, *Coleomegilla maculata*, and the bigeyed bug, *Geocoris punctipes*, was reduced by high trichome density, resulting in less predation of *Heliothis zea* eggs (Barbour et al., 1993, 1997). Increasing pubescence on poinsettia leaves by just 15% reduced oviposition and whitefly predation by *Delphastus catalinae* and other

Herbivores are in a constant evolutionary arms race with plants to overcome resistance traits (Ehrlich & Raven, 1964). It is not surprising then that pests have developed physiological resistance to genetically modified and conventional plant resistance traits (Gould, 1998). For example, certain soybean aphid biotypes are resistant to Rag1 or Rag2 genes that confer resistance to soybean plants (Hill et al., 2009, 2010). Evidence from

intensely studied (Gould, 1998; O'Callaghan et al., 2005; Shelton et al., 2002).

**2.1.1 Interaction of plant resistance traits and biological control** 

resistance, can improve pest management dramatically (Cai et al., 2009).

Gerling, 2008).

predators (Heinz & Parrella, 1994).

**2.1.2 Herbivore resistance to plant resistance traits** 

theoretical and empirical research suggests that multiple resistance traits or genes and a combination of different modes of action such as antibiosis and antixenosis should confer more stable resistance to crops. In addition, mixing resistant and susceptible varieties in the same field can reduce evolution of resistance by insect pest populations (Gould, 1986, 1998).

#### **2.2 Cultural control via fertility management**

Plant fertility and water stress play a major role in plant susceptibility to herbivore feeding, tolerance to herbivore damage, and herbivore population growth. Nitrogen can be a limiting nutrient for herbivorous insects due to the nitrogen-poor quality of their host plants (Mattson, 1980). Therefore, increasing nitrogen concentration within plants by applying fertilizer has a tendency to increase plant quality for herbivores (Mattson, 1980). Increasing foliar nitrogen can reduce pest development time and increase survival and fecundity, leading to more rapid population growth (Mattson, 1980). Research in potato crops has found that increasing nitrogen fertilization increases leaf consumption, reduces development time, and increases abundance of Colorado potato beetles (Boiteau et al., 2008). Likewise, in greenhouse ornamental production, increasing fertilization increases the fecundity, body size, and development rate of citrus mealybug (Hogendorp et al., 2006), and through similar mechanisms increases population growth rates of whiteflies, thrips, aphids, and spider mites (Bentz et al., 1995; Chau et al., 2005; Chau & Heinz, 2006; Chow et al., 2009).

In ornamental landscapes, fertilizer is often applied to improve the growth of trees and other plants and increase their resistance to abiotic and biotic stress, including herbivore feeding. However, nitrogen fertilization of trees has been shown to reduce plant resistance to many arthropod pests (Herms, 2002; Kytö et al., 1996). This reduced resistance occurs through a combination of fertilizer effects on plant nutrition for herbivores and defense against herbivores (Herms & Mattson, 1992). Herms & Mattson (1992) hypothesized that as nitrogen fertilization stimulates rapid plant growth, carbon available for production of defensive compounds is limited. Thus, over-fertilization of trees, shrubs, and other plants provides a dual benefit to many herbivores via increased nitrogen availability and decreased defensive compounds (Raupp et al., 2010).

#### **2.3 Cultural control via pesticide selection and management**

Pesticide applications are often an essential aspect of plant culture. Managing the type and frequency of applications is a cultural control tactic with well-documented implications. Insecticides can disrupt natural enemy communities and biological control via several mechanisms. Direct toxicity of pyrethroids and organophosphates to natural enemies has been documented frequently (Desneux et al., 2004b; see Galvan et al., 2005). Direct toxicity of insecticides to natural enemies results in smaller natural enemy populations on crop and landscape plants (Frank & Sadof, *in press*; Raupp et al., 2001). Insecticides also cause sublethal effects in parasitoids and predators. For example, the pyrethroid, lambdacyhalothrin, disrupts the host location and oviposition behavior of *Aphidius ervi*, resulting in lower parasitism rates of aphids (Desneux et al., 2004a).

Non-target impacts on natural enemy communities are not limited to contact insecticides. Systemic neonicotinoids such as imidacloprid and thiamethoxam have lethal and sublethal effects on natural enemy development, fitness, and efficacy (Cloyd & Bethke, 2009; Desneux et al., 2007). These compounds can reduce survival of developing parasitoids and intoxicate

Alternatives to Chemical Control of Insect Pests 177

and Northern corn rootworm *Diabrotica barberi*, damage in corn (Levine & Oloumi-Sadeghi, 1991; Spencer et al., 2009). Corn rootworm eggs overwinter in corn fields and larvae are present to feed on corn roots the following year (Spencer et al., 2009). Therefore, rotating to a different crop such as soybeans denies food to hatching rootworm larvae (Spencer et al., 2009). Likewise, corn planted after soybeans or other crops has less rootworm damage because the field is free of overwintering eggs and larvae. However, Western and Northern corn rootworm populations eventually developed resistance to this strategy (Gray et al., 2009; Levine et al., 2002; Spencer & Levine, 2008). Northern corn rootworms circumvent crop rotation by prolonging egg diapause for two winters instead of one (Chiang, 1965; Levine et al., 1992). Therefore, larvae hatch when fields are replanted in corn two years after the eggs were laid. Western corn rootworm has become resistant to crop rotation by a behavioral rather than physiological mechanism. Western corn rootworm adults move from corn fields to soybean and other crop fields, feeding on soybean leaves and ovipositing in soybean fields (Levine et al., 2002). Selection pressure imposed by rotation of two primary crops, corn and soybeans, strongly rewarded female beetles that strayed from corn for

Other planting practices such as delayed planting dates can also benefit pest control. Hessian fly is an introduced pest of winter wheat that has been in the U.S. since the 1700's. Prior to the development of resistant wheat varieties, growers exploited the fly's life cycle to reduce damage to winter wheat crops. Hessian fly adults become active in the fall when they oviposit in wheat and other grasses. By planting after a "fly free date" when fly activity subsides, winter wheat is protected from oviposition from the fall hessian fly generation (Buntin et al., 1991). This is a perfect example of how simple changes in plant culture can reduce the need for insecticide applications, increase yield, and provide economic benefit to

Many definitions of biological control have been published in the literature since the term was first used by H.S. Smith more than 90 years ago (Caltagirone & Huffaker, 1980; Cook, 1987; Coppel & Mertins, 1977; DeBach & Rosen, 1991; Garcia et al., 1988; see Huffaker & Messenger, 1976; Perkins & Garcia, 1999; Rabb, 1972; Smith, 1919). In its strictest sense, biological control is the use of beneficial organisms to reduce the relative abundance of, and damage caused by, noxious ones. This definition attributes economic rather than biological characters to organisms that fall into two categories, beneficial and noxious, based on their positive or negative impact on human-valued resources. It is also important to distinguish *biological* from *natural* control, which does not require human intervention, and from similar methods of pest control that do not involve whole (living) organisms (Huffaker et al., 1984). In fact, biological control involves interspecific, population-level processes by way of predation, parasitism, competition, or a combination of these mechanisms (van Driesche & Bellows, 1996). In practice, the effectiveness and appropriateness of biological control methods rely on real-time evolutionary forces that shape the beneficial organism's genotype, phenotype, and performance. This is not the case for similar, biologically based methods such as the application of insecticides formulated with pathogens, antagonists, or their byproducts. Furthermore, in its strictest definition, biological control does not include the deployment of pest-tolerant organisms, regardless of the source or origin of the resistance-

oviposition.

growers (Buntin et al., 1992).

**3. Biological control of insect pests** 

conferring characters (e.g., Bt crops) (see Perkins & Garcia, 1999).

predators such as lady beetles and lacewing larvae exposed to the chemicals topically or by feeding on exposed prey (Moser & Obrycki, 2009; Papachristos & Milonas, 2008; Smith & Krischik, 1999; Szczepaniec et al., 2011). Parasitoids are also affected negatively via feeding on plant nectar or hosts exposed to the chemicals (Krischik et al., 2007; Rebek & Sadof, 2003). The consequence of disrupting natural enemy populations can be outbreaks of primary or secondary pests due to the loss of underlying biological control services (Raupp et al., 2010). Considerable work has documented this effect in field crops, orchards, vineyards, and landscape ornamentals (Penman & Chapman, 1988; Raupp et al., 2010). The effect is particularly prevalent among spider mites and scale insects that are not killed as easily as their natural enemies by insecticide applications. Pyrethroids and other broad-spectrum insecticides have direct and indirect effects on spider mites that can promote mite outbreaks. First, pyrethroids promote spider mite dispersal from treated to untreated areas of reduced competition (Iftner & Hall, 1983; Penman & Chapman, 1983). Spider mites have many predators including lady beetles, predatory bugs, lacewing larvae, and predatory mites. Pyrethroids can promote outbreaks of spider mites indirectly by killing the natural enemies that otherwise help suppress spider mite populations (Penman & Chapman, 1988).

Predatory mites in the family Phytoseiidae feed on spider mite eggs, juveniles, and adults and are effective at reducing spider mite abundance and damage on plants (McMurtry & Croft, 1997). In addition, phytoseiid mites often respond with a numerical increase to burgeoning spider mite populations via aggregation and increased reproduction. However, the abundance and efficacy of phytoseiid mites depends in large part on plant culture practices and plant characteristics. Phytoseiid mites are extremely susceptible to insecticides such as pyrethroids, organophosphates, and carbamates (Hardman et al., 1988). In many cases, phytoseiids have been found to be more vulnerable to these insecticides than spider mites (e.g., Sanford, 1967; Wong and Chapman, 1979). Therefore, by killing a disproportionate number of predatory mites compared to target pests, broad-spectrum insecticides frequently lead to spider mite outbreaks (Hardman et al., 1988). Similar dynamics have been demonstrated for scale insects, which are generally not killed by cover sprays of contact insecticides due to their protective cover. Moreover, by drastically reducing natural enemy abundance and efficacy, these insecticide applications create enemy-free space for scales, which can result in outbreak populations (McClure, 1977; Raupp et al., 2001).

Insecticide applications can directly benefit pest reproduction and survival through a process known as hormoligosis. Increased spider mite fecundity has been demonstrated after exposure to sublethal doses of pyrethroids (Iftner & Hall, 1984; Jones & Parrella, 1984). However, this is most evident in spider mites that frequently outbreak after applications of the neonicotinoid, imidacloprid (Gupta & Krischik, 2007; Raupp et al., 2004; Sclar et al., 1998; Szczepaniec et al., 2011). Outbreaks are triggered in part by negative effects on predators, but also by greater fecundity of spider mites that feed on imidacloprid-treated foliage (Szczepaniec et al., 2011). Although not commonly observed, this phenomenon points out another reason for proper insecticide management as a cultural control strategy.

#### **2.4 Cultural control via crop rotation and planting practices**

Exploiting the biological limitations of the pest to minimize insecticide applications is the essence of cultural control tactics such as crop rotation. This strategy has been used successfully to control corn rootworm for over 100 years (Forbes, 1883). Crop rotation has been highly effective as a tool to reduce Western corn rootworm, *Diabrotica virgifera virgifera,* 

predators such as lady beetles and lacewing larvae exposed to the chemicals topically or by feeding on exposed prey (Moser & Obrycki, 2009; Papachristos & Milonas, 2008; Smith & Krischik, 1999; Szczepaniec et al., 2011). Parasitoids are also affected negatively via feeding on plant nectar or hosts exposed to the chemicals (Krischik et al., 2007; Rebek & Sadof, 2003). The consequence of disrupting natural enemy populations can be outbreaks of primary or secondary pests due to the loss of underlying biological control services (Raupp et al., 2010). Considerable work has documented this effect in field crops, orchards, vineyards, and landscape ornamentals (Penman & Chapman, 1988; Raupp et al., 2010). The effect is particularly prevalent among spider mites and scale insects that are not killed as easily as their natural enemies by insecticide applications. Pyrethroids and other broad-spectrum insecticides have direct and indirect effects on spider mites that can promote mite outbreaks. First, pyrethroids promote spider mite dispersal from treated to untreated areas of reduced competition (Iftner & Hall, 1983; Penman & Chapman, 1983). Spider mites have many predators including lady beetles, predatory bugs, lacewing larvae, and predatory mites. Pyrethroids can promote outbreaks of spider mites indirectly by killing the natural enemies

that otherwise help suppress spider mite populations (Penman & Chapman, 1988).

Raupp et al., 2001).

Predatory mites in the family Phytoseiidae feed on spider mite eggs, juveniles, and adults and are effective at reducing spider mite abundance and damage on plants (McMurtry & Croft, 1997). In addition, phytoseiid mites often respond with a numerical increase to burgeoning spider mite populations via aggregation and increased reproduction. However, the abundance and efficacy of phytoseiid mites depends in large part on plant culture practices and plant characteristics. Phytoseiid mites are extremely susceptible to insecticides such as pyrethroids, organophosphates, and carbamates (Hardman et al., 1988). In many cases, phytoseiids have been found to be more vulnerable to these insecticides than spider mites (e.g., Sanford, 1967; Wong and Chapman, 1979). Therefore, by killing a disproportionate number of predatory mites compared to target pests, broad-spectrum insecticides frequently lead to spider mite outbreaks (Hardman et al., 1988). Similar dynamics have been demonstrated for scale insects, which are generally not killed by cover sprays of contact insecticides due to their protective cover. Moreover, by drastically reducing natural enemy abundance and efficacy, these insecticide applications create enemy-free space for scales, which can result in outbreak populations (McClure, 1977;

Insecticide applications can directly benefit pest reproduction and survival through a process known as hormoligosis. Increased spider mite fecundity has been demonstrated after exposure to sublethal doses of pyrethroids (Iftner & Hall, 1984; Jones & Parrella, 1984). However, this is most evident in spider mites that frequently outbreak after applications of the neonicotinoid, imidacloprid (Gupta & Krischik, 2007; Raupp et al., 2004; Sclar et al., 1998; Szczepaniec et al., 2011). Outbreaks are triggered in part by negative effects on predators, but also by greater fecundity of spider mites that feed on imidacloprid-treated foliage (Szczepaniec et al., 2011). Although not commonly observed, this phenomenon points out

Exploiting the biological limitations of the pest to minimize insecticide applications is the essence of cultural control tactics such as crop rotation. This strategy has been used successfully to control corn rootworm for over 100 years (Forbes, 1883). Crop rotation has been highly effective as a tool to reduce Western corn rootworm, *Diabrotica virgifera virgifera,* 

another reason for proper insecticide management as a cultural control strategy.

**2.4 Cultural control via crop rotation and planting practices** 

and Northern corn rootworm *Diabrotica barberi*, damage in corn (Levine & Oloumi-Sadeghi, 1991; Spencer et al., 2009). Corn rootworm eggs overwinter in corn fields and larvae are present to feed on corn roots the following year (Spencer et al., 2009). Therefore, rotating to a different crop such as soybeans denies food to hatching rootworm larvae (Spencer et al., 2009). Likewise, corn planted after soybeans or other crops has less rootworm damage because the field is free of overwintering eggs and larvae. However, Western and Northern corn rootworm populations eventually developed resistance to this strategy (Gray et al., 2009; Levine et al., 2002; Spencer & Levine, 2008). Northern corn rootworms circumvent crop rotation by prolonging egg diapause for two winters instead of one (Chiang, 1965; Levine et al., 1992). Therefore, larvae hatch when fields are replanted in corn two years after the eggs were laid. Western corn rootworm has become resistant to crop rotation by a behavioral rather than physiological mechanism. Western corn rootworm adults move from corn fields to soybean and other crop fields, feeding on soybean leaves and ovipositing in soybean fields (Levine et al., 2002). Selection pressure imposed by rotation of two primary crops, corn and soybeans, strongly rewarded female beetles that strayed from corn for oviposition.

Other planting practices such as delayed planting dates can also benefit pest control. Hessian fly is an introduced pest of winter wheat that has been in the U.S. since the 1700's. Prior to the development of resistant wheat varieties, growers exploited the fly's life cycle to reduce damage to winter wheat crops. Hessian fly adults become active in the fall when they oviposit in wheat and other grasses. By planting after a "fly free date" when fly activity subsides, winter wheat is protected from oviposition from the fall hessian fly generation (Buntin et al., 1991). This is a perfect example of how simple changes in plant culture can reduce the need for insecticide applications, increase yield, and provide economic benefit to growers (Buntin et al., 1992).

#### **3. Biological control of insect pests**

Many definitions of biological control have been published in the literature since the term was first used by H.S. Smith more than 90 years ago (Caltagirone & Huffaker, 1980; Cook, 1987; Coppel & Mertins, 1977; DeBach & Rosen, 1991; Garcia et al., 1988; see Huffaker & Messenger, 1976; Perkins & Garcia, 1999; Rabb, 1972; Smith, 1919). In its strictest sense, biological control is the use of beneficial organisms to reduce the relative abundance of, and damage caused by, noxious ones. This definition attributes economic rather than biological characters to organisms that fall into two categories, beneficial and noxious, based on their positive or negative impact on human-valued resources. It is also important to distinguish *biological* from *natural* control, which does not require human intervention, and from similar methods of pest control that do not involve whole (living) organisms (Huffaker et al., 1984). In fact, biological control involves interspecific, population-level processes by way of predation, parasitism, competition, or a combination of these mechanisms (van Driesche & Bellows, 1996). In practice, the effectiveness and appropriateness of biological control methods rely on real-time evolutionary forces that shape the beneficial organism's genotype, phenotype, and performance. This is not the case for similar, biologically based methods such as the application of insecticides formulated with pathogens, antagonists, or their byproducts. Furthermore, in its strictest definition, biological control does not include the deployment of pest-tolerant organisms, regardless of the source or origin of the resistanceconferring characters (e.g., Bt crops) (see Perkins & Garcia, 1999).

Alternatives to Chemical Control of Insect Pests 179

typically mass reared in an insectary, either to inoculate or inundate the target area of impact (Obrycki et al., 1997; Parrella et al., 1992; Ridgway, 1998). Inoculative releases involve relatively low numbers of natural enemies, typically when pest populations are low or at the beginning of a growth cycle or season. Inundation involves relatively high numbers of natural enemies released repeatedly throughout the growth cycle or season. Thus, inundative release of natural enemies is similar to insecticide use in that releases are made when pests achieve high enough density to cause economic harm to the crop. In both types of release, the objective is to inflict high mortality by synchronizing the life cycles of the pest and natural enemy. Hence, an effective monitoring program of pest populations is essential

Augmentation biological control has been used successfully against key pests of field and greenhouse crops. A well-known example of augmentation biological control is the use of the parasitoid, *Encarsia formosa*, for control of greenhouse whitefly (Hoddle et al., 1998). Indeed, augmentation plays an important role in greenhouse production, especially in Europe, and many natural enemies are commercially available for control of perennial greenhouse pests such as spider mites, aphids, scales, and whiteflies (Grant, 1997; Pottorff & Panter, 2009). The success of augmentative releases in greenhouses, and elsewhere, depends on the compatibility of cultural practices such as insecticide use with natural enemies (see Section 2.3). Greenhouses are often ideal sites for augmentation biological control because of the relative stability of the enclosed environment. In contrast, a critical review of augmentation biological control in field crops revealed that augmentation was typically less effective and more expensive than conventional control with pesticides (Collier & van Steenwyk, 2004). The authors found that the low success rate of augmentation biological control in field crops is influenced by ecological limitations such as unfavorable environmental conditions, natural enemy dispersal, and refuge for herbivores from released

Conservation biological control involves any practice that increases colonization, establishment, reproduction, and survival of native or previously established natural enemies (Landis et al., 2000). Conservation biological control can be achieved in two ways: modifying pesticide use and manipulating the growing environment in favor of natural enemies. Conservation practices have proven effective in a wide variety of growing situations ranging from small garden plots to large fields, agricultural to urban environments, and commercial to private settings (Frank & Shrewsbury, 2004; Landis et al.,

Modifications to pesticide regimens include reducing or eliminating insecticide use, using pest-specific insecticides when needed, making applications when beneficial arthropods are not active, and making treatment decisions based on monitoring and the presence of vulnerable life stages. While total independence from chemical control is not feasible for most situations, reductions in insecticide use are possible through IPM programs based on rigorous pest monitoring, established treatment thresholds, and/or insect population models (see Horn, 1988; Pimental, 1997). Thus, insecticides are used only when needed to prevent crop damage that results in economic loss. When insecticide use is warranted,

2000; Rebek et al., 2005, 2006; Sadof et al., 2004; Tooker & Hanks, 2000).

**3.3.1 Conserving natural enemies via modified pesticide use** 

to the success of augmentation biological control.

natural enemies.

**3.3 Conservation biological control** 

The history and origins of biological control have been extensively covered in previous volumes (Caltagirone & Doutt, 1989; DeBach & Rosen, 1991; van Driesche & Bellows, 1996) and is not the subject of this review. However, it is significant to note that early theory and application of biological control principles pre-date the modern insecticide era (Smith, 1919). Therefore, it is modern insecticides that became an alternative to biological control and not the other way around. In this context, biological control should not be viewed as a novel tactic but as the foundation of a successful pest management strategy involving, at minimum, the conservation of ecosystem resources to facilitate the process of pest-natural enemy colonization, host/prey finding, and ultimately, damage reduction. Although what constitutes biological control (or not) continues to be a subject of discussion and will likely evolve with new technologies, the recognition of three principal biological control methods remains unchanged. These three approaches are importation (a.k.a., classical biological control), augmentation, and conservation biological control (Smith, 1919).

#### **3.1 Importation biological control**

Importation biological control is the oldest of the three approaches (hence its alternative name, 'classical'). The first successful case of importation biological control occurred over a century ago in the control of cottony cushion scale in California citrus following importation of the vedalia beetle (Horn, 1988). The classical approach involves re-establishing the interspecific interactions (and their impact on population regulation) between pests and their natural enemies (i.e., predators, parasitoids, or insect-killing pathogens) as they occur in the pest's endemic range (Howarth, 1983). The need to re-establish these interactions arises because pests are commonly introduced into areas outside their native range where they lack natural enemies, or those that are present do not significantly impact the pest's abundance and local distribution. Since its inception, importation biological control has been used with varying degrees of success against noxious pests like cassava mealybug in Africa, Rhodesgrass mealybug in Texas, walnut aphid in California, and southern green stink bug in Australia, New Zealand, and Hawaii (Hokkanen, 1997).

The technical expertise, time commitment, and considerable expense necessary to carry out importation biological control require the involvement of specially trained university and government scientists. Importation is highly regulated in many countries, largely due to growing concern over the introduction of exotic, invasive species into new environments. In the U.S., the Animal and Plant Health Inspection Service (APHIS) oversees and coordinates importation biological control programs. The agency's charge is to preserve the safety and effectiveness of biological control primarily through post-release monitoring of biological control agents (USDA APHIS, 2011). Although there are a few documented cases of introduced biological control agents causing economic or ecological harm, societal perceptions that importation biological control is too risky are often influenced by subjectivity and misinformation (Delfosse, 2005). To minimize risk, researchers must provide evidence that introduced natural enemies are unlikely to harm crops, humans, and ecosystems. This requires substantial analysis of host feeding preference and other biological traits of prospective biological control agents (see Briese, 2005).

#### **3.2 Augmentation biological control**

The aim of augmentation biological control is to improve the numerical ratio between pest and natural enemy to increase pest mortality. It involves the release of natural enemies,

The history and origins of biological control have been extensively covered in previous volumes (Caltagirone & Doutt, 1989; DeBach & Rosen, 1991; van Driesche & Bellows, 1996) and is not the subject of this review. However, it is significant to note that early theory and application of biological control principles pre-date the modern insecticide era (Smith, 1919). Therefore, it is modern insecticides that became an alternative to biological control and not the other way around. In this context, biological control should not be viewed as a novel tactic but as the foundation of a successful pest management strategy involving, at minimum, the conservation of ecosystem resources to facilitate the process of pest-natural enemy colonization, host/prey finding, and ultimately, damage reduction. Although what constitutes biological control (or not) continues to be a subject of discussion and will likely evolve with new technologies, the recognition of three principal biological control methods remains unchanged. These three approaches are importation (a.k.a., classical biological

Importation biological control is the oldest of the three approaches (hence its alternative name, 'classical'). The first successful case of importation biological control occurred over a century ago in the control of cottony cushion scale in California citrus following importation of the vedalia beetle (Horn, 1988). The classical approach involves re-establishing the interspecific interactions (and their impact on population regulation) between pests and their natural enemies (i.e., predators, parasitoids, or insect-killing pathogens) as they occur in the pest's endemic range (Howarth, 1983). The need to re-establish these interactions arises because pests are commonly introduced into areas outside their native range where they lack natural enemies, or those that are present do not significantly impact the pest's abundance and local distribution. Since its inception, importation biological control has been used with varying degrees of success against noxious pests like cassava mealybug in Africa, Rhodesgrass mealybug in Texas, walnut aphid in California, and southern green stink bug

The technical expertise, time commitment, and considerable expense necessary to carry out importation biological control require the involvement of specially trained university and government scientists. Importation is highly regulated in many countries, largely due to growing concern over the introduction of exotic, invasive species into new environments. In the U.S., the Animal and Plant Health Inspection Service (APHIS) oversees and coordinates importation biological control programs. The agency's charge is to preserve the safety and effectiveness of biological control primarily through post-release monitoring of biological control agents (USDA APHIS, 2011). Although there are a few documented cases of introduced biological control agents causing economic or ecological harm, societal perceptions that importation biological control is too risky are often influenced by subjectivity and misinformation (Delfosse, 2005). To minimize risk, researchers must provide evidence that introduced natural enemies are unlikely to harm crops, humans, and ecosystems. This requires substantial analysis of host feeding preference and other

The aim of augmentation biological control is to improve the numerical ratio between pest and natural enemy to increase pest mortality. It involves the release of natural enemies,

control), augmentation, and conservation biological control (Smith, 1919).

in Australia, New Zealand, and Hawaii (Hokkanen, 1997).

biological traits of prospective biological control agents (see Briese, 2005).

**3.1 Importation biological control** 

**3.2 Augmentation biological control** 

typically mass reared in an insectary, either to inoculate or inundate the target area of impact (Obrycki et al., 1997; Parrella et al., 1992; Ridgway, 1998). Inoculative releases involve relatively low numbers of natural enemies, typically when pest populations are low or at the beginning of a growth cycle or season. Inundation involves relatively high numbers of natural enemies released repeatedly throughout the growth cycle or season. Thus, inundative release of natural enemies is similar to insecticide use in that releases are made when pests achieve high enough density to cause economic harm to the crop. In both types of release, the objective is to inflict high mortality by synchronizing the life cycles of the pest and natural enemy. Hence, an effective monitoring program of pest populations is essential to the success of augmentation biological control.

Augmentation biological control has been used successfully against key pests of field and greenhouse crops. A well-known example of augmentation biological control is the use of the parasitoid, *Encarsia formosa*, for control of greenhouse whitefly (Hoddle et al., 1998). Indeed, augmentation plays an important role in greenhouse production, especially in Europe, and many natural enemies are commercially available for control of perennial greenhouse pests such as spider mites, aphids, scales, and whiteflies (Grant, 1997; Pottorff & Panter, 2009). The success of augmentative releases in greenhouses, and elsewhere, depends on the compatibility of cultural practices such as insecticide use with natural enemies (see Section 2.3). Greenhouses are often ideal sites for augmentation biological control because of the relative stability of the enclosed environment. In contrast, a critical review of augmentation biological control in field crops revealed that augmentation was typically less effective and more expensive than conventional control with pesticides (Collier & van Steenwyk, 2004). The authors found that the low success rate of augmentation biological control in field crops is influenced by ecological limitations such as unfavorable environmental conditions, natural enemy dispersal, and refuge for herbivores from released natural enemies.

#### **3.3 Conservation biological control**

Conservation biological control involves any practice that increases colonization, establishment, reproduction, and survival of native or previously established natural enemies (Landis et al., 2000). Conservation biological control can be achieved in two ways: modifying pesticide use and manipulating the growing environment in favor of natural enemies. Conservation practices have proven effective in a wide variety of growing situations ranging from small garden plots to large fields, agricultural to urban environments, and commercial to private settings (Frank & Shrewsbury, 2004; Landis et al., 2000; Rebek et al., 2005, 2006; Sadof et al., 2004; Tooker & Hanks, 2000).

#### **3.3.1 Conserving natural enemies via modified pesticide use**

Modifications to pesticide regimens include reducing or eliminating insecticide use, using pest-specific insecticides when needed, making applications when beneficial arthropods are not active, and making treatment decisions based on monitoring and the presence of vulnerable life stages. While total independence from chemical control is not feasible for most situations, reductions in insecticide use are possible through IPM programs based on rigorous pest monitoring, established treatment thresholds, and/or insect population models (see Horn, 1988; Pimental, 1997). Thus, insecticides are used only when needed to prevent crop damage that results in economic loss. When insecticide use is warranted,

Alternatives to Chemical Control of Insect Pests 181

Lenteren, 1980). In practice, the successful application of biological control usually requires a combination of at least two of the three approaches, importation, augmentation, and conservation of natural enemies (DeBach & Rosen, 1991; van Driesche & Bellows, 1996). What drives the success or failure of biological control programs in plant crops has been the subject of many analyses, either using historical records or theoretical approaches (Andow et al., 1997; Murdoch et al., 1985; Murdoch & Briggs, 1996; van Lenteren, 1980). In general terms, biological control programs are more likely to succeed under certain production systems and environmental conditions (Clausen, 1978; van Driesche & Heinz, 2004). Biological control has been more successful in crops that: 1) are perennial versus annual; 2) grow in areas with few pests versus many pests; 3) the harvested portion is not damaged by the target pest; 4) the target pest is not a disease vector; and 5) the aesthetic damage is

Failures in biological control programs, especially those recorded in the literature, also involve cases where the biology and ecology of the natural enemy or the pests are not well understood or altogether unknown. Historically, failures in importation biological control have occurred after errors in identification of a pest or natural enemy at the level of species, biotype, or even local strain; a mismatch in micro-environmental requirements for natural enemy growth and development; incorrectly timing natural enemy release when the production system is not conducive to establishment; or when socioeconomic or regulatory barrier have prevented adoption or implementation (Clausen, 1978; Greathead, 1976; Hall & Ehler, 1979; Knutson, 1981). Similarly, failures in augmentation and conservation biological control, although not commonly recorded in the literature, may be due to a lack of understanding of the basic biology and ecology of the species involved, the basic requirements of the production system, and any socioeconomic barriers including real or perceived costs and benefits (Murdoch et al., 1985; Perkins & Garcia, 1999; Collier & van Steenwyk, 2004). The success of biological control programs involves integrated efforts at many levels ranging from biology to economics, from research to implementation and

Plant health can benefit greatly from preventing or limiting injury from arthropod pests from the start. Indeed, the cornerstone of an effective IPM program is prevention, which can be achieved, in part, through physical control. Physical control strategies include methods for excluding pests or limiting their access to crops, disrupting pest behavior, or causing direct mortality (Vincent et al., 2009). Physical control methods can be categorized as active and passive (Vincent et al., 2009). Active methods involve the removal of individual pests by hand, pruning out infested plant tissues, and rogueing out heavily infested plants. Passive methods usually include the use of a device or tool for excluding or removing pests from a crop. Typically, these devices serve as barriers between plants and insect pests, thus protecting plants from injury and damage. Other passive tools include repellents and traps. While traps are often used for monitoring pest abundance and distribution, many are designed as "attract and kill" technologies, which attract insect pests through color, light,

The greatest disadvantage to physical control is that these methods can be laborious and time consuming, especially for crops grown in large areas. Also, a moderate degree of specialization or training is often required due to the highly technical nature of some

acceptable (e.g., some food and fiber crops versus ornamentals).

experience, and from the farm to the community and region.

shape, texture, and scent, or a combination of these factors.

**4. Physical control strategies to reduce pest incidence** 

adverse effects on natural enemies can be minimized by using selective, pest-specific products that are only effective against the target pest and its close relatives. Selective chemistries include microbial insecticides, insect growth regulators, botanicals, and novel insecticides with specific modes of action against target insects. Alternatively, insecticide applications can be timed so they not coincide with natural enemy activity; dormant or inactive predators and parasitoids are not exposed to broad-spectrum insecticides applied when they are dormant or inactive (van Driesche & Bellows, 1996). This strategy requires a thorough understanding of the crop, agroecosystem, and the biology and life cycle of important natural enemies in the system.

#### **3.3.2 Conserving natural enemies via habitat manipulation**

Natural enemies are attracted to habitats rich in food, shelter, and nesting sites (Landis et al., 2000; Rabb et al., 1976). Many perennial plants can provide these resources when incorporated into the system. Ellis et al. (2005) and Rebek et al. (2005) independently observed significantly enhanced parasitism of two key ornamental pests, bagworm and euonymus scale, in experimental plots containing nectar and pollen sources (i.e., resource plants). Resource plants also served as refuge for vertebrate predators of bagworms as evidenced by increased predation rates (Ellis et al., 2005). Resource plants can harbor alternative prey/host species, which sustain adult and immature natural enemies when primary prey/hosts are scarce. For example, many studies have focused on the influence of banker plants, which contain alternative prey species, on natural enemy effectiveness (see Frank, 2010).

Resource plants provide more than food to enhance natural enemy abundance in impoverished landscapes. Suitable changes in microclimate are afforded by many plants, tempering environmental extremes by providing improved conditions for natural enemy survival (Rabb et al., 1976). Candidate plants include small trees, shrubs, bushy perennials, and tall ornamental grasses with dense canopies or complex architecture. Similarly, organic mulches and ground cover plants can support large numbers of ground-dwelling predators like spiders and ground beetles (Bell et al., 2002; Mathews et al., 2004; Rieux et al., 1999; Snodgrass & Stadelbacher, 1989), which may enhance biological control of key pests (Brust, 1994). Finally, resource plants can enhance reproduction of natural enemies and provide refuge from their own enemies (Landis et al., 2000; Rabb et al., 1976).

The effectiveness of habitat manipulation to improve biological control requires careful planning and selection of plant attributes that are appropriate for the natural enemy complex present in the system (Landis et al., 2000). For example, flower morphology can significantly impact nectar accessibility by foraging parasitoids (Patt et al., 1997; Wäckers, 2004). Also important is coincidence of floral bloom with natural enemy activity. Selected resource plants should overlap in blooming periods to ensure a continuous supply of nectar and pollen to natural enemies (Bowie et al., 1995; Rebek et al., 2005). Other considerations that exceed the scope of this chapter include the influence of landscape-level attributes on biological control at different spatial scales (Kruess & Tscharntke, 1994; Marino & Landis, 1996; Roland & Taylor, 1997).

#### **3.4 Factors affecting success of biological control**

While there have been some tremendous successes, the worldwide rate of effective biological control is estimated to be between 16-25% (Hall et al., 1980; Horn, 1988; van

adverse effects on natural enemies can be minimized by using selective, pest-specific products that are only effective against the target pest and its close relatives. Selective chemistries include microbial insecticides, insect growth regulators, botanicals, and novel insecticides with specific modes of action against target insects. Alternatively, insecticide applications can be timed so they not coincide with natural enemy activity; dormant or inactive predators and parasitoids are not exposed to broad-spectrum insecticides applied when they are dormant or inactive (van Driesche & Bellows, 1996). This strategy requires a thorough understanding of the crop, agroecosystem, and the biology and life cycle of

Natural enemies are attracted to habitats rich in food, shelter, and nesting sites (Landis et al., 2000; Rabb et al., 1976). Many perennial plants can provide these resources when incorporated into the system. Ellis et al. (2005) and Rebek et al. (2005) independently observed significantly enhanced parasitism of two key ornamental pests, bagworm and euonymus scale, in experimental plots containing nectar and pollen sources (i.e., resource plants). Resource plants also served as refuge for vertebrate predators of bagworms as evidenced by increased predation rates (Ellis et al., 2005). Resource plants can harbor alternative prey/host species, which sustain adult and immature natural enemies when primary prey/hosts are scarce. For example, many studies have focused on the influence of banker plants, which contain alternative prey species, on natural enemy effectiveness (see

Resource plants provide more than food to enhance natural enemy abundance in impoverished landscapes. Suitable changes in microclimate are afforded by many plants, tempering environmental extremes by providing improved conditions for natural enemy survival (Rabb et al., 1976). Candidate plants include small trees, shrubs, bushy perennials, and tall ornamental grasses with dense canopies or complex architecture. Similarly, organic mulches and ground cover plants can support large numbers of ground-dwelling predators like spiders and ground beetles (Bell et al., 2002; Mathews et al., 2004; Rieux et al., 1999; Snodgrass & Stadelbacher, 1989), which may enhance biological control of key pests (Brust, 1994). Finally, resource plants can enhance reproduction of natural enemies and provide

The effectiveness of habitat manipulation to improve biological control requires careful planning and selection of plant attributes that are appropriate for the natural enemy complex present in the system (Landis et al., 2000). For example, flower morphology can significantly impact nectar accessibility by foraging parasitoids (Patt et al., 1997; Wäckers, 2004). Also important is coincidence of floral bloom with natural enemy activity. Selected resource plants should overlap in blooming periods to ensure a continuous supply of nectar and pollen to natural enemies (Bowie et al., 1995; Rebek et al., 2005). Other considerations that exceed the scope of this chapter include the influence of landscape-level attributes on biological control at different spatial scales (Kruess & Tscharntke, 1994; Marino & Landis,

While there have been some tremendous successes, the worldwide rate of effective biological control is estimated to be between 16-25% (Hall et al., 1980; Horn, 1988; van

important natural enemies in the system.

Frank, 2010).

1996; Roland & Taylor, 1997).

**3.4 Factors affecting success of biological control** 

**3.3.2 Conserving natural enemies via habitat manipulation** 

refuge from their own enemies (Landis et al., 2000; Rabb et al., 1976).

Lenteren, 1980). In practice, the successful application of biological control usually requires a combination of at least two of the three approaches, importation, augmentation, and conservation of natural enemies (DeBach & Rosen, 1991; van Driesche & Bellows, 1996). What drives the success or failure of biological control programs in plant crops has been the subject of many analyses, either using historical records or theoretical approaches (Andow et al., 1997; Murdoch et al., 1985; Murdoch & Briggs, 1996; van Lenteren, 1980). In general terms, biological control programs are more likely to succeed under certain production systems and environmental conditions (Clausen, 1978; van Driesche & Heinz, 2004). Biological control has been more successful in crops that: 1) are perennial versus annual; 2) grow in areas with few pests versus many pests; 3) the harvested portion is not damaged by the target pest; 4) the target pest is not a disease vector; and 5) the aesthetic damage is acceptable (e.g., some food and fiber crops versus ornamentals).

Failures in biological control programs, especially those recorded in the literature, also involve cases where the biology and ecology of the natural enemy or the pests are not well understood or altogether unknown. Historically, failures in importation biological control have occurred after errors in identification of a pest or natural enemy at the level of species, biotype, or even local strain; a mismatch in micro-environmental requirements for natural enemy growth and development; incorrectly timing natural enemy release when the production system is not conducive to establishment; or when socioeconomic or regulatory barrier have prevented adoption or implementation (Clausen, 1978; Greathead, 1976; Hall & Ehler, 1979; Knutson, 1981). Similarly, failures in augmentation and conservation biological control, although not commonly recorded in the literature, may be due to a lack of understanding of the basic biology and ecology of the species involved, the basic requirements of the production system, and any socioeconomic barriers including real or perceived costs and benefits (Murdoch et al., 1985; Perkins & Garcia, 1999; Collier & van Steenwyk, 2004). The success of biological control programs involves integrated efforts at many levels ranging from biology to economics, from research to implementation and experience, and from the farm to the community and region.

#### **4. Physical control strategies to reduce pest incidence**

Plant health can benefit greatly from preventing or limiting injury from arthropod pests from the start. Indeed, the cornerstone of an effective IPM program is prevention, which can be achieved, in part, through physical control. Physical control strategies include methods for excluding pests or limiting their access to crops, disrupting pest behavior, or causing direct mortality (Vincent et al., 2009). Physical control methods can be categorized as active and passive (Vincent et al., 2009). Active methods involve the removal of individual pests by hand, pruning out infested plant tissues, and rogueing out heavily infested plants. Passive methods usually include the use of a device or tool for excluding or removing pests from a crop. Typically, these devices serve as barriers between plants and insect pests, thus protecting plants from injury and damage. Other passive tools include repellents and traps. While traps are often used for monitoring pest abundance and distribution, many are designed as "attract and kill" technologies, which attract insect pests through color, light, shape, texture, and scent, or a combination of these factors.

The greatest disadvantage to physical control is that these methods can be laborious and time consuming, especially for crops grown in large areas. Also, a moderate degree of specialization or training is often required due to the highly technical nature of some

Alternatives to Chemical Control of Insect Pests 183

crop and estimate their relative abundance in order to properly time an insecticide application or natural enemy release. Pheromones and other olfactory stimuli are receiving increased attention as repellents and attractants in push-pull strategies for modifying pest behavior (see Cook et al., 2007). Repellents include synthetic chemicals (e.g., DEET), nonhost volatiles that mask host plant odors (e.g., essential oils), anti-aggregation and alarm pheromones, anti-feedants (e.g., neem oil), and oviposition deterrents (e.g., ovipositiondeterring pheromones) (Cook et al., 2007). Herbivore-induced plant volatiles are host plant semiochemicals that induce plant defense from herbivores and attract natural enemies (James, 2003). Non-chemical repellents include reflective mulches, which have been shown to reduce damage and population density of tarnished plant bug in strawberry fields (Rhainds et al., 2001). Attractants include sex and aggregation pheromones, host plant volatiles, and feeding stimulants (e.g., baits), and oviposition stimulants (Cook et al., 2007). Other attractants are based on visual cues. For example, apple maggots are effectively controlled in apple orchards with 8-cm, red, spherical traps covered in adhesive. The attractiveness of these traps is enhanced by adding butyl hexanoate and ammonium acetate,

Another common tactic is to use sex pheromones for mating disruption. Many insect pests rely on a species-specific, sex pheromone produced by females for mate location and recognition. Mating disruption is achieved by flooding the crop environment with the chemical signal, thus confusing males and reducing mate-finding success. This approach has been used with varying degrees of success for management of orchard and vineyard pests including codling moth, oriental fruit moth, grape berry moth, and peachtree borer (see

Insects can be killed directly through mechanical, thermal, or other means. Vincent et al. (2009) list several strategies that inflict mortality on pests including freezing, heating, flaming, crushing, and irradiating. One of the most common mechanical methods requires no specialized equipment – many gardeners derive great satisfaction from hand picking pests from a plant and crushing them. Hand removal can be used effectively for a myriad of relatively sessile landscape pests including bagworms, tent caterpillars, and sawfly larvae. Galls, egg masses, and web-making insects can also be pruned out of infested landscape plants (Potter, 1986). However, this tactic may be impractical for large trees or shrubs and dense populations of the pest. Other mechanical control options require specialized machinery. Pneumatic control involves removing pests from crops by use of a vacuum or blower and subsequently destroying them. Field crop pests such as Colorado potato beetle and lygus bug have been controlled in this manner, although care must be taken to avoid negatively impacting natural enemies (Vincent et al., 2003, 2009). Another example of mechanized destruction is the entoleter, an impact machine that is used in mills to remove

Modifying the microclimate can be effective in killing many insect pests, which cannot survive outside of optimal temperature and humidity ranges. Heat has been shown to be a very effective control method for bed bugs, which are difficult to control and are becoming more prevalent in domestic dwellings worldwide (Pereira et al., 2009). A wide variety of stored product pests can be controlled by pumping hot or cold air into the food storage facility, or modifying the storage environment with elevated temperatures and carbon

synthetic olfactory stimulants (Prokopy et al., 1994).

**4.3 Physical control via pest destruction** 

and kill all life stages of insect pests (Vincent et al., 2003).

Zalom, 1997).

physical control methods. Physical control methods may also be difficult or practically impossible in some crops like large trees grown in extensive monocultures (e.g., timber production). For many crops, however, physical control of certain pests can be incorporated into established routines for managing crops. Despite the drawbacks and considering the costs, regulations, and limitations of insecticide use, physical control methods are likely candidates for inclusion in many pest management programs, especially for high-value crops (see Vincent et al. 2003). Here, we discuss briefly some examples of physical control classified by their primary function: exclusion, behavior modification, and destruction of pests.

#### **4.1 Physical control via exclusion**

Pest exclusion is a key factor in preventing pests from accessing crops, thereby reducing the economic impact of insects. Both passive and active exclusion methods have been implemented in various agricultural systems including fields, greenhouses, and postharvest facilities. Physical control via exclusion devices is perhaps most important in protected environments such as greenhouses and grain bins, where optimal temperatures and humidity, a readily available food source, and a general lack of natural enemies contribute to the proliferation of pest populations. Screens are common passive exclusion devices used in greenhouse production. Screens can prevent pest migration into greenhouses through vents and other openings, especially when insect populations build up in weeds and crops in the surrounding environment (Gill et al., 2006; Pottorff & Panter, 2009). However, screen mesh size is an important concern as fine materials with small openings inhibit entry of tiny arthropods such as thrips and mites but also restrict air flow for cooling (Pottorff & Panter, 2009). Other active methods of physical control are necessary components of greenhouse IPM. Specifically, crops should be inspected for pests prior to moving new plant materials into production areas; discovered pests are removed by hand, pruned out, or discarded and destroyed with heavily infested plants.

In the field, floating row covers can exclude important vegetable pests such as cabbage maggot, flea beetles, and cabbageworm (Rekika et al., 2008; Theriault et al., 2009). Adhesives and burlap have been used to trap caterpillar pests such as gypsy moth and cankerworms as they migrate vertically along tree trunks (Potter, 1986). Other barriers include fences, ditches, moats, or trenches. For example, V-shaped trenches have been used around potato fields to prevent movement of Colorado potato beetle into the crop from adjacent, overwintering habitat (Boiteau & Vernon, 2001; Misener et al., 1993; see Vincent et al., 2003). Efficacy of this technique relies on trench design and knowledge of the pest, specifically, the population size and the ratio of crawling to flying individuals (Weber et al., 1994; Vincent et al., 2003).

#### **4.2 Physical control via behavior modification**

IPM programs often consist of physical control methods that alter the behavior of insect pests. Behaviors such as reproduction, aggregation, oviposition, feeding, alarm, and defense can be modified in two ways: "push-pull" strategies and mating disruption (Cook et al., 2007; Zalom, 1997). The former are designed to repel (push) or attract (pull) insect pests away from a crop by exploiting their reproductive, feeding, or aggregation behavior. Although many repellents and attractants are chemically based, here we treat their use in IPM as a form of non-chemical (non-insecticidal) control.

Pheromones, or chemical lures, are used in IPM programs to monitor pest populations and modify their behavior. Specifically, pheromone traps are used to detect pest activity in a

physical control methods. Physical control methods may also be difficult or practically impossible in some crops like large trees grown in extensive monocultures (e.g., timber production). For many crops, however, physical control of certain pests can be incorporated into established routines for managing crops. Despite the drawbacks and considering the costs, regulations, and limitations of insecticide use, physical control methods are likely candidates for inclusion in many pest management programs, especially for high-value crops (see Vincent et al. 2003). Here, we discuss briefly some examples of physical control classified by their primary function: exclusion, behavior modification, and destruction of

Pest exclusion is a key factor in preventing pests from accessing crops, thereby reducing the economic impact of insects. Both passive and active exclusion methods have been implemented in various agricultural systems including fields, greenhouses, and postharvest facilities. Physical control via exclusion devices is perhaps most important in protected environments such as greenhouses and grain bins, where optimal temperatures and humidity, a readily available food source, and a general lack of natural enemies contribute to the proliferation of pest populations. Screens are common passive exclusion devices used in greenhouse production. Screens can prevent pest migration into greenhouses through vents and other openings, especially when insect populations build up in weeds and crops in the surrounding environment (Gill et al., 2006; Pottorff & Panter, 2009). However, screen mesh size is an important concern as fine materials with small openings inhibit entry of tiny arthropods such as thrips and mites but also restrict air flow for cooling (Pottorff & Panter, 2009). Other active methods of physical control are necessary components of greenhouse IPM. Specifically, crops should be inspected for pests prior to moving new plant materials into production areas; discovered pests are removed by hand, pruned out, or discarded and

In the field, floating row covers can exclude important vegetable pests such as cabbage maggot, flea beetles, and cabbageworm (Rekika et al., 2008; Theriault et al., 2009). Adhesives and burlap have been used to trap caterpillar pests such as gypsy moth and cankerworms as they migrate vertically along tree trunks (Potter, 1986). Other barriers include fences, ditches, moats, or trenches. For example, V-shaped trenches have been used around potato fields to prevent movement of Colorado potato beetle into the crop from adjacent, overwintering habitat (Boiteau & Vernon, 2001; Misener et al., 1993; see Vincent et al., 2003). Efficacy of this technique relies on trench design and knowledge of the pest, specifically, the population size

IPM programs often consist of physical control methods that alter the behavior of insect pests. Behaviors such as reproduction, aggregation, oviposition, feeding, alarm, and defense can be modified in two ways: "push-pull" strategies and mating disruption (Cook et al., 2007; Zalom, 1997). The former are designed to repel (push) or attract (pull) insect pests away from a crop by exploiting their reproductive, feeding, or aggregation behavior. Although many repellents and attractants are chemically based, here we treat their use in

Pheromones, or chemical lures, are used in IPM programs to monitor pest populations and modify their behavior. Specifically, pheromone traps are used to detect pest activity in a

and the ratio of crawling to flying individuals (Weber et al., 1994; Vincent et al., 2003).

pests.

**4.1 Physical control via exclusion** 

destroyed with heavily infested plants.

**4.2 Physical control via behavior modification** 

IPM as a form of non-chemical (non-insecticidal) control.

crop and estimate their relative abundance in order to properly time an insecticide application or natural enemy release. Pheromones and other olfactory stimuli are receiving increased attention as repellents and attractants in push-pull strategies for modifying pest behavior (see Cook et al., 2007). Repellents include synthetic chemicals (e.g., DEET), nonhost volatiles that mask host plant odors (e.g., essential oils), anti-aggregation and alarm pheromones, anti-feedants (e.g., neem oil), and oviposition deterrents (e.g., ovipositiondeterring pheromones) (Cook et al., 2007). Herbivore-induced plant volatiles are host plant semiochemicals that induce plant defense from herbivores and attract natural enemies (James, 2003). Non-chemical repellents include reflective mulches, which have been shown to reduce damage and population density of tarnished plant bug in strawberry fields (Rhainds et al., 2001). Attractants include sex and aggregation pheromones, host plant volatiles, and feeding stimulants (e.g., baits), and oviposition stimulants (Cook et al., 2007). Other attractants are based on visual cues. For example, apple maggots are effectively controlled in apple orchards with 8-cm, red, spherical traps covered in adhesive. The attractiveness of these traps is enhanced by adding butyl hexanoate and ammonium acetate, synthetic olfactory stimulants (Prokopy et al., 1994).

Another common tactic is to use sex pheromones for mating disruption. Many insect pests rely on a species-specific, sex pheromone produced by females for mate location and recognition. Mating disruption is achieved by flooding the crop environment with the chemical signal, thus confusing males and reducing mate-finding success. This approach has been used with varying degrees of success for management of orchard and vineyard pests including codling moth, oriental fruit moth, grape berry moth, and peachtree borer (see Zalom, 1997).

#### **4.3 Physical control via pest destruction**

Insects can be killed directly through mechanical, thermal, or other means. Vincent et al. (2009) list several strategies that inflict mortality on pests including freezing, heating, flaming, crushing, and irradiating. One of the most common mechanical methods requires no specialized equipment – many gardeners derive great satisfaction from hand picking pests from a plant and crushing them. Hand removal can be used effectively for a myriad of relatively sessile landscape pests including bagworms, tent caterpillars, and sawfly larvae. Galls, egg masses, and web-making insects can also be pruned out of infested landscape plants (Potter, 1986). However, this tactic may be impractical for large trees or shrubs and dense populations of the pest. Other mechanical control options require specialized machinery. Pneumatic control involves removing pests from crops by use of a vacuum or blower and subsequently destroying them. Field crop pests such as Colorado potato beetle and lygus bug have been controlled in this manner, although care must be taken to avoid negatively impacting natural enemies (Vincent et al., 2003, 2009). Another example of mechanized destruction is the entoleter, an impact machine that is used in mills to remove and kill all life stages of insect pests (Vincent et al., 2003).

Modifying the microclimate can be effective in killing many insect pests, which cannot survive outside of optimal temperature and humidity ranges. Heat has been shown to be a very effective control method for bed bugs, which are difficult to control and are becoming more prevalent in domestic dwellings worldwide (Pereira et al., 2009). A wide variety of stored product pests can be controlled by pumping hot or cold air into the food storage facility, or modifying the storage environment with elevated temperatures and carbon

Alternatives to Chemical Control of Insect Pests 185

Barbour, J. D., Farrar, R. R., Jr., & Kennedy, G. G. (1997). Populations of predaceous natural

Benbrook, C. M., Groth, E., III, Halloran, J. M., Hansen, M. K., & Marquardt, S. (1996). *Pest* 

Bentz, J. A., Reeves, J., Barbosa, P., & Francis, B. (1995). Nitrogen fertilizer effect on selection,

Boiteau, G., Lynch, D. H., & Martin, R. C. (2008). Influence of fertilization on the Colorado

*Entomology*, Vol. 37, No. 2, (April 2008), pp. 575-585, ISSN 0046-225X Boiteau, G., & Vernon, R. S. (2001). Physical Barriers for the Control of Insect Pests, In:

Lessard (Eds.), pp. 224-247, Springer, ISBN 3540645624, Berlin, Germany Bowie, M. H., Wratten, S. D., & White, A. J. (1995). Agronomy and phenology of

Briese, D. T. (2005). Translating host-specificity test results into the real world: the need to

Brust, G. E. (1994). Natural enemies in straw-mulch reduce Colorado potato beetle

Buntin, G. D., & Hudson, R. D. (1991). Spring control of the hessian fly (Diptera:

Buntin, G. D., Ott, S. L., & Johnson, J. W. (1992). Integration of plant resistance, insecticides,

Cai, Q. N., Ma, X. M., Zhao, X., Cao, Y. Z., & Yang, X. Q. (2009). Effects of host plant

35, No. 3, (December 2005), pp. 208-214, ISSN 1049-9644

Vol. 84, No. 6, (December 1991), pp. 1913-1919, ISSN 0022-0493

*Biological Control*, Vol. 9, No. 3, (July 1997), pp. 173–184, ISSN 1049-9644 Bell, J. R., Johnson, P. J., Hambler, C., Haughton, A. J., Smith, H., Feber, R. E., Tattersall, F.

8703

2002), pp. 295-304, ISSN 0167-8809

423-427, ISSN 0114-0671

163-169, ISSN 1049-9644

538, ISSN 0022-0493

0890439001, New York, New York, USA

1, (February 1995), pp. 40-45, ISSN 0046-225X

*Experimentalis et Applicata*, Vol. 68, No. 2, (August 1993), pp. 143–155, ISSN 0013-

enemies developing on insect-resistant and susceptible tomato in North Carolina.

H., Hart, B. H., Manley, W., & Macdonald, D. W. (2002). Manipulating the abundance of *Lepthyphantes tenuis* (Araneae: Linyphiidae) by field margin management. *Agriculture, Ecosystems and Environment*, Vol. 93, No. 1-3, (December

*Management at the Crossroads*. Consumers Union of the United States, ISBN

acceptance, and suitability of *Euphorbia pulcherrima* (Euphorbiaceae) as a host plant to *Bemisia tabaci* (Homoptera: Aleyrodidae). *Environmental Entomology*, Vol. 24, No.

potato beetle, *Leptinotarsa decemlineata*, in organic potato production. *Environmental* 

*Physical Control Methods in Plant Protection*, C. Vincent, B. Panneton, & F. Fleurat-

"companion plants" of potential for enhancement of insect biological control. *New Zealand Journal of Crop and Horticultural Science*, Vol. 23, No. 4, (December 1995), pp.

harmonize the yin and yang of current testing procedures. *Biological Control*, Vol.

populations and damage in potato. *Biological Control*, Vol. 4, No. 2, (June 1994), pp.

Cecidomyiidae) in winter wheat using insecticides. *Journal of Economic Entomology*,

and planting date for management of the hessian fly (Diptera: Cecidomyiidae) in winter wheat. *Journal of Economic Entomology*, Vol. 85, No. 2, (April 1992), pp. 530-

resistance on insect pests and its parasitoid: a case study of wheat-aphid-parasitoid system. *Biological Control*, Vol. 49, No. 2, (May 2009), pp. 134–138, ISSN 1049-9644 Caltagirone, L. E. & Huffaker, C. B. (1980). Benefits and Risks of Using Natural Enemies for

Controlling Pests, In: *Environmental Protection and Biological Forms of Control of Pest* 

dioxide (Vincent et al., 2003, 2009). Hot-water immersion, flaming, steaming, and solar heating are other thermal control options (Vincent et al., 2003).

Electromagnetic energy has been studied for its effectiveness at killing insects (Vincent et al., 2009). Ionizing radiation has been used in quarantine facilities to treat fruit and other commodities suspected of carrying serious agricultural pests (Vincent et al., 2003). Targets of other electromagnetic methods, especially microwave treatments, include stored product pests. However, electromagnetic treatments may be limited by government regulations, cost, and the need for specialized equipment and training (Vincent et al., 2009).

#### **5. Conclusions**

Crop culture sets the stage for interactions between plants, pests, and natural enemies, and has a strong influence on the outcome of these interactions. In many cases, implementing effective cultural controls can be the most economical pest management tactic available to growers because labor and expense are incurred regardless of whether an effective cultural tactic is used. Understanding and implementing cultural practices can reduce other production expenses such as insecticides and fertilizer. Cultural control can be compatible with biological control if the myriad interactions among plants, pests, and natural enemies are well defined. Improving the predictability of biological control will rely on elevating the discipline to its proper place in applied evolutionary ecology and further refinement of the art and practice of biological control (van Lenteren, 1980; Heinz et al., 1993; Heinz, 2005). Fortunately, the organic and sustainable agriculture movements that are gaining both societal and political momentum seem to embrace the art and science of biological pest control (Edwards, 1990; Raynolds, 2000). While various physical control techniques have been used successfully in production systems, this strategy is limited by the significant labor, time, cost, and specialization required for successful control (Vincent et al., 2009). Further refinements and developments in physical control technologies hold promise for enhanced efficacy, compatibility with cultural and biological control, and profits.

As we move into the future of pest management, new challenges await. Crops are now genetically modified to produce their own "insecticides" for protection. Newly registered insecticides tend to be more target specific and often, more expensive. Older chemistries are being removed both voluntarily and involuntarily from the market. There is increasing demand for organically grown food, or food perceived as "safe" for consumption. Yet we must still feed a growing human population. More than ever, IPM researchers need to develop programs that use effective alternatives to insecticides whenever possible. We also must intensify efforts to truly integrate insecticides selectively into our IPM programs, so that they are not the predominant tool in our IPM toolbox. As such, we need to further develop principles and methods of cultural, biological, and physical control as relevant pest management tools for sustainable agricultural production.

#### **6. References**

Andow, D. A., Ragsdale, D. W., & Nyvall, R. F. (Eds.) (1997). *Ecological Interactions and Biological Control*. Intercept Ltd., ISBN 0813387582, Andover, Hants, UK

Barbour, J. D., Farrar, R. R., Jr., & Kennedy, G. G. (1993). Interaction of *Manduca sexta*  resistance in tomato with insect predators of *Helicoverpa zea*. *Entomologia* 

dioxide (Vincent et al., 2003, 2009). Hot-water immersion, flaming, steaming, and solar

Electromagnetic energy has been studied for its effectiveness at killing insects (Vincent et al., 2009). Ionizing radiation has been used in quarantine facilities to treat fruit and other commodities suspected of carrying serious agricultural pests (Vincent et al., 2003). Targets of other electromagnetic methods, especially microwave treatments, include stored product pests. However, electromagnetic treatments may be limited by government regulations,

Crop culture sets the stage for interactions between plants, pests, and natural enemies, and has a strong influence on the outcome of these interactions. In many cases, implementing effective cultural controls can be the most economical pest management tactic available to growers because labor and expense are incurred regardless of whether an effective cultural tactic is used. Understanding and implementing cultural practices can reduce other production expenses such as insecticides and fertilizer. Cultural control can be compatible with biological control if the myriad interactions among plants, pests, and natural enemies are well defined. Improving the predictability of biological control will rely on elevating the discipline to its proper place in applied evolutionary ecology and further refinement of the art and practice of biological control (van Lenteren, 1980; Heinz et al., 1993; Heinz, 2005). Fortunately, the organic and sustainable agriculture movements that are gaining both societal and political momentum seem to embrace the art and science of biological pest control (Edwards, 1990; Raynolds, 2000). While various physical control techniques have been used successfully in production systems, this strategy is limited by the significant labor, time, cost, and specialization required for successful control (Vincent et al., 2009). Further refinements and developments in physical control technologies hold promise for

cost, and the need for specialized equipment and training (Vincent et al., 2009).

enhanced efficacy, compatibility with cultural and biological control, and profits.

management tools for sustainable agricultural production.

As we move into the future of pest management, new challenges await. Crops are now genetically modified to produce their own "insecticides" for protection. Newly registered insecticides tend to be more target specific and often, more expensive. Older chemistries are being removed both voluntarily and involuntarily from the market. There is increasing demand for organically grown food, or food perceived as "safe" for consumption. Yet we must still feed a growing human population. More than ever, IPM researchers need to develop programs that use effective alternatives to insecticides whenever possible. We also must intensify efforts to truly integrate insecticides selectively into our IPM programs, so that they are not the predominant tool in our IPM toolbox. As such, we need to further develop principles and methods of cultural, biological, and physical control as relevant pest

Andow, D. A., Ragsdale, D. W., & Nyvall, R. F. (Eds.) (1997). *Ecological Interactions and Biological Control*. Intercept Ltd., ISBN 0813387582, Andover, Hants, UK Barbour, J. D., Farrar, R. R., Jr., & Kennedy, G. G. (1993). Interaction of *Manduca sexta* 

resistance in tomato with insect predators of *Helicoverpa zea*. *Entomologia* 

heating are other thermal control options (Vincent et al., 2003).

**5. Conclusions** 

**6. References** 

*Experimentalis et Applicata*, Vol. 68, No. 2, (August 1993), pp. 143–155, ISSN 0013- 8703


Alternatives to Chemical Control of Insect Pests 187

Desneux, N., Pham-Delègue, M. H., & Kaiser, L. (2004a). Effect of a sublethal and lethal dose

Desneux N., Rafalimanana, H., Kaiser, L. (2004b). Dose-response relationship in lethal and

*Chemosphere*, Vol. 54, No. 5, (February 2004), pp. 619–627, ISSN 0045-6535 Diaz-Montano, J., Reese, J. C., Louis, J., Campbell, L. R., & Schapaugh, W. T. (2007). Feeding

Diaz-Montano, J., Reese, J. C., Schapaugh, W. T., & Campbell, L. R. (2006). Characterization

Edwards, C. A., Lal, R., Madden, P., Miller, R. H., & House, G. (Eds.) (1990). *Sustainable* 

Ehrlich, P. R., & Raven, P. H. (1964). Butterflies and plants: a study in coevolution. *Evolution*,

Ellis, J. A., Walter, A. D., Tooker, J. F., Ginzel, M. D., Reagel, P. F., Lacy, E. S., Bennett, A. B.,

Farid A., Johnson, J. B., Shafii, B., & Quisenberry, S. S. (1998). Tritrophic studies of Russian

Forbes , S. A. (1883). The corn root-worm. (*Diabrotica longicornis* Say) Order Coleoptera. Family Chrysomelidae. *Illinois State Entomologist Annual Report*, Vol. 12, pp. 10–31 Frank, S. D. (2010). Biological control of arthropod pests using banker plant systems: past

Frank, S. D., & Sadof, C. S. *In press*. Reducing insecticide volume and non-target effects of

Frank, S. D., & Shrewsbury, P. M. (2004). Effect of conservation strips on the abundance and

Frisbie, R. E., & Smith, J. W., Jr. (1989). Biologically Intensive Integrated Pest Management:

(December 2004), pp. 1662-1672, ISSN 0046-225X

Vol. 18, No. 4, (December 1964), pp. 586-608, ISSN 0014-3820

pp. 381–389, ISSN 1526-498X

(June 2007), pp. 984–989, ISSN 0022-0493

2006), pp. 1884-1889, ISSN 0022-0493

Ankeny, Iowa, USA

9644

1049-9644

*Entomology*.

pp. 8-16, ISSN 1049-9644

Lanham, Maryland, USA

of lambdacyhalothrin on oviposition experience and host searching behaviour of a parasitic wasp, *Aphidius ervi*. *Pest Management Science*, Vol. 60, No. 4, (April 2004),

behavioural effects of different insecticides on the parasitic wasp *Aphidius ervi*.

behavior by the soybean aphid (Hemiptera: Aphididae) on resistant and susceptible soybean genotypes. *Journal of Economic Entomology*, Vol. 100, No. 3,

of antibiosis and antixenosis to the soybean aphid (Hemiptera: Aphididae) in several soybean genotypes. *Journal of Economic Entomology*, Vol. 99, No. 5, (October

*Agricultural Systems*, Soil and Water Conservation Society, ISBN 093573421X,

Grossman, E. M., & Hanks, L. M. (2005). Conservation biological control in urban landscapes: manipulating parasitoids of bagworm (Lepidoptera: Psychidae) with flowering forbs. *Biological Control*, Vol. 34, No. 1, (July 2005), pp. 99-107, ISSN 1049-

wheat aphid, a parasitoid, and resistant and susceptible wheat over three parasitoid generations. *Biological Control*, Vol. 12, No. 1, (May 1998), pp. 1–6, ISSN

progress and future direction. *Biological Control*, Vol. 52, No. 1, (January 2010),

ambrosia beetle management in nurseries. Submitted to *Journal of Economic* 

distribution of natural enemies and predation of *Agrotis ipsilon* (Lepidoptera: Noctuidae) on golf course fairways. *Environmental Entomology*, Vol. 33, No. 6,

the Future. In: *Progress and Perspectives for the 21st Century*, J. J. Menn & A. L. Stienhauer (Eds.), pp. 156-184, Entomological Society of America, ISBN 0938522361,

*Organisms*, Ecological Bulletins No. 31, B. Lundsholm & M. Stackerud (Eds.), pp. 103-109, Swedish Natural Science Research Council, Stockholm, Sweden.


103-109, Swedish Natural Science Research Council, Stockholm, Sweden. Caltagirone, L. E., & Doutt, R. L. (1989). The history of the vedalia beetle importation to

Chau, A., & Heinz, K. M. (2006). Manipulating fertilization: a management tactic against

Chiang, H. C. (1965). Survival of northern corn rootworm eggs through one and two

Chow, A., Chau, A., & Heinz, K. M. (2009). Reducing fertilization for cut roses: effect on

Clausen, C. P., (Ed.) (1978). *Introduced Parasites and Predators of Arthropod Pests and Weeds: a* 

Cloyd, R. A., & Bethke, J. A. 2009. Pesticide use in ornamental production: what are the

Collier, T., & van Steenwyk, R. (2004). A critical evaluation of augmentative biological

Cook, R. J. (1987). *Report of the Research Briefing Panel on Biological Control in Managed* 

Cook, S. M., Kahn, Z. R., & Pickett, J. A. (2007). The use of push-pull strategies in integrated

Coppel, H. C., & Mertins, J. W. (1977). *Biological Insect Pest Suppression,* Advanced Series in

DeBach, P. D., & Rosen, D. (1991). *Biological Control by Natural Enemies* (2nd edition),

De Barro, P. J., Hart, P. J., & Morton, R. (2000). The biology of two *Eretmocerus* spp.

Delfosse, E. S. (2005). Risk and ethics in biological control. *Biological Control*, Vol. 35, No. 3,

Desneux, N., Decourtye, A., & Delpuech, J. M. (2007). The sublethal effects of pesticides on

United States Department of Agriculture, Washington, D.C., USA

*Ecosystems*. National Academy Press, Washington, D.C., USA

Agricultural Sciences, Vol. 4, Springer-Verlag., Berlin, Germany

*Applicata*, Vol. 94, No. 1, (January 2000), pp. 93–102, ISSN 0013-8703

Cambridge University Press, ISBN 0521391911, London, UK

(December 2005), pp. 319-329, ISSN 1049-9644

*Applicata*, Vol. 120, No. 3, (September 2006), pp. 201-209, ISSN 0013-8703 Chau, A., Heinz, K. M., & Davies, F. T. (2005). Influences of fertilization on *Aphis gossypii*

Carson, R. (1962). *Silent Spring*. Houghton Mifflin Co., New York, New York, USA

*Entomology*, Vol. 34, pp. 1–16, ISSN 0066-4170

pp. 89-97, ISSN 0931-2048

1896-1907, ISSN 0022-0493

ISSN 0022-0493

1526-498X

9644

4170

4170

*Organisms*, Ecological Bulletins No. 31, B. Lundsholm & M. Stackerud (Eds.), pp.

California and its impact on the development of biological control. *Annual Review of* 

*Frankliniella occidentalis* on potted chrysanthemum. *Entomologia Experimentalis et* 

and insecticide usage. *Journal of Applied Entomology*, Vol. 129, No. 2, (March 2005),

winters. *Journal of Economic Entomology*, Vol. 58, No. 3, (April 1965), pp. 470–472,

crop productivity and twospotted spider mite abundance, distribution, and management. *Journal of Economic Entomology*, Vol. 102, No. 5, (October 2009), pp.

*World Review*, Agriculture Handbook No. 480, Agricultural Research Service,

benefits? *Pest Management Science*, Vol. 65, No. 4, (April 2009), pp. 345-350, ISSN

control. *Biological Control*, Vol. 31, No. 2, (October 2004), pp. 245-256, ISSN 1049-

pest management. *Annual Review of Entomology*, Vol. 52, pp. 375-400. ISSN 0066-

(Haldeman) and three *Encarsia* spp. Forster and their potential as biological control agents of *Bemisia tabaci* biotype B in Australia. *Entomologia Experimentalis et* 

beneficial arthropods. *Annual Review of Entomology*, Vol. 52, pp. 81–106, ISSN 0066-


Alternatives to Chemical Control of Insect Pests 189

Heinz, K. M., & Parrella, M. P. (1994). Poinsettia (*Euphorbia pulcherrima* Willd. ex Koltz.)

Heinz, K. M., & Zalom, F. G. (1995). Variation in trichome-based resistance to *Bemisia* 

*Entomology*, Vol. 88, No. 5, (October 1995), pp. 1494–1502, ISSN 0022-0493 Heinz, K. M. (2005). Evolutionary Pest Management: an Approach to the Twenty-First

Herms, D. A. (2002). Effects of fertilization on insect resistance of woody ornamental plants:

Herms, D. A., & Mattson, W. J. (1992). The dilemma of plants - to grow or defend. *Quarterly Review of Biology*, Vol. 67, No. 3, (September 1992), pp. 283-335, ISSN 0033-5770 Hill, C. B., Crull, L., Herman, T., Voegtlin, D. J., & Hartman, G. L. (2010). A new soybean

Hill, C. B., Kim, K. S., Crull, L., Diers, B. W., & Hartman G. L. (2009). Inheritance of

Hill, C. B., Li, Y., & Hartman, G. L. (2004). Resistance of *Glycine* species and various

Hogendorp, B. K., Cloyd, R. A., & Swiader, J. M. (2006). Effect of nitrogen fertility on

Hokkanen, H. M. T. (1997). Role of Biological Control and Transgenic Crops in Reducing

Horn, D. J. (1988). *Ecological Approach to Pest Management*. The Guilford Press, ISBN

Horne, P., & Page, J. (2008). *Integrated Pest Management for Crops and Pastures*. Landlinks

Vol. 4, No. 4, (December 1994), pp. 305–318, ISSN 1049-9644

(December 2002), pp. 923-933, ISSN 0046-225X

Vol. 103, No. 2, (April 2010), pp. 509–515, ISSN 0022-0493

and Sons, ISBN 0471968382, Chichester, West Sussex, UK

0898623022, New York, New York, USA

Press, ISBN 0643092579, Collingwood, Australia

(July/August 2009), pp. 1193–1200, ISSN 0011-183X

1217-1233, ISSN 0046-225X

USA

0066-4170

201-211, ISSN 0046-225X

Chrysanthemums. *Environmental Entomology*, Vol. 22, No. 6, (December 1993), pp.

cultivar-mediated differences in performance of five natural enemies of *Bemisia argentifolii* Bellows and Perring, n. sp. (Homoptera: Aleyrodidae). *Biological Control*,

*argentifolii* (Homoptera: Aleyrodidae) oviposition on tomato. *Journal of Economic* 

Century, In: *Entomology at the Land Grant University: Perspectives from the Texas A&M University Department Centenary*, K. M. Heinz, R. E. Frisbie, & C. E. Bográn (Eds.), pp. 305-315 , Texas A&M University Press, ISBN 1585444324 College Station, Texas,

reassessing an entrenched paradigm. *Environmental Entomology*, Vol. 31, No. 6,

aphid (Hemiptera: Aphididae) biotype identified. *Journal of Economic Entomology*,

resistance to the soybean aphid in soybean PI 200538. *Crop Science*, Vol. 49, No. 4,

cultivated legumes to the soybean aphid (Homoptera: Aphididae). *Journal of Economic Entomology*, Vol. 97, No. 3, (June 2004), pp. 1071-1077, ISSN 0022-0493 Hoddle, M. S., van Driesche, R. G., & Sanderson, J. P. (1998). Biology and use of the whitefly

parasitoid *Encarsia formosa*. *Annual Review of Entomology*, Vol. 43, pp. 645-669, ISSN

reproduction and development of citrus mealybug, *Planococcus citri* Risso (Homoptera: Pseudococcidae), feeding on two colors of coleus, *Solenostemon scutellarioides* L. Codd. *Environmental Entomology*, Vol. 35, No. 2, (April 2006), pp.

Use of Chemical Pesticides for Crop Protection, In: *Techniques for Reducing Pesticide Use: Economic and Environmental Benefits*, D. Pimental (Ed.), pp. 103-127, John Wiley


Galvan, T. L., Koch, R. L., & Hutchison, W. D. (2005). Toxicity of commonly used

Garcia, R., Caltagirone, L. E., & Gutierrez, A. P. (1988). Comments on a redefinition of

Gentile, A. G., & Stoner, A. K. (1968). Resistance in *Lycopersicon* and *Solanum* species to

Gill, S., Cloyd, R. A., Baker, J. R., Clement, D. L., & Dutky, E., (2006). *Pests and Diseases of* 

Gould, F. (1986). Simulation models for predicting durability of insect-resistant germ plasm

Gray, M. E., Sappington, T. W., Miller, N. J., Moeser, J., & Bohn, M. O. (2009). Adaptation

pest. *Annual Review of Entomology*, Vol. 54, pp. 303-321, ISSN 0066-4170 Greathead, D. J., (Ed.) (1976). *A Review of Biological Control in Western and Southern Europe*,

*Entomology*, Vol. 100, No. 3, (June 2007), pp. 830–837, ISSN 0022-0493 Hall, R. W., & Ehler, L. E. (1979). Rate of establishment of natural enemies in classical

Hall, R. W., Ehler, L. E., & Bisabri-Ershadi, B. (1980). Rate of success in classical biological

Hardman, J. M., Rogers, R. E. L., & MacLellan, C. R. (1988). Advantages and disadvantages

Headrick, D. H., Bellows, T. S., Jr., & Perring, T. M. (1996). Behaviors of female *Eretmocerus* 

Heinz, K. M., Nunney, L., & Parrella, M. P. (1993). Towards predictable biological control of

Vol. 81, No. 6, (December 1988), pp. 1737–1749, ISSN 0022-0493

*Entomology*, Vol. 15, No. 1, (February 1986), pp. 11-23, ISSN 0046-225X Gould, F. (1998). Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. *Annual Review of Entomology*, Vol. 43, pp. 701-726, ISSN 0066-4170 Grant, J. A. (1997). IPM techniques for greenhouse crops. In: *Techniques for Reducing Pesticide* 

and Sons, ISBN 0471968382, Chichester, West Sussex, UK

(15 December 1979), pp. 280-282, ISSN 0013-8754

(15 June 1980), pp. 111-114, ISSN 0013-8754

64–75, ISSN 1049-9644

2005), pp. 780-789, ISSN 0022-0493

ISSN 0006-3568

Batavia, IL, USA

1152–54, ISSN 0022-0493

insecticides in sweet corn and soybean to multicolored Asian lady beetle (Coleoptera: Coccinellidae). *Journal of Economic Entomology*, Vol. 98, No. 3, (June

biological control. *BioScience*, Vol. 38, No. 10, (November 1988), pp. 692-694,

potato aphid. *Journal of Economic Entomology*, Vol. 61, No. 5, (October 1968), pp.

*Herbaceous Perennials: the Biological Approach*. Ball Publishing, ISBN 1883052508,


*Use: Economic and Environmental Benefits*, D. Pimental (Ed.), pp. 399-406, John Wiley

and invasiveness of western corn rootworm: intensifying research on a worsening

Technical Communication Vol. 7, pp. 52-64, Commonweath Institute for Biological Control, Commonwealth Agricultural Bureau, ISBN 0851983693, Slough, UK Gupta, G., & Krischik, V. A. (2007). Professional and consumer insecticides for the

management of adult Japanese beetle on hybrid tea rose. *Journal of Economic* 

biological control. *Bulletin of the Entomological Society of America*, Vol. 25, No. 4,

control of arthropods. *Bulletin of the Entomological Society of America*, Vol. 26, No. 2,

of using pyrethroids in Nova Scotia apple orchards. *Journal of Economic Entomology*,

sp. nr. *californicus* (Hymenoptera: Aphelinidae) attacking *Bemisia argentifolii*  (Homoptera: Aleyrodidae) on cotton, *Gossypium hirsutum* (Malavaceae), and melon, *Cucumis melo* (Cucurbitaceae). *Biological Control*, Vol. 6, No. 1, (February 1996), pp.

*Liriomyza trifolii* (Diptera: Agromyzidae) infesting greenhouse cut

Chrysanthemums. *Environmental Entomology*, Vol. 22, No. 6, (December 1993), pp. 1217-1233, ISSN 0046-225X


Alternatives to Chemical Control of Insect Pests 191

Kaufman, W. C., & Kennedy, G. G. (1989b). Toxicity of allelochemicals from the wild insect-

Kennedy, G. G. (2003). Tomato, pests, parasitoids, and predators: tritrophic interactions

Kennedy, G. G., & Sorenson, C. F. (1985). Role of glandular trichomes in the resistance of

Kennedy, G. G., & Barbour, J. D. (1992). Resistance Variation in Natural and Managed

Knutson, L. (1981). Symbiosis on Biosystematics and Biological Control, In: *Biological Control* 

Kogan, M. (1998). Integrated pest management: historical perspectives and contemporary developments. *Annual Review of Entomology*, Vol. 43, pp. 243-270, ISSN 0066-4170 Koul, O., & Cuperus, G. W. (2007). *Ecologically Based Integrated Pest Management*. CAB

Krischik, V. A., Landmark, A. L., & Heimpel, G. E. (2007). Soil-applied imidacloprid is

Kruess, A., & Tscharntke, T. (1994). Habitat fragmentation, species loss, and biological control. *Science*, Vol. 264, No. 5165, (10 June 1994), pp. 1581-1584, ISSN 0036-8075 Kytö, M., Niemela, P., & Larsson, S. (1996). Insects on trees: population and individual

Landis, D. A., Wratten, S. D., & Gurr, G. M. (2000). Habitat management to conserve natural

Levine, E. & Oloumi-Sadeghi, H. (1991). Management of diabroticite rootworms in corn.

Levine, E., Oloumi-Sadeghi, H., & Fisher, J. R. (1992). Discovery of multiyear diapause in

*Entomology*, Vol. 85, No. 1, (February 1992), pp. 262–267, ISSN 0022-0493 Levine, E., Spencer, J. L., Isard, S. A., Onstad, D. W., & Gray, M. E. (2002). Adaptation of the

*Annual Review of Entomology*, Vol. 36, 229–255, ISSN 0066-4170

pp. 2051–60, ISSN 0098-0331

ISBN 0226265536, Chicago, Illinois, USA

2007), pp. 1238–1245, ISSN 0046-225X

175-201, ISSN 0066-4170

International, ISBN 1845930649, Wallingford, UK

ISSN 0066-4170

551, ISSN 0022-0493

London, UK

1299

2821

resistant tomato *Lycopersicon hirsutum f. glabratum* to *Campoletis sonorensis*, a parasitoid of *Helicoverpa zea. Journal of Chemical Ecology*, Vol. 15, No. 7, (July 1989),

involving the genus *Lycopersicon*. *Annual Review of Entomology*, Vol. 48, pp. 51-72,

*Lycopersicon hirsutum f. glabratum* to Colorado potato beetle (Coleoptera: Chrysomelidae). *Journal of Economic Entomology*, Vol. 78, No. 3, (June 1985), pp. 547-

Systems, In: *Plant Resistance to Herbivores and Pathogens: Ecology, Evolution, and Genetics*, R. S. Fritz & E. L. Simms (Eds.), pp. 13-41, University of Chicago Press,

*in Crop Production*, G. C. Papavizas (Ed.), pp. 61-78, Allanheld, Osmun Publishers,

translocated to nectar and kills nectar-feeding *Anagyrus pseudococci* (Girault) (Hymenoptera: Encyrtidae). *Environmental Entomology*, Vol. 36, No. 5, (October

response to fertilization. *Oikos*, Vol. 75, No. 2, (March 1996), pp. 148-159, ISSN 0030-

enemies of arthropod pests in agriculture. *Annual Review of Entomology*, Vol. 45, pp.

Illinois and South Dakota northern corn rootworm (Coleoptera: Chrysomelidae) eggs and incidence of the prolonged diapause trait in Illinois. *Journal of Economic* 

western corn rootworm to crop rotation: evolution of a new strain in response to a management practice. *American Entomologist*, Vol. 48, No. 2, pp. 94–107, ISSN 1046-


Hoskins, W.M., Borden, A. D., & Michelbacher, A.E. (1939). Recommendations for a more

Howarth, F. G. (1983). Classical biological control: panacea or Pandora's box? *Proceedings of the Hawaiian Entomological Society*, Vol. 24, pp. 239-244, ISSN 0073-134X Huffaker, C. B., Berryman, A. A., & Laing, J. E. (1984). Natural Control of Insect

Huffaker, C. B., & Messenger, P. S. (Eds.) (1976). *Theory and Practice of Biological Control*.

Iftner, D. C., & Hall, F. R. (1983). Effects of fenvalerate and permethrin on *Tetranychus urticae*

Iftner, D. C., & Hall, F. R. (1984). The effects of fenvalerate and permethrin residues on

Inbar, M., & Gerling, D. (2008). Plant-mediated interactions between whiteflies, herbivores,

Insecticide Resistance Action Committee (IRAC) (2010). 03.08.11, Available from

Jacobsen, B. J. (1997). Role of plant pathology in integrated pest management. *Annual Review* 

James, D. G. (2003). Synthetic herbivore-induced plant volatiles as field attractants for

Johnson, M. T., & Gould, F. (1992). Interaction of genetically engineered host plant-

Jones, V. P., & Parella, M. P. (1984). The sublethal effects of selected insecticides on life table

Kashyap, R. K., Kennedy, G. G., & Farrar, R. R., Jr. (1991a). Behavioral response of

Kashyap, R. K., Kennedy, G. G., & Farrar, R. R., Jr. (1991b). Mortality and inhibition of

Kaufman, W. C., & Kennedy, G. G. (1989a). Inhibition of *Campoletis sonorensis* parasitism of

398, Wiley, ISBN 0471064939, New York, New York, USA

No. 6, (December 1983), pp. 1782-1786, ISSN 0046-225X

*of Phytopathology*, Vol. 35, pp. 373-391, ISSN 0066-4286

*Entomology*, Vol. 1, pp. 191-200, ISSN 0735-939X

http://www.irac-online.org/about/irac/

116, pp. 1033-1040, ISSN 0008-347X

2381–2395, ISSN 0098-0331

1919–1930, ISSN 0098-0331

No. 3, (March 1991), pp. 543–556, ISSN 0098-0331

Academic Press, ISBN 0123603501, New York, New York, USA

Vol. 5, pp. 119-123

4170

982, ISSN 0046-225X

0046-225X

discriminating use of insecticides, In: *Proceedings of the 6th Pacific Science Congress*,

Populations, In: *Ecological Entomology,* C. B. Huffaker & R.L. Rabb (Eds.), pp. 359-

Koch (Acari: Tetranychidae) dispersal behavior. *Environmental Entomology*, Vol. 12,

*Tetranychus urticae* Koch fecundity and rate of development. *Journal of Agricultural* 

and natural enemies. *Annual Review of Entomology*, Vol. 53, pp. 431-448, ISSN 0066-

beneficial insects. *Environmental Entomology*, Vol. 32, No. 5, (October 2003), pp. 977-

resistance and natural enemies of *Heliothis virescens* (Lepidoptera: Noctuidae) in tobacco. *Environmental Entomology*, Vol. 21, No. 3, (June 1992), pp. 587-597, ISSN

parameters of *Panonychus citri* (Acari: Tetranychidae). *Canadian Entomologist*, Vol.

*Trichogramma pretiosum* Riley and *Telenomus sphingis* (Ashmead) to trichmone/ methyl ketone-mediated resistance in tomato. *Journal of Chemical Ecology*, Vol. 17,

*Helicoverpa zea* egg parasitism rates by *Trichogramma* in relation to trichome/ methyl ketone-mediated insect resistance of *Lycopersicon hirsutum f. glabratum*  accession PI134417. *Journal of Chemical Ecology*, Vol. 17, No. 12, (December 1991), pp.

*Heliothis zea* and of parasitoid development by 2-tridecanone mediated insect resistance of wild tomato. *Journal of Chemical Ecology*, Vol. 15, No. 6, (June 1989), pp.


Alternatives to Chemical Control of Insect Pests 193

Papachristos, D. P., & Milonas, P. G. (2008). Adverse effects of soil applied insecticides on

*Biological Control*, Vol. 47, No. 1, (October 2008), pp. 77–81, ISSN 1049-9644 Painter, R. H. (1951). *Insect Resistance in Crop Plants*. The MacMillan Company, New York,

Parrella, M. P., Heinz, K. M., & Nunney, L. (1992). Biological control through augmentative

Patt, J. M., Hamilton, G. C., & Lashomb, J. H. (1997). Foraging success of parasitoid wasps

Penman, D. R., & Chapman, R. B. (1983). Fenvalerate-induced distributional imbalances of 2-

Penman, D. R., & Chapman, R. B. (1988). Pesticide-induced mite outbreaks: pyrethroids and

Pereira, R. M., Walker, W., Pfeister, M., & Koehler, P. G. (2009). Lethal effects of heat and use

Pimental, D. (Ed.) (1997). *Techniques for Reducing Pesticide Use: Economic and Environmental Benefits*. John Wiley and Sons, ISBN 0471968382, Chichester, West Sussex, UK Potter, D. A. (1986). Urban Landscape Pest Management, In: *Advances in Urban Pest* 

Pottorff, L. P., & Panter, K. L. (2009). Integrated pest management and biological control in

Prokopy, R. J., Cooley, D. R., Autio, W. R., & Coli, W. M. (1994). Second-level integrated pest

Rabb, R. L. (1972). Principles and Concepts of Pest Management, In: *Implementing Practical* 

Ragsdale, D. W., Landis, D. A., Brodeur, J., Heimpel, G. E., & Desneux, N. (2011). Ecology

*Workshop*, pp. 6-29, Purdue University, West Lafayette, Indiana, USA Rabb, R. L., Stinner, R. E., & van den Bosch, R. (1976). Conservation and Augmentation of

Reinhold Co., ISBN 0442209606, New York, New York, USA

*Agriculture*, Vol. 9, No. 4, pp. 148-156, ISSN 0889-1893

*Entomology*, Vol. 56, pp. 375-399, ISSN 0066-4170

*Entomology*, Vol. 102, No. 3, (June 2009), pp. 1182-1188, ISSN 0022-0493 Perkins, J. H., & Garcia, R. (1999). Social and Economic Factors Affecting Research and

New York, USA

30, ISSN 0013-8703

Diego, California, USA

61-65, ISSN 1063-0198

York, USA

Vol. 38, No. 3, pp. 172–179, ISSN 1046-2821

No. 1, (January 1983), pp. 71-78, ISSN 0013-8703

the predatory coccinellid *Hippodamia undecimnotata* (Coleoptera: Coccinellidae).

releases of natural enemies: a strategy whose time has come. *American Entomologist*,

on flowers: interplay of insect morphology, floral architecture and searching behavior. *Entomologia Experimentalis et Applicata*, Vol. 83, No. 1, (April 1997), pp. 21-

spotted spider-mite on bean plants. *Entomologia Experimentalis et Applicata*, Vol. 33,

spider mites. *Experimental and Applied Acarology*, Vol. 4, pp. 265-276, ISSN 0168-8162

of localized heat treatment for control of bed bug infestations. *Journal of Economic* 

Implementation of Biological Control, In: *Handbook of Biological Control*, T. S. Bellows & T. W. Fisher (Eds.), pp. 993-1008, Academic Press, ISBN 0122573056, San

*Management*, G. W. Bennett & J. M. Owens (Eds.), pp. 219-251, Van Nostrand

high tunnel production. *HortTechnology*, Vol. 19, No. 1, (January-March 2009), pp.

management in commercial apple orchards. *American Journal of Alternative* 

*Pest Management Strategies: Proceedings of a National Extension Pest Management* 

Natural Enemies, In: *Theory and Practice of Biological Control*, C. B. Huffaker & P. S. Messenger (Eds.), pp. 233-254, Academic Press, ISBN 0123603501, New York, New

and management of the soybean aphid in North America. *Annual Review of* 


Marino, P. C., & Landis, D. A. (1996). Effect of landscape structure on parasitoid diversity

Mathews, C. R., Bottrell, D. G., & Brown, M. W. (2004). Habitat manipulation of the apple

Mattson, W. J. (1980). Herbivory in relation to plant nitrogen content. *Annual Review of* 

McClure, M. S. (1977). Resurgence of the scale, *Fiorinia externa* (Homoptera: Diaspididae), on

McMurtry, J. A., & Croft, B. A. (1997). Life-styles of phytoseiid mites and their role in

Metcalf, R. L. (1989). Insect resistance to insecticides. *Pesticide Science*, Vol. 26, pp. 333-358,

Misener, G. C., Boiteau, G., & McMillan, L. P. (1993). A plastic-lining trenching device for

Moser, S. E., & Obrycki, J. J. (2009). Non-target effects of neonicotinoid seed treatments;

Murdoch, W. W., Chesson, J., & Chesson, P. L. (1985). Biological control in theory and

Murdoch, W. W., & Briggs, C. J. (1996). Theory for biological control: recent developments. *Ecology*, Vol. 77, No. 7, (October 1996), pp. 2001-2013, ISSN 0012-9658 National Research Council (NRC). (1996). *Ecologically Based Pest Management: New Solutions* 

Nihoul, P. (1993a). Controlling glasshouse climate influences the interaction between tomato

Nihoul, P. (1993b). Do light intensity, temperature and photoperiod affect the entrapment of

Obrycki, J. J., Lewis, L. C., & Orr, D. B. (1997). Augmentative releases of entomophagous

Obrycki, J. J., & Tauber, M. J. (1984). Natural enemy activity on glandular pubescent potato

Vol. 17, No. 9, (September 1993), pp. 709–718, ISSN 0168-8162

*Ecology and Systematics*, Vol. 11, pp. 119-161, ISSN 0066-4162

70, No. 12, (December 1993), pp. 903–908, ISSN 0003-0589

No. 3, (December 2009), pp. 487–492, ISSN 1049-9644

(September 1993), pp. 443–447, ISSN 0261-2194

*Entomology*, Vol. 50, pp. 271-292, ISSN 0066-4170

pp. 30- 36, ISSN 1049-9644

1996), pp. 276-284, ISSN 1051-0761

2004), pp. 265-273, ISSN 1049-9644

ISSN 0031-613X

0147

USA

(June 1977), pp. 480–484, ISSN 0046-225X

and parasitism in agroecosystems. *Ecological Applications*, Vol. 6, No. 1, (February

orchard floor to increase ground-dwelling predators and predation of *Cydia pomonella* (L.) (Lepidoptera: Tortricidae). *Biological Control*, Vol. 30, No. 2, (June

hemlock following insecticide application. *Environmental Entomology*, Vol. 6, No. 3,

biological control. *Annual Review of Entomology*, Vol. 42, pp. 291-321, ISSN 0066-4170

the control of Colorado potato beetle: beetle excluder. *American Potato Journal*, Vol.

mortality of coccinellid larvae related to zoophytophagy. *Biological Control*, Vol. 51,

practice. *American Naturalist*, Vol. 125, No. 3, (March 1985), pp. 344-366, ISSN 0003-

*for a New Century*, National Academy Press, ISBN 0309053307, Washington, D.C.,

glandular trichome, spider mite and predatory mite. *Crop Protection*, Vol. 12, No. 6,

mites on glandular hairs of cultivated tomatoes? *Experimental and Applied Acarology*,

species in annual systems. *Biological Control*, Vol. 10, No. 1, (September 1997),

plants in the greenhouse: an unreliable predictor of effects in the field. *Environmental Entomology*, Vol. 13, No. 3, (June 1984), pp. 679–83, ISSN 0046-225X O'Callaghan, M., Glare, T. R., Burgess, E. P. J., & Malone, L. A. (2005). Effects of plants

genetically modified for insect resistance on non-target organisms. *Annual Review of* 


Alternatives to Chemical Control of Insect Pests 195

Sanford, K. H. (1967). The influence of spray programs on the fauna of apple orchards in

Sclar, D. C., Gerace, D., & Cranshaw, W. S. (1998). Observations of population increase and

Shelton, A. M., Zhao, J. Z., & Roush, R. T. (2002). Economic, ecological, food safety, and

Simmons, A. T., & Gurr, G. M. (2005). Trichomes of *Lycopersicon* species and their hybrids:

Smith, H. S. (1919). On some phases of insect control by the biological method. *Journal of Economic Entomology*, Vol. 12, No. 4, (August 1919), pp. 288-292, ISSN 0022-0493 Smith, S. F., & Krischik, V. A. (1999). Effects of systemic imidacloprid on *Coleomegilla* 

Snodgrass, G. L., & Stadelbacher, E. A. (1989). Effect of different grass and legume

Spencer, J. L., Hibbard, B. E., Moeser, J., & Onstad, D. W. (2009). Behaviour and ecology of

*Forest Entomology*, Vol. 11, No. 1, (February 2009), pp. 9-27, ISSN 1461-9555 Spencer, J. L., & Levine, E. (2008). Resistance to Crop Rotation, In: *Insect Resistance* 

Stern, V. M., Smith, R. F., van den Bosch, R., & Hagen, K. S. (1959). The integration of

Szczepaniec, A., Creary, S. F., Laskowski, K. L., Nyrop, J. P., & Raupp, M. J. (2011).

Theriault, F., Sequin, P., & Stewart, K. A. (2009). Incidence of *Pieris rapae* in organic broccoli

Tooker, J. F., & Hanks, L. M. (2000). Influence of plant community structure on natural

Academic Press, ISBN 9780123738585, New York, New York, USA

control concept. *Hilgardia*, Vol. 29, pp. 81-101, ISSN 0073-2230

*Vegetable Science*, Vol. 15, No. 3, pp. 218-225, ISSN 1931-5260

doi:10.1371/journal.pone.0020018, ISSN 1932-6203

0046-225X

154, ISSN 1063-0198

99, pp. 197-201, ISSN 0008-347X

*Entomology*, Vol. 47, pp. 845-881, ISSN 0066-4170

4, (November 2004), pp. 265-276, ISSN 1461-9555

(December 1999), pp. 1189–95, ISSN 0046-225X

Vol. 18, No. 4, (August 1989), pp. 575-581, ISSN 0046-225X

250–255, ISSN 0022-0493

biological control. *HortTechnology*, Vol. 14, No. 1, (January-March 2004), pp. 149-

Nova Scotia. XVII. Effects on some predaceous mites. *Canadian Entomologist*, Vol.

injury by spider mites (Acari: Tetranychidae) on ornamental plants treated with imidacloprid. *Journal of Economic Entomology*, Vol. 91, No. 1, (February 1998), pp.

social consequences of the deployment of Bt transgenic plants. *Annual Review of* 

effects on pests and natural enemies. *Agricultural and Forest Entomology*, Vol. 7, No.

*maculata* (Coleoptera: Coccinellidae). *Environmental Entomology*, Vol. 28, No. 6,

combinations on spider (Araneae) and ground beetle (Coleoptera: Carabidae) populations in roadside habitats in the Mississippi Delta. *Environmental Entomology*,

the western corn rootworm (*Diabrotica virgifera virgifera* LeConte). *Agricultural and* 

*Management: Biology, Economics and Prediction,* D. W. Onstad (Ed.), pp. 153–183,

chemical and biological control of the spotted alfalfa aphid. I. The integrated

Neonicotinoid insecticide imidacloprid causes outbreaks of spider mites on elm trees in urban landscapes. *PLoS ONE*, Vol. 6, No. 5, e20018.

grown with living mulches under floating row cover. *International Journal of* 

enemies of pine needle scale (Homoptera: Diaspididae) in urban landscapes, *Environmental Entomology*, Vol. 29, No. 6, (December 2000), pp. 1305–1311, ISSN


Raupp, M. J., Shrewsbury, P. M., & Herms, D. A. (2010) Ecology of herbivorous arthropods

Raupp, M. J., Holmes, J. J., Sadof, C., Shrewsbury, P., & Davidson, J. A. (2001). Effects of

Raupp, M. J., Webb, R., Szczepaniec, A., Booth, D., & Ahern, R. (2004). Incidence,

Raynolds, L. T. (2000). Re-embedding global agriculture: the international organic and fair

Rebek, E. J., & Sadof, C. S. (2003). Effects of pesticide applications on the euonymus scale

Rebek, E. J., Sadof, C. S., & Hanks, L. M. (2005). Manipulating the abundance of natural

Rebek, E. J., Sadof, C. S., & Hanks, L. M. (2006). Influence of floral resource plants on control

Rekika, D., Stewart, K. A., Boivin, G., & Jenni, S. (2008). Reduction of insect damage in

Ridgway, R. L. (Ed.) (1998). *Mass-Reared Natural Enemies: Application, Regulation, and Needs*.

Rieux, R., Simon, S., & Defrance, H. (1999). Role of hedgerows and ground cover

*and Environment*, Vol. 73, No. 2, (April 1999), pp. 119-127, ISSN 0167-8809 Rhainds, M., Kovach, J., Dosa, E. L., & English-Loeb, G., (2001). Impact of reflective mulch

Roland, J., & Taylor, P. (1997). Insect parasitoid species respond to forest structure at

Royer, T. A., Mulder, P. G., & Cuperus, G. W. (1999). Renaming (redefining) integrated pest

Sadof, C. S., O'Neil, R. J., Heraux, F. M., & Wiedenmann, R. N. (2004). Reducing insecticide

Vol. 33, No. 2, (May 2005), pp. 203-216, ISSN 1049-9644

America, ISBN 0938522663, Lanham, Maryland, USA

4170

5226

9644

0028-0836

139, ISSN 1046-2821

0278-5226

pp. 297–309, ISSN 0889-048X

No. 2, pp. 177-193, ISSN 1931-5260

2001), pp. 1477-1484, ISSN 0022-0493

452, ISSN 0022-0493

in urban landscapes. *Annual Review of Entomology*, Vol. 55, pp. 19-38, ISSN 0066-

cover sprays and residual pesticides on scale insects and natural enemies in urban forests. *Journal of Arboriculture*, Vol. 27, No. 4, (July 2001), pp. 203-214, ISSN 0278-

abundance, and severity of mites on hemlocks following applications of imidacloprid. *Journal of Arboriculture*, Vol. 30, No. 2, (March 2004), pp. 108–13, ISSN

trade movements. *Agriculture and Human Values*, Vol. 17, No. 3, (September 2007),

(Homoptera: Diaspididae) and its parasitoid, *Encarsia citrina* (Hymenoptera: Aphelinidae). *Journal of Economic Entomology*, Vol. 96, No. 2, (April 2003), pp. 446-

enemies in ornamental landscapes with floral resource plants. *Biological Control*,

of an armored scale pest by the parasitoid *Encarsia citrina* (Craw.) (Hymenoptera: Aphelinidae). *Biological Control*, Vol. 37, No. 3, (June 2006), pp. 320-328, ISSN 1049-

radish with floating row covers. *International Journal of Vegetable Science*, Vol. 14,

Thomas Say Publications in Entomology: Proceedings. Entomological Society of

management on arthropod populations in pear orchards. *Agriculture, Ecosystems* 

on yield of strawberry plants and incidence of damage by tarnished plant bug (Heteroptera: Miridae). *Journal of Economic Entomology*, Vol. 94, No. 6, (December

different spatial scales. *Nature*, Vol. 386, No. 6626, (17 April 1997), pp. 710-713, ISSN

management: fumble, pass, or play? *American Entomologist*, Vol. 45, No. 3, pp. 136-

use in home gardens: effects of training and volunteer research on adoption of

biological control. *HortTechnology*, Vol. 14, No. 1, (January-March 2004), pp. 149- 154, ISSN 1063-0198


**Part 2** 

**Further Applications** 


## **Part 2**

**Further Applications** 

196 Insecticides – Basic and Other Applications

United States Department of Agriculture, Animal and Plant Health Inspection Service

van Driesche, R. G., & Heinz, K.M. (2004). An Overview of Biological Control in Protected

Parrella (Eds.), pp. 1-24, Ball Publishing, ISBN 1883052394, Batavia, IL, USA van Haren, R. J. F., Steenhuis, M. M., Sabelis, M. W., & de Ponti, O. M. B. (1987). Tomato

van Lenteren, J. C. (1980). Evaluation of control capabilities of natural enemies: does art

van Lenteren, J. C., Hua, L. Z., Kamerman, J. W., & Xu, R. (1995). The parasite-host

Vol. 119, No. 1-5, (January/December 1995), pp. 553–559, ISSN 0931-2048 Vincent, C., Hallman, G., Panneton, B, & Fleurat-Lessard, F. (2003). Management of

Vincent, C., Weintraub, P., & Hallman, G. (2009). Physical Control of Insect Pests, In:

Wäckers, F. L. (2004). Assessing the suitability of flowering herbs as parasitoid food sources:

Ware, G. W., & Whitacre, D. M. (2004). *The Pesticide Book* (6th edition), MeisterPro,

Weber, D. C., Ferro, D. N., Buonaccorsi, J., & Hazzard, R. V. (1994). Disrupting spring

Wong, S. W., & Chapman, R. B. (1979). Toxicity of synthetic pyrethroid insecticides to

Zalom, F. (1997). IPM Practices for Reducing Insecticide Use in U.S. Fruit Crops, In:

Academic Press, ISBN 9780123741448, San Diego, California, USA

http://www.aphis.usda.gov/plant\_health/plant\_pest\_info/biocontrol van Driesche, R. G., & Bellows, T. S. (1996). *Biological Control*. Chapman & Hall, ISBN

0412028611, New York, New York, USA

Vol. 48, pp. 261-281, ISSN 0066-4170

(March 2004), pp. 307-314, ISSN 1049-9644

*Research*, Vol. 30, No. 3, pp. 497-501, ISSN 0004-9409

Willoughby, Ohio, USA

West Sussex, UK

0168-8162

0028-2960

(8 July, 2011). Biological Control Program, 03.08.11, Available from

Culture, In: *Biocontrol in Protected Culture*, K. M. Heinz, R. G. Van Driesche, & M.

stem trichomes and dispersal success of *Phytoseiulus persimilis* relative to its prey *Tetranychus urticae*. *Experimental and Applied Acarology*, Vol. 3, pp. 115–121, ISSN

have to become science? *Netherlands Journal of Zoology*, Vol. 30, pp. 369-381, ISSN

relationship between *Encarsia formosa* (Hymenoptera: Aphelinidae) and *Trialeurodes vaporariorum* (Homoptera: Aleyrodidae). XXVI. Leaf hairs reduce the capacity of *Encarsia* to control greenhouse whitefly on cucumber. *Journal of Applied Entomology*,

agricultural insects with physical control methods. *Annual Review of Entomology*,

*Encyclopedia of Insects* (2nd edition), V. H. Resh & R. T. Cardé (Eds.), pp. 794-798,

flower attractiveness and nectar accessibility. *Biological Control*, Vol. 29, No. 3,

colonization of Colorado potato beetle to nonrotated potato elds. *Entomologia Experimentalis et Applicata*, Vol. 73, No. 1, (October 1994), pp. 39–50, ISSN 0013-8703

predaceous phytoseiid mites and their prey. *Australian Journal of Agricultural* 

*Techniques for Reducing Pesticide Use: Economic and Environmental Benefits,* D. Pimental (Ed.), pp. 317-342, John Wiley and Sons, ISBN 0471968382, Chichester,

**10** 

*India* 

**Proteomic Profiling of** *Escherichia coli* **in** 

Amritha G. Kulkarni and B. B. Kaliwal

*Karnatak University,* 

*P.G. Department of Studies in Biotechnology and Microbiology* 

**Response to Carbamate Pesticide - Methomyl** 

Since decades, there has been mounting concern regarding the adverse health effects of environmental contaminants in general and carbamate in particular. Methomyl is a carbamate and widely used throughout the world since it is effective as "contact insecticide" as well as "systemic insecticide" for fruits and vegetables and is well known established cholinesterase inhibitor 1. Methomyl has been classified as a pesticide of category-I toxicity 2. Methomyl is a metabolite of thiodicarb and acetimidate is suspected oncogen, which is a metabolite in animal tissues 3. It has been classified by the WHO, EPA (Environmental Protection Agency, USA), and EC (European Commission) as a very toxic and hazardous pesticide 4. Methomyl is highly soluble in water and can therefore, easily cause ground water contamination in agricultural areas 5. Bonatti *et al*., 6 have shown genotoxic effects of methomyl in *in vitro* studies. Methomyl is potent genotoxic and is capable of inducing

Prokaryotic cells respond to environmental or chemical stress by inducing specific sites of proteins characteristic to each stress 8. Studies on stress response and survival strategies of enteric bacteria have evolved a range of complex mechanisms, which use different regulatory structures and genetic components for their survival and virulence 9. The stress protein induced in response to four different pesticides viz. cypermethrin, zeta-cypermethrin, carbofuran and bifenthrin were analyzed by protein profiling of *Escherichia coli* by Asghar *et al.,* 10. Mechanisms of cellular adaptation and compensation against different kinds of toxic metals have been proposed. However, the molecular mechanisms and underlying responses of

Proteomics is a technique used to investigate whole proteins expressed by an organism, tissue or a cell at a specific time point under defined environmental conditions. Nowadays, proteomics has been used for many research purposes e.g. disease diagnosis, drug target and biomarkers of pollutants 12,13 . Proteomics, transcriptomics and metabolomics are powerful tools for acquiring information on gene/protein function and regulatory networks28. Using proteomics, one can determine protein expression profiles related to research for both microbial isolates and communities. Proteomics provides a global view of the protein complement of biological systems and, in combination with other omics technologies, has an important role in helping uncover the mechanisms of these cellular

processes and thereby advance the development of environmental biotechnologies29.

structural and numerical chromosomal aberration in mammalian cells 7.

cells against various pesticides are not yet completely understood 11.

**1. Introduction** 

### **Proteomic Profiling of** *Escherichia coli* **in Response to Carbamate Pesticide - Methomyl**

Amritha G. Kulkarni and B. B. Kaliwal

*P.G. Department of Studies in Biotechnology and Microbiology Karnatak University, India* 

#### **1. Introduction**

Since decades, there has been mounting concern regarding the adverse health effects of environmental contaminants in general and carbamate in particular. Methomyl is a carbamate and widely used throughout the world since it is effective as "contact insecticide" as well as "systemic insecticide" for fruits and vegetables and is well known established cholinesterase inhibitor 1. Methomyl has been classified as a pesticide of category-I toxicity 2. Methomyl is a metabolite of thiodicarb and acetimidate is suspected oncogen, which is a metabolite in animal tissues 3. It has been classified by the WHO, EPA (Environmental Protection Agency, USA), and EC (European Commission) as a very toxic and hazardous pesticide 4. Methomyl is highly soluble in water and can therefore, easily cause ground water contamination in agricultural areas 5. Bonatti *et al*., 6 have shown genotoxic effects of methomyl in *in vitro* studies. Methomyl is potent genotoxic and is capable of inducing structural and numerical chromosomal aberration in mammalian cells 7.

Prokaryotic cells respond to environmental or chemical stress by inducing specific sites of proteins characteristic to each stress 8. Studies on stress response and survival strategies of enteric bacteria have evolved a range of complex mechanisms, which use different regulatory structures and genetic components for their survival and virulence 9. The stress protein induced in response to four different pesticides viz. cypermethrin, zeta-cypermethrin, carbofuran and bifenthrin were analyzed by protein profiling of *Escherichia coli* by Asghar *et al.,* 10. Mechanisms of cellular adaptation and compensation against different kinds of toxic metals have been proposed. However, the molecular mechanisms and underlying responses of cells against various pesticides are not yet completely understood 11.

Proteomics is a technique used to investigate whole proteins expressed by an organism, tissue or a cell at a specific time point under defined environmental conditions. Nowadays, proteomics has been used for many research purposes e.g. disease diagnosis, drug target and biomarkers of pollutants 12,13 . Proteomics, transcriptomics and metabolomics are powerful tools for acquiring information on gene/protein function and regulatory networks28. Using proteomics, one can determine protein expression profiles related to research for both microbial isolates and communities. Proteomics provides a global view of the protein complement of biological systems and, in combination with other omics technologies, has an important role in helping uncover the mechanisms of these cellular processes and thereby advance the development of environmental biotechnologies29.

Proteomic Profiling of *Escherichia coli* in Response to Carbamate Pesticide - Methomyl 201

M of methomyl for a period of 96 hrs and at regular intervals of 24 hrs, the proteins induced were analyzed. The protein expression was observed at 29, 45, 48, 55, 63, 92 and 114 kDa at 24 hrs **(Fig. 1)**. On exposure to methomyl for 48 hrs the bands were observed at 29, 45, 48, 55, 63, 92 and 114 kDa **(Fig. 2)**. The methomyl treated for 72 hrs showed expression at 29, 39, 45, 66 and 92 kDa **(Fig. 3)** and for 96 hrs the expressions was observed at 29, 35, 39, 45, 55, 63, and 92 kDa **(Fig. 4)** respectively. The expression of proteins were more conspicuous in our result which was obligatory, since the free *Escherichia coli* cells possess antioxidant enzymes,

which are induced in response to the stress and are directly exposed to methomyl 18.

Fig. 1. Protein profile of *Esherichia coli* induced by methomyl for 24 hours.

Fig. 2. Protein profile of *Esherichia coli* induced by methomyl for 48 hours.

The polyacrylamide gel electrophoresis has been used extensively for the separation of proteins in yeast, bacteria and higher organisms with the successful separation of whole cell extracts or specific proteins under selected conditions. This is an excellent method to attempt a global depiction of the cells protein profile. Thus, this technique is being extensively used to determine the *in vivo* amount of protein, its rate of synthesis, and rate its rate of degradation 13. SDS-PAGE is an important molecular technique used for the identification of whole cell proteins and it has the advantage of being fairly simple and rapid to perform 14. Therefore, the present investigation was undertaken to study the proteomic profiling of *Escherichia coli* on dose and durational exposure to methomyl by gel electrophoresis.

#### **2. Materials and methods**

#### **2.1 Preparation of stock solution of methomyl**

The sample of methomyl (Lannate ®) used in the experiment was supplied by E.I. Dupont India Pvt. Ltd., Haryana obtained. The stock solution of 1 M of methomyl was prepared and further diluted to give different required molar concentrations.

#### **2.2 Maintenance and propagation of culture**

The organism *Escherichia coli* was procured from NCL, Pune and the bacteria was maintained at 4°C on nutrient agar formulated by Lapage and Shelton 15 and sub cultured very fortnight.

#### **2.3 Medium used for the study**

Synthetic sewage medium (S-medium) formulated by Babich and Stotzky 16 was used as the medium for toxicity testing.

#### **2.4 Preparation of inoculum for free cells**

Pre-inoculum was prepared by inoculating a loopful of bacteria from the overnight incubated nutrient agar slant cultures on a 100 ml sterilized synthetic sewage medium and incubated for 18-24 hours at 37°C under static conditions depending on the exponential phases of bacteria under test.

#### **2.5 Experimental procedures**

**Free cells:** Five ml of the pre-inoculum was inoculated to 250 ml Erlenmeyer's flask containing 100 ml of sterilized S-medium amended with different molar concentrations of heavy metals. The flasks were incubated at 37°C for 96 hours under shaking conditions at 120 rpm on a rotary shaker (REMI – CIS-24). At regular intervals sample was taken out from each flask aseptically for analysis.

#### **2.6 Isolation of protein**

The bacterial cell pellet was dissolved in 100µl of lysis buffer and incubated at 37ºc for 15 min. the tubes were centrifuged and the supernatant was used as protein sample. PAGE according to Laemmli 17 analyzed these protein samples.

#### **3. Results and discussion**

The present investigation was attempted to elucidate the protein profiling in *Escherichia coli cells* that were exposed to different concentrations of methomyl ranging from 10-7 M to 10–3

The polyacrylamide gel electrophoresis has been used extensively for the separation of proteins in yeast, bacteria and higher organisms with the successful separation of whole cell extracts or specific proteins under selected conditions. This is an excellent method to attempt a global depiction of the cells protein profile. Thus, this technique is being extensively used to determine the *in vivo* amount of protein, its rate of synthesis, and rate its rate of degradation 13. SDS-PAGE is an important molecular technique used for the identification of whole cell proteins and it has the advantage of being fairly simple and rapid to perform 14. Therefore, the present investigation was undertaken to study the proteomic profiling of *Escherichia coli* on

The sample of methomyl (Lannate ®) used in the experiment was supplied by E.I. Dupont India Pvt. Ltd., Haryana obtained. The stock solution of 1 M of methomyl was prepared and

The organism *Escherichia coli* was procured from NCL, Pune and the bacteria was maintained at 4°C on nutrient agar formulated by Lapage and Shelton 15 and sub cultured very fortnight.

Synthetic sewage medium (S-medium) formulated by Babich and Stotzky 16 was used as the

Pre-inoculum was prepared by inoculating a loopful of bacteria from the overnight incubated nutrient agar slant cultures on a 100 ml sterilized synthetic sewage medium and incubated for 18-24 hours at 37°C under static conditions depending on the exponential

**Free cells:** Five ml of the pre-inoculum was inoculated to 250 ml Erlenmeyer's flask containing 100 ml of sterilized S-medium amended with different molar concentrations of heavy metals. The flasks were incubated at 37°C for 96 hours under shaking conditions at 120 rpm on a rotary shaker (REMI – CIS-24). At regular intervals sample was taken out from

The bacterial cell pellet was dissolved in 100µl of lysis buffer and incubated at 37ºc for 15 min. the tubes were centrifuged and the supernatant was used as protein sample. PAGE

The present investigation was attempted to elucidate the protein profiling in *Escherichia coli cells* that were exposed to different concentrations of methomyl ranging from 10-7 M to 10–3

dose and durational exposure to methomyl by gel electrophoresis.

further diluted to give different required molar concentrations.

**2.1 Preparation of stock solution of methomyl** 

**2.2 Maintenance and propagation of culture** 

**2.4 Preparation of inoculum for free cells** 

**2. Materials and methods** 

**2.3 Medium used for the study** 

medium for toxicity testing.

phases of bacteria under test.

**2.5 Experimental procedures** 

each flask aseptically for analysis.

**3. Results and discussion** 

according to Laemmli 17 analyzed these protein samples.

**2.6 Isolation of protein** 

M of methomyl for a period of 96 hrs and at regular intervals of 24 hrs, the proteins induced were analyzed. The protein expression was observed at 29, 45, 48, 55, 63, 92 and 114 kDa at 24 hrs **(Fig. 1)**. On exposure to methomyl for 48 hrs the bands were observed at 29, 45, 48, 55, 63, 92 and 114 kDa **(Fig. 2)**. The methomyl treated for 72 hrs showed expression at 29, 39, 45, 66 and 92 kDa **(Fig. 3)** and for 96 hrs the expressions was observed at 29, 35, 39, 45, 55, 63, and 92 kDa **(Fig. 4)** respectively. The expression of proteins were more conspicuous in our result which was obligatory, since the free *Escherichia coli* cells possess antioxidant enzymes, which are induced in response to the stress and are directly exposed to methomyl 18.

Fig. 1. Protein profile of *Esherichia coli* induced by methomyl for 24 hours.

Fig. 2. Protein profile of *Esherichia coli* induced by methomyl for 48 hours.

Proteomic Profiling of *Escherichia coli* in Response to Carbamate Pesticide - Methomyl 203

other cases, proteins, which are associated with one stimulon, can be induced during other stresses, such as various heat shock proteins in *Escherichia coli*. These proteins are also synthesized when the cells are exposed to different physical and chemical stress. In some stimulons, exposure to non-lethal levels of a stress agent can confer protection against subsequent exposure to lethal levels of the same stress agent 19. Similarly, in the present study, the proteins expressed at 29 and 45 kDa could be unique or could be observed in the protein profiling of other micro-organisms exposed to various physical or chemical stress. It has been suggested that the analysis of many proteins produced during the transition into stationery phase and under stress conditions demonstrated that a number of novel proteins were induced in common to each stress and could be the reason for cross protection in bacterial cells. It is necessary to investigate the synthesis of these proteins during different stress conditions 20. Similarly it has been mentioned that when organisms or cells are exposed to low levels of certain harmful physical and chemical agents, the organisms acquire an induced tolerance against the adverse effects 7. Hence, in the present study the high molecular weight proteins of 114 kDa at 24 and 48 hrs respectively observed in all the doses of exposure in comparison to their corresponding controls may be ascertained to the protein selective proteolytic degradation that appears to be rather significant in homeostasis maintaining and metabolism regulation in the cell 21. It has been reported that along with short-lived regulatory proteins, the polypeptide chains with disrupted or changed structures are selectively hydrolyzed. Such defects might arise from inaccuracy during protein biosynthesis, chemical or physical damage 22 and moreover, the extracts of *Escherichia coli* have been shown to degrade rapidly the damaged enzyme, but not the native protein, and several preliminary reports have appeared concerning the *Escherichia coli* protease that may be responsible for selective degradation of the modified

Although it has been reported that the starvation for individual nutrients and other stress induce a unique and individual profile of protein expression, some proteins are common to different starvation and stress factors in *Escherichia coli.* However, the proteins of one stimulon do not respond coordinately to all the starvation and stress treatments and relatively few of the starvation- inducible proteins have been found to overlap with those induced by stress. This suggests that despite the regulation of a few specific proteins being interconnected, there are major difference in the regulatory pathways controlling the expression of starvation and different stress proteins 24. Studies in the micro-organisms have provided evidence for increased longevity, cell division rate and survival when exposed to stress 25. Similarly in the present study, the types of stress patterns observed with the dose and duration of exposure of methomyl were identical which agreed with the earlier reports 10 that the stress proteins produced in response to two different classes of pesticides showed that the same stress patterns were obtained for different substituent chemical groups within the same class and two different classes, indicating that the gene or set of genes responsible for stress expressions were the same irrespective of the class or nature of substituent's on the

Further, an increase in the intensity in protein expression observed in the present study may be due to the fact that the major protein modification is observed due to stress, loss of catalytic activity, amino acid modification, carbonyl group formation, increase in acidity, decrease in thermal stability, change in viscosity, fluorescence, fragmentation, formation of protein protein crosslink's, s-s bridges and increased susceptibility to proteolysis 3. It has been revealed that the secretion of extra cellular proteins, including toxins and cellular

proteins 23.

pesticide.

Fig. 3. Protein profile of *Esherichia coli* induced by methomyl for 72 hours.

Fig. 4. Protein profile of *Esherichia coli* induced by methomyl for 96 hours.

The protein profiles were compared with the dose and duration of exposure of methomyl in *Escherichia coli* and the results revealed that the intensity of the proteins expressed increased with an increase in the dose and duration of exposure of methomyl when compared with those of the corresponding parameters of the control, indicating that the pesticide methomyl induces stress. Our results agreed with the observations made by Asghar *et al.,* 10 who analyzed the stress proteins of *Escherichia coli* induced in response to the pesticides cypermethrin, zeta-cypermethrin, carbofuran and bifenthrin.

The over expressions of some of the proteins observed in the present study at 29 and 45 kDa at all the dose and duration of exposure could be due to the fact that prokaryotic cells respond to environmental or chemical stress by inducing specific sets of proteins characteristic to each stress. It has been reported that the proteins in each set of their coding genes constitute a stimulon, such as heat shock, SOS response and oxidation stress. In some

Fig. 3. Protein profile of *Esherichia coli* induced by methomyl for 72 hours.

Fig. 4. Protein profile of *Esherichia coli* induced by methomyl for 96 hours.

cypermethrin, zeta-cypermethrin, carbofuran and bifenthrin.

The protein profiles were compared with the dose and duration of exposure of methomyl in *Escherichia coli* and the results revealed that the intensity of the proteins expressed increased with an increase in the dose and duration of exposure of methomyl when compared with those of the corresponding parameters of the control, indicating that the pesticide methomyl induces stress. Our results agreed with the observations made by Asghar *et al.,* 10 who analyzed the stress proteins of *Escherichia coli* induced in response to the pesticides

The over expressions of some of the proteins observed in the present study at 29 and 45 kDa at all the dose and duration of exposure could be due to the fact that prokaryotic cells respond to environmental or chemical stress by inducing specific sets of proteins characteristic to each stress. It has been reported that the proteins in each set of their coding genes constitute a stimulon, such as heat shock, SOS response and oxidation stress. In some other cases, proteins, which are associated with one stimulon, can be induced during other stresses, such as various heat shock proteins in *Escherichia coli*. These proteins are also synthesized when the cells are exposed to different physical and chemical stress. In some stimulons, exposure to non-lethal levels of a stress agent can confer protection against subsequent exposure to lethal levels of the same stress agent 19. Similarly, in the present study, the proteins expressed at 29 and 45 kDa could be unique or could be observed in the protein profiling of other micro-organisms exposed to various physical or chemical stress.

It has been suggested that the analysis of many proteins produced during the transition into stationery phase and under stress conditions demonstrated that a number of novel proteins were induced in common to each stress and could be the reason for cross protection in bacterial cells. It is necessary to investigate the synthesis of these proteins during different stress conditions 20. Similarly it has been mentioned that when organisms or cells are exposed to low levels of certain harmful physical and chemical agents, the organisms acquire an induced tolerance against the adverse effects 7. Hence, in the present study the high molecular weight proteins of 114 kDa at 24 and 48 hrs respectively observed in all the doses of exposure in comparison to their corresponding controls may be ascertained to the protein selective proteolytic degradation that appears to be rather significant in homeostasis maintaining and metabolism regulation in the cell 21. It has been reported that along with short-lived regulatory proteins, the polypeptide chains with disrupted or changed structures are selectively hydrolyzed. Such defects might arise from inaccuracy during protein biosynthesis, chemical or physical damage 22 and moreover, the extracts of *Escherichia coli* have been shown to degrade rapidly the damaged enzyme, but not the native protein, and several preliminary reports have appeared concerning the *Escherichia coli* protease that may be responsible for selective degradation of the modified proteins 23.

Although it has been reported that the starvation for individual nutrients and other stress induce a unique and individual profile of protein expression, some proteins are common to different starvation and stress factors in *Escherichia coli.* However, the proteins of one stimulon do not respond coordinately to all the starvation and stress treatments and relatively few of the starvation- inducible proteins have been found to overlap with those induced by stress. This suggests that despite the regulation of a few specific proteins being interconnected, there are major difference in the regulatory pathways controlling the expression of starvation and different stress proteins 24. Studies in the micro-organisms have provided evidence for increased longevity, cell division rate and survival when exposed to stress 25. Similarly in the present study, the types of stress patterns observed with the dose and duration of exposure of methomyl were identical which agreed with the earlier reports 10 that the stress proteins produced in response to two different classes of pesticides showed that the same stress patterns were obtained for different substituent chemical groups within the same class and two different classes, indicating that the gene or set of genes responsible for stress expressions were the same irrespective of the class or nature of substituent's on the pesticide.

Further, an increase in the intensity in protein expression observed in the present study may be due to the fact that the major protein modification is observed due to stress, loss of catalytic activity, amino acid modification, carbonyl group formation, increase in acidity, decrease in thermal stability, change in viscosity, fluorescence, fragmentation, formation of protein protein crosslink's, s-s bridges and increased susceptibility to proteolysis 3. It has been revealed that the secretion of extra cellular proteins, including toxins and cellular

Proteomic Profiling of *Escherichia coli* in Response to Carbamate Pesticide - Methomyl 205

[5] Laura L., Eerd. V., Hoagland. R. E., Zablotowicz. R M.., Hall C. J., Pesticide metabolism

[6] Benjamin I.J., McMillan D. R., Stress (heat shock) proteins: molecular chaperones in

[7] Flahaut S., Hartke A., Giard J. Nystrom, T., Olsgon, R.M., and Kjelleberg, S. Survival,

[8] Ronan, O.T., Marjan J., Smeulders, Marian, C., Blokpoel, Emily, J. Kay, Kathryn

[9] Kappke. J., da Silva E.R.,.Schelin H.R., Pashchuk S.A. and de Oliveira. A.., Evaluation of

[10] Asghar. M. N., Ashfaq. M., Ahmad. Z., Khan I. U., 2-D PAGE analysis of pesticideinduced stress proteins of E. coli. *Anal Bioanal Chem*. 384: 946–950 (2006). [11] Patcharee. Isarankura-Na-Ayudhya., Virapong Prachayasittikul., Proteomic profiling

[12] Mahashi Nakayama., Kyoko Ishizawa., Jiro Nakajima., Akiko Kawamura and Takako

*Memoirs of Osaka Kyoiku University, Ser. III,* Vol. 45, No. 1 pp. 81-91 (1996). [13] Khemika Lomthaisong., Kanchanit Boonmaleerat and Aphinya Wongpia Proteomic

[14] Leisner J.J., Millan H. H., Huss and Larsen L. M., Production of histamine and

[15] Lapage S. P. and Shelton J. E., In Methods in Microbiology, (ed. Norris J. R.and

[16] Babich and Stotzky.. Reduction in the toxicity of cadmium to micro-organisms by clay

[17] Laemmli, U. K. : Cleavage of structural proteins during the assembly of the head of

[18] Kulkarni A. G. and Kaliwal B. B., Studies on methomyl induced stress in free and

[19] Nystrom T., Olsgon R.M. and Kjelleberg S., Survival, stress resistance and alteration in

[20] Jamshid Raheb., Shamim Naghdi1 and Ken P. Flint., The Effect of Starvation Stress on

[21] Beckwith and Strauch., Periplasmic protease mutants of *Escherichia coli*, *World* 

protein expression in the marine *Vibrio* sp. Strain S14 during starvation for different

the Protein Profiles in *Flexibacter chinensis*. *Iranian Biomedical Journal* 12 (2): 67-75.

Ribbons D. W.), academic Press. New York, N Y. pp, 1,3A (1970).

immobilized *Escherichia coli. Proceedings of ISBT* 419-423 (2008).

individual nutrients. *Appl environ. Microbial.* 58*.* 55-65*.* (1992).

minerals. *Appl. Environ. Microbiol.*, 33, 696-705. (1977).

bacteriophage T4, *Nature* 227: 680-685 (1970).

*intellectual property organization,* 5. 819-821 (1988).

stress resistance, and alteration in protein expression in the marine *Vibrio* sp. strain S14 during starvation for different individual nutrients. *App. Environ. Microbiol.* 

Lougheed, and Huw, D. Williams., A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in *Mycobacterium* 

*Escherichia coli* cells damages induced by ultraviolet and proton beam radiation.,

of *Escherichia coli* in response to heavy metal stress. *European Journal of Scientific* 

Umino: Cellular Protein Profile of Halobacteriurn Halophilic halobium, Archaea.

study of recombinant *Escherichia coli* expressing *Beauveria bassiana* Chitinase Gene,

tyramine by Lactic acid bactria isolated from vacuum packed sugar-salted fish. *J.* 

in plants and micro-organisms. *Weed science;* 51, 472-495 (2003)

cardiovascular biology and disease. *Circ Res;* 83,117–132 (1998)

*smegmatis*. *J. Bacteriol*. 185 (5): 1543-1554. (2003)

*Brazilian journal of Physics,* 35, 3B. (2005).

*Research*. Vol.25. No 4. Pp 679-688 (2009).

*Chiang Mai J. Sci*. 35(2) : 324-330. (2008)

*Appl. Bacteriol,* 76. 417-423 (1994).

(2008).

58, 55-65 (1992).

effectors, is one of the key contributing factors in a bacterium's ability to thrive in diverse environments 26. Hence, the present study indicates that the protein expressions are dose and duration dependent. It has been suggested that there are many protein synthesized in common with many stress in *Escherichia coli* and some of these proteins may play a major role in the stability of the cells under different stresses. The fact that specific patterns of proteins are expressed for a particular stress has led to the use of stress proteins to monitor environmental samples for the presence of particular pollutants 27. It has been suggested that the analysis of such stress proteins will aid in the development of more sensitive techniques for the pollutant analysis. The unique proteins could be purified and raised to enable quick detection, which could be used as biomarkers of xenobiotics in the environment 11.

#### **4. Conclusions**

The present study indicated the molecular weights of the various stress proteins induced in response to the dose and durational exposure of methomyl. Further, it indicates that the stress protein analysis is a promising alternative and more sensitive method for measuring toxic effects on the organisms at sub lethal levels. The study suggests that the proteomic profiling is a sensitive tool for environmental stress diagnosis, and that the stress proteins could be used as biomarkers for environmental pollution identification. The specific patterns of the proteins that are expressed in response to the stress induced by methomyl could be used to monitor the environmental samples for the presence of such pollutants. Although the application of gene and protein expression analysis to ecotoxicology is still at an early stage, this holistic approach seems to have several potentials in different fields of ecological risk assessment. It can be concluded that such extensive work on proteomics can be performed in understanding the proteomic/genomic response and tolerance of the microorganisms to the extreme environment.

#### **5. Acknowledgements**

The authors are grateful to the Post Graduate Department of Studies in Microbiology and Biotechnology, Karnatak University Dharwad for providing the necessary facilities.

#### **6. References**


effectors, is one of the key contributing factors in a bacterium's ability to thrive in diverse environments 26. Hence, the present study indicates that the protein expressions are dose and duration dependent. It has been suggested that there are many protein synthesized in common with many stress in *Escherichia coli* and some of these proteins may play a major role in the stability of the cells under different stresses. The fact that specific patterns of proteins are expressed for a particular stress has led to the use of stress proteins to monitor environmental samples for the presence of particular pollutants 27. It has been suggested that the analysis of such stress proteins will aid in the development of more sensitive techniques for the pollutant analysis. The unique proteins could be purified and raised to enable quick detection, which could be used as biomarkers of xenobiotics in the

The present study indicated the molecular weights of the various stress proteins induced in response to the dose and durational exposure of methomyl. Further, it indicates that the stress protein analysis is a promising alternative and more sensitive method for measuring toxic effects on the organisms at sub lethal levels. The study suggests that the proteomic profiling is a sensitive tool for environmental stress diagnosis, and that the stress proteins could be used as biomarkers for environmental pollution identification. The specific patterns of the proteins that are expressed in response to the stress induced by methomyl could be used to monitor the environmental samples for the presence of such pollutants. Although the application of gene and protein expression analysis to ecotoxicology is still at an early stage, this holistic approach seems to have several potentials in different fields of ecological risk assessment. It can be concluded that such extensive work on proteomics can be performed in understanding the proteomic/genomic response and tolerance of the

The authors are grateful to the Post Graduate Department of Studies in Microbiology and

[1] Barakat .K.K., Effect of Certain Insecticides on the Stabilization And Lysis of Human and

[2] Tamimi M., Qourzal S., Assabbane A., Chovelon J. M., Ferronato C., Emmelin C., Ait-

[4] Bonatti.S., Bolognesi. C., Degan. P., Abbondandolo. A., Genotoxic effect of the carbamate

Fish Erythocyte *Research Journal of Agriculture and Biological Sciences* ; 1(2), 195-199

Ichou Y., Photocatalytic degradation of pesticide methomyl Determination of the reaction pathway and identification of intermediate Products*. Photochem. Photobiol.* 

insecticide methomyl. In vitro studies with pure compound and the technical formulation " Lannate 25". *Environmental and molecular mutagenesis*; vol 23, p.306-

Biotechnology, Karnatak University Dharwad for providing the necessary facilities.

[3] Stadtman E. R., protein oxidation and ageing. *Science* 257, 1220-1224 (1992)

environment 11.

**4. Conclusions** 

microorganisms to the extreme environment.

**5. Acknowledgements** 

**6. References** 

(2005)

*Sci;* 5, 477-48 (2006)

311. (2006)


**11** 

*Nigeria* 

**in Wistar Rats** 

*2Department of Veterinary Anatomy Ahmadu Bello University, Zaria,* 

**Ameliorative Effect of Vitamin E on** 

Suleiman F. Ambali1, Joseph O. Ayo1, Muftau Shittu1,

Mohammed U. Kawu1 and Suleiman O. Salami2 *1Department of Veterinary Physiology and Pharmacology* 

**Sensorimotor and Cognitive Changes** 

**Induced by Chronic Chlorpyrifos Exposure** 

The use of pesticides is inevitable in contemporary world because of their role in the improvement of food production through increase in crop yields and quality, reduction of farm labour requirements hence lowering cost of production, and improving public health through control of vector and vector-borne diseases (Weiss et al., 2004). Despite all these benefits, pesticides constitute menace to the health of man, animals and even the environment. This is because they are poorly selective and are toxic to non-target species, including humans. The segments of the population that are at the greatest risk of exposure are those that are occupationally exposed, such as agricultural workers. Despite the strict measures put in place concerning its commercialization and use, pesticides sales has increased in recent years (Carlock et al., 1999). The World Health Organization (WHO) estimated that about 3 million cases of acute intoxication and 220,000 deaths are attributable to pesticides each year with majority of these cases occurring in less developed countries (He, 2000; Clegg & van Gemert, 1999), particularly in Africa, Asia, Central America, and South America (Pancetti et al., 2007). Although many pesticides cause neurotoxicity, insecticides are the most acutely neurotoxic to humans and other non-target species compared to other pesticides (Costa et al., 2008). Association between acute exposure to pesticides and neurotoxicity is well known (Lotti, 2000) but the potential effects of chronic low-level exposure are less well established (Alavanja et al., 2004; Ambali et al., 2010a;

Organophosphate (OP) compounds are one of the most widely used constituting about 50% global insecticide use (Casida & Quistad, 2004). Studies in humans showed neurological, cognitive and psychomotor impairments following cumulative exposure to OPs and organochlorines in people from agricultural communities, without history of acute poisoning (Kamel & Hoppin 2004; Kamel et al., 2007). Neurobehavioural changes following low-dose OP exposure have been reported in sheep farmers (Stephens et al., 1995),

**1. Introduction** 

Ambali & Aliyu, 2012).


### **Ameliorative Effect of Vitamin E on Sensorimotor and Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats**

Suleiman F. Ambali1, Joseph O. Ayo1, Muftau Shittu1, Mohammed U. Kawu1 and Suleiman O. Salami2 *1Department of Veterinary Physiology and Pharmacology 2Department of Veterinary Anatomy Ahmadu Bello University, Zaria, Nigeria* 

#### **1. Introduction**

206 Insecticides – Basic and Other Applications

[22] Vasilyeva.O.V., Potapenko. N.A., Ovchinnikova.T.V: Limited proteolysis of *Escherichia* 

[23] Young.S.Lee., Sang.C.Park., Alfred.L.Goldberg., and Chin.Ha.Chung., Protease so from

synthetase.m, *the Journal of Biological Chemistry.,*263(14)., 6643-6646 (1988). [24] Lambert N. H., Abshire k., Blanmenhorn D. and Slonczewski J. L. proteins induced in

[25] Smith S.J., Barbee A.S., Exercise stress response as an adaptive tolerance strategie.

[27] Sanders H.M., Martin L.S., Stress proteins as biomarkers of contaminant exposure in archived environmental samples. *Science Total Environ.*139/140. 459-470 (1993). [28] Phelps, T.J., Palumbo, A.V., Beliaev, A.S., Metabolomics and microarrays for improved

[29] Carla, M. R., Lacerda and Kenneth F., Reardon Environmental proteomics: applications

*Escherichia coli* by benzoic acid. *J. Bacteriol*. 179. 7595-7599 (1997).

[26] Werner G. and Stephen L. *Current opinion in Microbiology,* 9, 123-126. (2006).

environmental constraints., *Curr Opin Biotechnol*, 13, 20–24 (2002).

*in functional genomics and proteomics*., pp 1-13(2009).

*Environ Health Persp* 106. 325-330. (1998).

(2000).

*coli* ATP-Dependent protease ion., *vestnik Moskovskogo Universtiteta, Khimiya.,*41., 6.

*Escherichia coli* preferentially degradesOxidatively damaged Glutathione

understanding of phenotypic characteristics controlled by both genomics and

of proteome profiling in environmental microbiology and biotechnology., *briefings* 

The use of pesticides is inevitable in contemporary world because of their role in the improvement of food production through increase in crop yields and quality, reduction of farm labour requirements hence lowering cost of production, and improving public health through control of vector and vector-borne diseases (Weiss et al., 2004). Despite all these benefits, pesticides constitute menace to the health of man, animals and even the environment. This is because they are poorly selective and are toxic to non-target species, including humans. The segments of the population that are at the greatest risk of exposure are those that are occupationally exposed, such as agricultural workers. Despite the strict measures put in place concerning its commercialization and use, pesticides sales has increased in recent years (Carlock et al., 1999). The World Health Organization (WHO) estimated that about 3 million cases of acute intoxication and 220,000 deaths are attributable to pesticides each year with majority of these cases occurring in less developed countries (He, 2000; Clegg & van Gemert, 1999), particularly in Africa, Asia, Central America, and South America (Pancetti et al., 2007). Although many pesticides cause neurotoxicity, insecticides are the most acutely neurotoxic to humans and other non-target species compared to other pesticides (Costa et al., 2008). Association between acute exposure to pesticides and neurotoxicity is well known (Lotti, 2000) but the potential effects of chronic low-level exposure are less well established (Alavanja et al., 2004; Ambali et al., 2010a; Ambali & Aliyu, 2012).

Organophosphate (OP) compounds are one of the most widely used constituting about 50% global insecticide use (Casida & Quistad, 2004). Studies in humans showed neurological, cognitive and psychomotor impairments following cumulative exposure to OPs and organochlorines in people from agricultural communities, without history of acute poisoning (Kamel & Hoppin 2004; Kamel et al., 2007). Neurobehavioural changes following low-dose OP exposure have been reported in sheep farmers (Stephens et al., 1995),

Ameliorative Effect of Vitamin E on Sensorimotor and

minimise tissue damage.

chronic CPF exposure in Wistar rats.

**2.1 Experimental animals and housing** 

reconstituted in soya oil (100% v/v) prior to daily use.

**2. Materials and methods** 

**2.3 Animal treatment schedule** 

**2.2 Chemicals** 

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 209

death (Kehrer, 1993; Sally et al., 2003). The body is however endowed with cellular defence systems to combat the menace posed by the oxidants to the body. These defensive systems are accomplished by the activities of both the enzymatic and non-enzymatic antioxidants which mitigate the toxic effect of oxidants. However, under increased ROS production, the antioxidant cellular defensive systems are overwhelmed, resulting in oxidative stress. Under this type of condition, exogenous supplementation of antioxidants becomes imperative to

Vitamin E is nature's major lipid soluble chain breaking antioxidant that protects biological membranes and lipoproteins from oxidative stress (Osfor et al., 2010). The main biological function of vitamin E is its direct influence on cellular responses to oxidative stress through modulation of signal transduction pathway (Hsu & Guo, 2002). Vitamin E primarily scavenges peroxyl radicals and is a major inhibitor of the free radical chain reaction of lipid peroxidation (Maxwell, 1995; Halliwell & Gutteridge, 1999). We have earlier demonstrated the mitigating effect of vitamin E on short-term neurobehavioural changes induced by acute CPF exposure (Ambali & Aliyu, 2012). The present study was therefore aimed at evaluating the ameliorative effect of vitamin E on sensorimotor and cognitive changes induced by

Twenty 10 week old male Wistar rats (104±4.2) used for this study were obtained from the Laboratory Animal House of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The animals were housed in plastic cages and allowed to acclimatize for at least two weeks in the laboratory prior to the commencement of the experiment. They were fed on standard rat pellets and water was provided *ad libitum*.

Commercial grade CPF (20% EC, Termicot®, Sabero Organics, Gujarat limited, India), was prepared by reconstituting in soya oil (Grand Cereals and Oil Mills Ltd., Jos, Nigeria) to make 10% stock solution. Vitamin E (100 mg/capsule; Pharco Pharmaceuticals, Egypt) was

The rats were weighed and then assigned at random into 4 groups of 5 rats in each group. Group I (S/oil) served as the control and was given only soya oil (2mL/kg b.w.) while group II (VE) was dosed with vitamin E [75 mg/kg b.w. (Ambali et al., 2010b)]. Group III (CPF) was administered with CPF only [10.6 mg/kg b.w. ~1/8th LD50 of 85 mg/kg b.w., as determined by Ambali (2009)]. Group IV (VE+CPF) was pretreated with vitamin E (75 mg/kg b.w.), and then dosed with CPF (10.6 mg/kg b.w.), 30 min later. The regimens were administered once daily by oral gavage for a period of 17 weeks. During this period, the animals were monitored for clinical signs and death. Furthermore, at various intervals during the study period, the animals were evaluated for neurobehavioural parameters measuring motor coordination, neuromuscular coordination, and motor strength, efficiency of locomotion, learning and memory using the appropriate neurobehavioural devices. In order to avoid bias, the neurobehavioural parameters were evaluated by two trained observers blinded to the treatment schedules. At the end of the dosing period,

greenhouse workers (Bazylewicz-Walczak et al. 1999), tree-fruit workers (Fiedler et al., 1997), and farm workers (Kamel et al., 2003). These studies have found deficits in measures of sustained attention, information processing, motor speed and coordination. The principal mode of insecticidal action of OPs relates to phosphorylation and subsequent inactivation of the esteratic sites of the acetylcholinesterase (AChE) enzyme. The classical role of AChE is to hydrolyze the neurotransmitter acetylcholine (ACh), effectively clearing it from the neuronal synapse and terminating impulse conduction (Farag et al., 2010). Inactivation of AChE results in the accumulation of ACh in the neuronal synapses in the central and peripheral nervous system, thereby overstimulating the nicotinic, muscarinic and central cholinergic receptors with consequent neurotoxicity. Thus, the acute neurotoxic effect of OP results in muscarinic, nicotinic and central cholinergic symptoms (Abou-Donia, 1992). However, toxicity has been reported at doses below the threshold required for inhibition of AChE (Pope, 1999; Slotkin, 2004, 2005) prompting search for other mechanisms. The induction of oxidative stress as one of the other molecular mechanisms involved in OPinduced neurotoxicity has received tremendous attention in recent years (Gultekin et al., 2007; Prendergast et al., 2007; El-Hossary et al., 2009; Ambali et al., 2010a, Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). Indeed, the enhanced production of reactive oxygen species (ROS) by pesticides has been used to explain the multiple types of responses associated with its toxic exposure (Bagchi et al., 1995; Verma et al., 2007).

Chlorpyrifos (*O*,*O*-diethyl-*O*-[3,5,6-trichloro-2-pyridyl] phosphorothioate) is a chlorinated OP insecticide that exhibit a broad spectrum of activity against arthropod pests of plants, animals, and humans, and has wide applications in both agricultural and commercial pest control (Rack, 1993). It is one of the most widely used insecticides and is applied about 20 million times per year in US to houses and lawns (Kingston et al., 1999) with 82% of adults having detectable levels of the 3,5,6-trichloro-2-pyridinol, the metabolite of CPF in their urine (Hill et al., 1995). However, the United States Environmental Protection Agency in 2000 placed ban on some its residential uses in 2000 because of the danger posed to children's health. However, CPF is still widely used as its residues have been detected in citrus fruits in some parts of the world (Iwasaki et al., 2007). Studies have shown that CPF induces neurobehavioural alterations following acute (Caňadas et al., 2005; Ambali et al., 2010a, Ambali & Aliyu, 2012) and repeated low-dose (Stamper et al., 1988; Sanchez-Santed et al., 2004; Ambali & Ayo, 2011a, 2011b) exposure. Similarly, CPF is a developmental neurotoxicant (Qiao et al., 2003; Dietrich et al., 2005; Colborn, 2006; Slotkin et al., 2006;) impairing children mental and behavioral health (Lizardi et al., 2008). Although, CPF like the other OP compounds phosphorylates and subsequently inactivate AChE, neurobehavioural and cognitive deficits have however been observed following repeated low-dose CPF exposure that cannot be attributed to the usual AChE inhibition and muscarinic receptor binding (Pope et al., 1992; Chakraborti et al., 1993; Saulsbury et al., 2009). Earlier studies have shown the involvement of oxidative stress in the neurotoxicity induced by CPF exposure (Gultekin et al., 2007; Ambali et al., 2010a; Ambali & Aliyu, 2012; Ambali and Ayo, 2011a, 2011b).

Oxidative stress, defined as a disruption of the prooxidant-antioxidant balance in favor of the former causes damage to the body tissue (Sies, 1991). Oxidative stress results from an increase in ROS, impairment of antioxidant defense system or insufficient capacity to repair oxidative damage (Halliwell, 1994; Aly et al., 2010). Damage induced by ROS which alters cellular macromolecules such as membrane lipids, DNA, and proteins results in impaired cell functions through changes in intracellular calcium or pH, and consequently leads to cell death (Kehrer, 1993; Sally et al., 2003). The body is however endowed with cellular defence systems to combat the menace posed by the oxidants to the body. These defensive systems are accomplished by the activities of both the enzymatic and non-enzymatic antioxidants which mitigate the toxic effect of oxidants. However, under increased ROS production, the antioxidant cellular defensive systems are overwhelmed, resulting in oxidative stress. Under this type of condition, exogenous supplementation of antioxidants becomes imperative to minimise tissue damage.

Vitamin E is nature's major lipid soluble chain breaking antioxidant that protects biological membranes and lipoproteins from oxidative stress (Osfor et al., 2010). The main biological function of vitamin E is its direct influence on cellular responses to oxidative stress through modulation of signal transduction pathway (Hsu & Guo, 2002). Vitamin E primarily scavenges peroxyl radicals and is a major inhibitor of the free radical chain reaction of lipid peroxidation (Maxwell, 1995; Halliwell & Gutteridge, 1999). We have earlier demonstrated the mitigating effect of vitamin E on short-term neurobehavioural changes induced by acute CPF exposure (Ambali & Aliyu, 2012). The present study was therefore aimed at evaluating the ameliorative effect of vitamin E on sensorimotor and cognitive changes induced by chronic CPF exposure in Wistar rats.

#### **2. Materials and methods**

#### **2.1 Experimental animals and housing**

Twenty 10 week old male Wistar rats (104±4.2) used for this study were obtained from the Laboratory Animal House of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The animals were housed in plastic cages and allowed to acclimatize for at least two weeks in the laboratory prior to the commencement of the experiment. They were fed on standard rat pellets and water was provided *ad libitum*.

#### **2.2 Chemicals**

208 Insecticides – Basic and Other Applications

greenhouse workers (Bazylewicz-Walczak et al. 1999), tree-fruit workers (Fiedler et al., 1997), and farm workers (Kamel et al., 2003). These studies have found deficits in measures of sustained attention, information processing, motor speed and coordination. The principal mode of insecticidal action of OPs relates to phosphorylation and subsequent inactivation of the esteratic sites of the acetylcholinesterase (AChE) enzyme. The classical role of AChE is to hydrolyze the neurotransmitter acetylcholine (ACh), effectively clearing it from the neuronal synapse and terminating impulse conduction (Farag et al., 2010). Inactivation of AChE results in the accumulation of ACh in the neuronal synapses in the central and peripheral nervous system, thereby overstimulating the nicotinic, muscarinic and central cholinergic receptors with consequent neurotoxicity. Thus, the acute neurotoxic effect of OP results in muscarinic, nicotinic and central cholinergic symptoms (Abou-Donia, 1992). However, toxicity has been reported at doses below the threshold required for inhibition of AChE (Pope, 1999; Slotkin, 2004, 2005) prompting search for other mechanisms. The induction of oxidative stress as one of the other molecular mechanisms involved in OPinduced neurotoxicity has received tremendous attention in recent years (Gultekin et al., 2007; Prendergast et al., 2007; El-Hossary et al., 2009; Ambali et al., 2010a, Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). Indeed, the enhanced production of reactive oxygen species (ROS) by pesticides has been used to explain the multiple types of responses

associated with its toxic exposure (Bagchi et al., 1995; Verma et al., 2007).

Ambali and Ayo, 2011a, 2011b).

Chlorpyrifos (*O*,*O*-diethyl-*O*-[3,5,6-trichloro-2-pyridyl] phosphorothioate) is a chlorinated OP insecticide that exhibit a broad spectrum of activity against arthropod pests of plants, animals, and humans, and has wide applications in both agricultural and commercial pest control (Rack, 1993). It is one of the most widely used insecticides and is applied about 20 million times per year in US to houses and lawns (Kingston et al., 1999) with 82% of adults having detectable levels of the 3,5,6-trichloro-2-pyridinol, the metabolite of CPF in their urine (Hill et al., 1995). However, the United States Environmental Protection Agency in 2000 placed ban on some its residential uses in 2000 because of the danger posed to children's health. However, CPF is still widely used as its residues have been detected in citrus fruits in some parts of the world (Iwasaki et al., 2007). Studies have shown that CPF induces neurobehavioural alterations following acute (Caňadas et al., 2005; Ambali et al., 2010a, Ambali & Aliyu, 2012) and repeated low-dose (Stamper et al., 1988; Sanchez-Santed et al., 2004; Ambali & Ayo, 2011a, 2011b) exposure. Similarly, CPF is a developmental neurotoxicant (Qiao et al., 2003; Dietrich et al., 2005; Colborn, 2006; Slotkin et al., 2006;) impairing children mental and behavioral health (Lizardi et al., 2008). Although, CPF like the other OP compounds phosphorylates and subsequently inactivate AChE, neurobehavioural and cognitive deficits have however been observed following repeated low-dose CPF exposure that cannot be attributed to the usual AChE inhibition and muscarinic receptor binding (Pope et al., 1992; Chakraborti et al., 1993; Saulsbury et al., 2009). Earlier studies have shown the involvement of oxidative stress in the neurotoxicity induced by CPF exposure (Gultekin et al., 2007; Ambali et al., 2010a; Ambali & Aliyu, 2012;

Oxidative stress, defined as a disruption of the prooxidant-antioxidant balance in favor of the former causes damage to the body tissue (Sies, 1991). Oxidative stress results from an increase in ROS, impairment of antioxidant defense system or insufficient capacity to repair oxidative damage (Halliwell, 1994; Aly et al., 2010). Damage induced by ROS which alters cellular macromolecules such as membrane lipids, DNA, and proteins results in impaired cell functions through changes in intracellular calcium or pH, and consequently leads to cell Commercial grade CPF (20% EC, Termicot®, Sabero Organics, Gujarat limited, India), was prepared by reconstituting in soya oil (Grand Cereals and Oil Mills Ltd., Jos, Nigeria) to make 10% stock solution. Vitamin E (100 mg/capsule; Pharco Pharmaceuticals, Egypt) was reconstituted in soya oil (100% v/v) prior to daily use.

#### **2.3 Animal treatment schedule**

The rats were weighed and then assigned at random into 4 groups of 5 rats in each group. Group I (S/oil) served as the control and was given only soya oil (2mL/kg b.w.) while group II (VE) was dosed with vitamin E [75 mg/kg b.w. (Ambali et al., 2010b)]. Group III (CPF) was administered with CPF only [10.6 mg/kg b.w. ~1/8th LD50 of 85 mg/kg b.w., as determined by Ambali (2009)]. Group IV (VE+CPF) was pretreated with vitamin E (75 mg/kg b.w.), and then dosed with CPF (10.6 mg/kg b.w.), 30 min later. The regimens were administered once daily by oral gavage for a period of 17 weeks. During this period, the animals were monitored for clinical signs and death. Furthermore, at various intervals during the study period, the animals were evaluated for neurobehavioural parameters measuring motor coordination, neuromuscular coordination, and motor strength, efficiency of locomotion, learning and memory using the appropriate neurobehavioural devices. In order to avoid bias, the neurobehavioural parameters were evaluated by two trained observers blinded to the treatment schedules. At the end of the dosing period,

Ameliorative Effect of Vitamin E on Sensorimotor and

**2.8 Assessment of the effect of treatments on learning** 

**2.9 Assessment of the effect of treatments on short-term memory** 

2 min was counted as maximum memory retention (ceiling response).

**2.11 Effect of treatments on brain lipoperoxidation** 

**2.10 Brain tissue preparation** 

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 211

The effect of treatments on learning task in rats was assessed 48h to the final termination of the study in week 17 using the step-down inhibitory avoidance learning task as described by Zhu et al. (2001). The apparatus used was an acrylic chamber 40 x 25 x 25 cm consisting of a floor made of parallel 2-mm-caliber stainless steel bars spaced 1 cm apart. An electric shock was delivered through the floor bars. A 2.5-cm-high, 8 x 25 cm wooden platform was placed on the left extreme of the chamber. Each rat was gently placed on the platform. Upon stepping down, the rat immediately received a single 1.5 amp foot shock through the floor bars. If the animal did not return to the platform, the foot shock was repeated every 5s. A rat was considered to have learned the avoidance task if it remained on the platform for more than 2 min. The number of foot shocks was recorded as an index of learning acquisition.

Short-term memory was assessed in individual rat from each group using the step-down avoidance inhibitory task as described by Zhu et al. (2001) 24h after the assessment of learning. The apparatus used was the same used earlier for the assessment of learning. In this test, each rat was again placed gently on the platform and the time an animal remained on the platform was recorded as an index of memory retention. Staying on the platform for

The whole brain tissue was carefully dissected and a known weight of the brain sample from each animal was homogenized in a known volume of ice cold phosphate buffer to obtain a 10% homogenate. This was then centrifuged at 3000 × g for 10 min to obtain the supernatant. The supernatant was then used to assess the levels of protein, malonaldehyde

The level of thiobarbituric acid reactive substance, malonaldehyde (MDA) as an index of lipid peroxidation was evaluated on the brain sample using the method of Draper & Hadley (1990) as modified (Freitas et al., 2005). The principle of the method was based on spectrophotometric measurement of the colour developed during reaction of thiobarbituric acid (TBA) with malonadehyde (MDA). The MDA concentration in each sample was calculated by the absorbance coefficient of MDA-TBA complex 1.56 x 105/cm/M and expressed as nmol/mg of tissue protein. The concentration of protein in the brain

**2.12 Evaluation of the effect of treatments on brain superoxide dismutase activity**  Superoxide dismutase activity was evaluated using NWLSSTM superoxide dismutase activity assay kit (Northwest Life Science Specialities, Vancouver, WA 98662) as stated by

Catalase activity was evaluated using NWLSSTM catalase activity assay kit (Northwest Life Science Specialities, LLC, Vancouver, WA 98662) as stated by the manufacturer and was

(MDA), superoxide dismutase (SOD), catalase (CAT) and AChE in the brain sample.

homogenates was evaluated using the Lowry method (Lowry et al*.,* 1951).

the manufacturer and was expressed as mMol/mg tissue protein.

expressed as mMol/mg tissue protein.

**2.13 Evaluation of the effect of treatments on brain catalse activity** 

each of the animals was sacrificed by jugular venesection and the brain dissected, removed and evaluated for the levels of oxidative stress parameters and AChE inhibition. The experiment was conducted with the permission of the Animals Research Ethics Committee of the Ahmadu Bello University, Zaria, Nigeria and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

#### **2.4 Evaluation of the effect of treatments on motor coordination**

The assessment of motor coordination was performed using the beam walk performance task as described in an earlier study (Ambali et al., 2010a) on day 0, weeks 8 and 16. Briefly, each of the rats was allowed to walk across a wooden black beam of 106-cm length, beginning at 17.2 cm width and ending at 1.0-cm width. Periodic widths were marked on the side of the apparatus. On each side of the narrowing beam, there was a 1.8 cm step-down to a 3.0-cm area where subjects may step if necessary. As the subject walked across from the 17.2 cm to the 1.0 cm width, the width at which they stepped down was recorded by one rater on each side, and this was repeated twice during each trial session.

#### **2.5 Evaluation of the effect of treatments on motor strength**

The forepaw grip time was used to evaluate the motor strength of the rats, as described by Abou-Donia et al. (2001). This was conducted by having each of the rats hung down from a 5 mm diameter wooden dowel gripped with both forepaws. The time spent by each rat before releasing their grips was recorded in seconds. This parameter was evaluated on day 0, weeks 8 and 16.

#### **2.6 Effect of treatments on neuromuscular coordination**

The effect of treatments on neuromuscular coordination was assessed using the performance on incline plane as was described earlier (Ambali et al., 2010a). Briefly, each rat was placed on an apparatus made with an angled rough wooden plank with thick foam pad at its bottom end. The plank was first raised to an inclination of 35°, and thereafter gradually increased stepwise by 5° until the subject could no longer stay and be situated horizontally on the plank for 3s, without sliding down. Angles were measured and marked on the apparatus beforehand, and were obtained by propping the plank on a vertical bar with several notches. The test was performed with the head of the rat first facing left and then right hand side of the experimenter. The highest angle at which each rat stayed and stood horizontally, and facing each direction was recorded. Two trials were performed at 2 min apart for each animal. This procedure was carried out on each animal from all the groups on day 0, weeks 8 and 16 of the study.

#### **2.7 Evaluation of the effect of treatments on efficiency of locomotion**

The ladder walk was used to assess the efficiency of locomotion as described by Ambali and Aliyu (2012). Briefly, each rat was encouraged to walk across a black wooden ladder (106 cm x17 cm) with 0.8-cm diameter rungs, and 2.5-cm spaces between them. The number of times the rat missed a rung was counted by one rater on each side. The performance on ladder walk was evaluated on Day 0, weeks 3, 7 and 11. Two trials were performed for each testing session.

#### **2.8 Assessment of the effect of treatments on learning**

The effect of treatments on learning task in rats was assessed 48h to the final termination of the study in week 17 using the step-down inhibitory avoidance learning task as described by Zhu et al. (2001). The apparatus used was an acrylic chamber 40 x 25 x 25 cm consisting of a floor made of parallel 2-mm-caliber stainless steel bars spaced 1 cm apart. An electric shock was delivered through the floor bars. A 2.5-cm-high, 8 x 25 cm wooden platform was placed on the left extreme of the chamber. Each rat was gently placed on the platform. Upon stepping down, the rat immediately received a single 1.5 amp foot shock through the floor bars. If the animal did not return to the platform, the foot shock was repeated every 5s. A rat was considered to have learned the avoidance task if it remained on the platform for more than 2 min. The number of foot shocks was recorded as an index of learning acquisition.

#### **2.9 Assessment of the effect of treatments on short-term memory**

Short-term memory was assessed in individual rat from each group using the step-down avoidance inhibitory task as described by Zhu et al. (2001) 24h after the assessment of learning. The apparatus used was the same used earlier for the assessment of learning. In this test, each rat was again placed gently on the platform and the time an animal remained on the platform was recorded as an index of memory retention. Staying on the platform for 2 min was counted as maximum memory retention (ceiling response).

#### **2.10 Brain tissue preparation**

210 Insecticides – Basic and Other Applications

each of the animals was sacrificed by jugular venesection and the brain dissected, removed and evaluated for the levels of oxidative stress parameters and AChE inhibition. The experiment was conducted with the permission of the Animals Research Ethics Committee of the Ahmadu Bello University, Zaria, Nigeria and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication

The assessment of motor coordination was performed using the beam walk performance task as described in an earlier study (Ambali et al., 2010a) on day 0, weeks 8 and 16. Briefly, each of the rats was allowed to walk across a wooden black beam of 106-cm length, beginning at 17.2 cm width and ending at 1.0-cm width. Periodic widths were marked on the side of the apparatus. On each side of the narrowing beam, there was a 1.8 cm step-down to a 3.0-cm area where subjects may step if necessary. As the subject walked across from the 17.2 cm to the 1.0 cm width, the width at which they stepped down was recorded by one rater on each side, and this was repeated twice during each

The forepaw grip time was used to evaluate the motor strength of the rats, as described by Abou-Donia et al. (2001). This was conducted by having each of the rats hung down from a 5 mm diameter wooden dowel gripped with both forepaws. The time spent by each rat before releasing their grips was recorded in seconds. This parameter was evaluated on day

The effect of treatments on neuromuscular coordination was assessed using the performance on incline plane as was described earlier (Ambali et al., 2010a). Briefly, each rat was placed on an apparatus made with an angled rough wooden plank with thick foam pad at its bottom end. The plank was first raised to an inclination of 35°, and thereafter gradually increased stepwise by 5° until the subject could no longer stay and be situated horizontally on the plank for 3s, without sliding down. Angles were measured and marked on the apparatus beforehand, and were obtained by propping the plank on a vertical bar with several notches. The test was performed with the head of the rat first facing left and then right hand side of the experimenter. The highest angle at which each rat stayed and stood horizontally, and facing each direction was recorded. Two trials were performed at 2 min apart for each animal. This procedure was carried out on each animal from all the groups on

The ladder walk was used to assess the efficiency of locomotion as described by Ambali and Aliyu (2012). Briefly, each rat was encouraged to walk across a black wooden ladder (106 cm x17 cm) with 0.8-cm diameter rungs, and 2.5-cm spaces between them. The number of times the rat missed a rung was counted by one rater on each side. The performance on ladder walk was evaluated on Day 0, weeks 3, 7 and 11. Two trials were performed for each testing

**2.4 Evaluation of the effect of treatments on motor coordination** 

**2.5 Evaluation of the effect of treatments on motor strength** 

**2.6 Effect of treatments on neuromuscular coordination** 

**2.7 Evaluation of the effect of treatments on efficiency of locomotion** 

No. 85-23, revised 1985).

trial session.

0, weeks 8 and 16.

session.

day 0, weeks 8 and 16 of the study.

The whole brain tissue was carefully dissected and a known weight of the brain sample from each animal was homogenized in a known volume of ice cold phosphate buffer to obtain a 10% homogenate. This was then centrifuged at 3000 × g for 10 min to obtain the supernatant. The supernatant was then used to assess the levels of protein, malonaldehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and AChE in the brain sample.

#### **2.11 Effect of treatments on brain lipoperoxidation**

The level of thiobarbituric acid reactive substance, malonaldehyde (MDA) as an index of lipid peroxidation was evaluated on the brain sample using the method of Draper & Hadley (1990) as modified (Freitas et al., 2005). The principle of the method was based on spectrophotometric measurement of the colour developed during reaction of thiobarbituric acid (TBA) with malonadehyde (MDA). The MDA concentration in each sample was calculated by the absorbance coefficient of MDA-TBA complex 1.56 x 105/cm/M and expressed as nmol/mg of tissue protein. The concentration of protein in the brain homogenates was evaluated using the Lowry method (Lowry et al*.,* 1951).

#### **2.12 Evaluation of the effect of treatments on brain superoxide dismutase activity**

Superoxide dismutase activity was evaluated using NWLSSTM superoxide dismutase activity assay kit (Northwest Life Science Specialities, Vancouver, WA 98662) as stated by the manufacturer and was expressed as mMol/mg tissue protein.

#### **2.13 Evaluation of the effect of treatments on brain catalse activity**

Catalase activity was evaluated using NWLSSTM catalase activity assay kit (Northwest Life Science Specialities, LLC, Vancouver, WA 98662) as stated by the manufacturer and was expressed as mMol/mg tissue protein.

Ameliorative Effect of Vitamin E on Sensorimotor and

dynamic of beam walk performance in Wistar rats.

**3.3 Effect of treatments on grip time** 

group compared to that in VE group (Fig. 2).

**3.4 Effect of treatments on incline plane performance** 

**0**

**1**

**2**

**3**

**4**

**5**

**Width of slip off the beam (cm)**

**6**

**7**

**8**

**9**

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 213

**S/oil VE CPF VE+CPF**

**D0 Wk 8 Wk16**

Fig. 1. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the

There was no significant change (P>0.05) in the grip time in the S/oil and VE groups throughout the study period. There was a significant increase (P<0.01) in the grip time of CPF and VE+CPF groups at day 0 compared to that of week 8 or 16, but not between week 8 and that of week 16. At day 0, there was no significant change (P>0.05) in the grip time of rats in between the groups. At week 8, there was a significant decrease (P<0.01) in the grip time of CPF group compared to that in the S/oil and VE groups, but not that of VE+CPF group. There was a significant decrease (P<0.05) in the grip time in the VE+CPF group compared to that in S/oil or VE group. There was no significant change (P>0.05) in the grip time in the VE group compared to that in S/oil group. At week 16, there was a significant decrease (P<0.01) in the grip time in the CPF group compared to that in S/oil or VE group but no significant change (P<0.05) compared to that in VE+CPF group. There was no significant change (P>0.05) in the grip time in the VE+CPF group compared to that in S/oil or VE group. Similarly, there was no significant change (P>0.05) in the grip time of S/oil

There was no significant change (P>0.05) in the angle at which the S/oil and VE groups slipped off the incline plane throughout the study period. There was a significant decrease (P<0.05) in the angle at which the CPF group slipped off the incline plane at weeks 8 and 16, respectively, compared to that of day 0 but no significant change (P>0.05) at week 8 relative to that recorded in week 16. There was a significant decrease (P<0.01) in the angle at which VE+CPF group slipped off the incline plane at week 16 compared to that of day 0 but no

significant change (P>0.05) at week 8 relative to that recorded in day 0 or week 16.

#### **2.14 Evaluation of the effect of treatments on brain acetylcholinesterase activity**

Acetylcholinesterase activity was evaluated using the method of Ellman et al. (1961) with acetylthiocholine iodide as a substrate. Briefly, the whole brain of each animal was homogenized in a cold (0–4 °C) 20 m*M* phosphate buffer saline (PBS) incubated with 0.01M 5,5-dithio-bis(2-nitrobenzoic acid) in 0.1 *M* PBS, pH 7.0. Incubations were allowed to proceed at room temperature for 10 min. Then, acetylthiocholine iodide (0.075 *M* in 0.1 M PBS, pH 8.0) was added to each tube, and absorbance at 412 nm was measured continuously for 30 min using a UV spectrophotometer (T80+ UV/VIS spectrometer®, PG Instruments Ltd, Liicestershire, LE 175BE, United Kingdom). AChE activity was expressed as IU/g tissue.

#### **2.15 Statistical analysis**

Data were expressed as mean ± standard error of mean. Data obtained from the sensorimotor assessment were analyzed using repeated one-way analysis of variance followed by Tukey's posthoc test. The cognitive and biochemical parameters were analyzed using one-way analysis of variance followed by Tukey's posthoc test. Values of P < 0.05 were considered significant.

#### **3. Results**

#### **3.1 Effect of treatments on clinical signs**

There was no clinical manifestation recorded in the S/oil, VE and VE+CPF groups, while lacrimation, congested ocular mucous membranes and intermittent tremors were observed in the CPF group.

#### **3.2 Effect of treatments on beam walk performance**

There was no significant change (P>0.05) in the dynamics of beam walk performance in the S/oil group throughout the period of the study. There was a progressive decrease in the width at which VE group slipped off the beam (increase in beam walk length) throughout the study period. Although no significant change (P>0.05) was recorded in week 8 compared to day 0 or week 16, a significant decrease (P<0.05) in the width at which the VE group slipped off the beam in week 16 compared to that of day 0. There was a significant increase (P<0.01) in the width of slip off the beam (decrease in beam walk length) in the CPF group at weeks 8 and 16 when compared to that of day 0, and between week 16 and that recorded in week 8. There was no significant change (P>0.05) in the width at which VE+CPF group slipped off the beam at week 8 when compared to that recorded on day 0 or week 16 but a significant increase (P<0.01) was recorded at week 16 compared to that of day 0.

There was no significant change (P>0.05) in the width at which animals in all the groups slipped off the beam at day 0. At week 8, there was a significant increase (P<0.01) in the width at which the CPF group slipped off the beam compared to that of S/oil, VE or VE+CPF group. Similarly, there was a significant increase (P>0.05) in the width of slip in the VE+CPF group compare to that of VE group but no significant change (P>0.05) in the S/oil group compared to that of VE or VE+CPF group. At week 16, there was a significant increase (P<0.01) in the width of slip off the beam in the CPF group compared to the other groups but no significant change (P>0.05) in the S/oil group when compared to that of VE or VE+CPF group, and between VE group and that recorded in theVE+CPF group (Fig. 1).

Fig. 1. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the dynamic of beam walk performance in Wistar rats.

#### **3.3 Effect of treatments on grip time**

212 Insecticides – Basic and Other Applications

Data were expressed as mean ± standard error of mean. Data obtained from the sensorimotor assessment were analyzed using repeated one-way analysis of variance followed by Tukey's posthoc test. The cognitive and biochemical parameters were analyzed using one-way analysis of variance followed by Tukey's posthoc test. Values of P < 0.05

There was no clinical manifestation recorded in the S/oil, VE and VE+CPF groups, while lacrimation, congested ocular mucous membranes and intermittent tremors were observed

There was no significant change (P>0.05) in the dynamics of beam walk performance in the S/oil group throughout the period of the study. There was a progressive decrease in the width at which VE group slipped off the beam (increase in beam walk length) throughout the study period. Although no significant change (P>0.05) was recorded in week 8 compared to day 0 or week 16, a significant decrease (P<0.05) in the width at which the VE group slipped off the beam in week 16 compared to that of day 0. There was a significant increase (P<0.01) in the width of slip off the beam (decrease in beam walk length) in the CPF group at weeks 8 and 16 when compared to that of day 0, and between week 16 and that recorded in week 8. There was no significant change (P>0.05) in the width at which VE+CPF group slipped off the beam at week 8 when compared to that recorded on day 0 or week 16 but a significant increase (P<0.01) was recorded at week 16

There was no significant change (P>0.05) in the width at which animals in all the groups slipped off the beam at day 0. At week 8, there was a significant increase (P<0.01) in the width at which the CPF group slipped off the beam compared to that of S/oil, VE or VE+CPF group. Similarly, there was a significant increase (P>0.05) in the width of slip in the VE+CPF group compare to that of VE group but no significant change (P>0.05) in the S/oil group compared to that of VE or VE+CPF group. At week 16, there was a significant increase (P<0.01) in the width of slip off the beam in the CPF group compared to the other groups but no significant change (P>0.05) in the S/oil group when compared to that of VE or VE+CPF group, and between VE group and that recorded in theVE+CPF group (Fig. 1).

**2.14 Evaluation of the effect of treatments on brain acetylcholinesterase activity**  Acetylcholinesterase activity was evaluated using the method of Ellman et al. (1961) with acetylthiocholine iodide as a substrate. Briefly, the whole brain of each animal was homogenized in a cold (0–4 °C) 20 m*M* phosphate buffer saline (PBS) incubated with 0.01M 5,5-dithio-bis(2-nitrobenzoic acid) in 0.1 *M* PBS, pH 7.0. Incubations were allowed to proceed at room temperature for 10 min. Then, acetylthiocholine iodide (0.075 *M* in 0.1 M PBS, pH 8.0) was added to each tube, and absorbance at 412 nm was measured continuously for 30 min using a UV spectrophotometer (T80+ UV/VIS spectrometer®, PG Instruments Ltd, Liicestershire, LE 175BE, United Kingdom). AChE activity was expressed as IU/g

tissue.

**3. Results** 

in the CPF group.

**2.15 Statistical analysis** 

were considered significant.

compared to that of day 0.

**3.1 Effect of treatments on clinical signs** 

**3.2 Effect of treatments on beam walk performance** 

There was no significant change (P>0.05) in the grip time in the S/oil and VE groups throughout the study period. There was a significant increase (P<0.01) in the grip time of CPF and VE+CPF groups at day 0 compared to that of week 8 or 16, but not between week 8 and that of week 16. At day 0, there was no significant change (P>0.05) in the grip time of rats in between the groups. At week 8, there was a significant decrease (P<0.01) in the grip time of CPF group compared to that in the S/oil and VE groups, but not that of VE+CPF group. There was a significant decrease (P<0.05) in the grip time in the VE+CPF group compared to that in S/oil or VE group. There was no significant change (P>0.05) in the grip time in the VE group compared to that in S/oil group. At week 16, there was a significant decrease (P<0.01) in the grip time in the CPF group compared to that in S/oil or VE group but no significant change (P<0.05) compared to that in VE+CPF group. There was no significant change (P>0.05) in the grip time in the VE+CPF group compared to that in S/oil or VE group. Similarly, there was no significant change (P>0.05) in the grip time of S/oil group compared to that in VE group (Fig. 2).

#### **3.4 Effect of treatments on incline plane performance**

There was no significant change (P>0.05) in the angle at which the S/oil and VE groups slipped off the incline plane throughout the study period. There was a significant decrease (P<0.05) in the angle at which the CPF group slipped off the incline plane at weeks 8 and 16, respectively, compared to that of day 0 but no significant change (P>0.05) at week 8 relative to that recorded in week 16. There was a significant decrease (P<0.01) in the angle at which VE+CPF group slipped off the incline plane at week 16 compared to that of day 0 but no significant change (P>0.05) at week 8 relative to that recorded in day 0 or week 16.

Ameliorative Effect of Vitamin E on Sensorimotor and

group compared to that in the S/oil group (Fig. 4).

dynamics of locomotion efficiency in Wistar rats.

**3.6 Effect of treatments on learning acquisition** 

dynamics of incline plane performance in Wistar rats.

VE group (Fig. 5).

**0**

**10**

**20**

**30**

**40**

**Angle of slip off incline plane (degree)**

**50**

**60**

**70**

**80**

**Number of missed rungs**

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 215

Similarly, there was no significant change (P>0.05) in the number of missed rungs in the VE

**S/oil VE CPF VE+CPF**

**D0 Wk 8 Wk16**

Fig. 3. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the

There was a significant increase (P<0.01) in the number of footshocks applied to the CPF group relative to that recorded in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the number of footshocks in the VE+CPF group relative to that in S/oil or

**S/oil VE CPF VE+CPF**

**D0 Wk 8 Wk16**

Fig. 4. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the

Fig. 2. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the dynamic of grip time in Wistar rats.

At day 0, there was no significant change (P>0.05) in the angle of slip off the incline plane in between the groups. At week 8, there was a significant decrease in the angle of slip off the incline plane in the CPF group relative to that recorded in S/oil (P<0.05), VE (P<0.01) or VE+CPF group. No significant change (P>0.05) in the angle of slip in the VE+CPF group relative to that in S/oil or VE group, and between VE group and that of S/oil group. At week 16, there was a significant decrease in the angle of slip off the incline plane in the CPF group relative to that in S/oil (P<0.05) or VE (P<0.01) group. Although not significant, there was a 6.3% increase in the angle of slip off the incline plane in the VE+CPF group relative to that in CPF group. There was no significant change (P>0.05) in the angle of slip off the plane in the S/oil group compared to that in VE or VE+CPF group (Fig. 3).

#### **3.5 Effect of treatments on ladderwalk performance**

There was no significant change (P>0.05) in the dynamics of the number of missed rungs in the S/oil, VE and VE+CPF groups throughout the study period. There was a significant decrease (P<0.01) in the number of missed rungs in the CPF group at day 0 compared to that in week 8 or 16 but no significant change at week 8 compared to that of week 16.

There was no significant change (P>0.05) in the number of missed rungs in between the groups at day 0. At week 8, there was a significant decrease (P<0.01) in the number of missed rungs in the CPF group compared to that in S/oil or VE group. Although not significant (P>0.05), the mean number of missed rungs in the VE+CPF group was 26% higher relative to that recorded in the CPF group. There was a significant decrease (P<0.01) in the number of missed rungs in the VE+CPF group compared to that in S/oil or VE group. There was no significant change (P>0.05) in the number of missed rungs in the VE group compared to that in S/oil group. At week 16, there was a significant decrease (P<0.01) in the number of missed rungs in the CPF group compared to the VE group but no significant change (P>0.05) when compared to that recorded in S/oil or VE+CPF group. There was no significant change (P>0.05) in the VE+CPF group compared to that in S/oil or VE group.

**S/oil VE CPF VE+CPF**

**D0 Wk 8 Wk16**

Fig. 2. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the

in the S/oil group compared to that in VE or VE+CPF group (Fig. 3).

**3.5 Effect of treatments on ladderwalk performance** 

At day 0, there was no significant change (P>0.05) in the angle of slip off the incline plane in between the groups. At week 8, there was a significant decrease in the angle of slip off the incline plane in the CPF group relative to that recorded in S/oil (P<0.05), VE (P<0.01) or VE+CPF group. No significant change (P>0.05) in the angle of slip in the VE+CPF group relative to that in S/oil or VE group, and between VE group and that of S/oil group. At week 16, there was a significant decrease in the angle of slip off the incline plane in the CPF group relative to that in S/oil (P<0.05) or VE (P<0.01) group. Although not significant, there was a 6.3% increase in the angle of slip off the incline plane in the VE+CPF group relative to that in CPF group. There was no significant change (P>0.05) in the angle of slip off the plane

There was no significant change (P>0.05) in the dynamics of the number of missed rungs in the S/oil, VE and VE+CPF groups throughout the study period. There was a significant decrease (P<0.01) in the number of missed rungs in the CPF group at day 0 compared to that

There was no significant change (P>0.05) in the number of missed rungs in between the groups at day 0. At week 8, there was a significant decrease (P<0.01) in the number of missed rungs in the CPF group compared to that in S/oil or VE group. Although not significant (P>0.05), the mean number of missed rungs in the VE+CPF group was 26% higher relative to that recorded in the CPF group. There was a significant decrease (P<0.01) in the number of missed rungs in the VE+CPF group compared to that in S/oil or VE group. There was no significant change (P>0.05) in the number of missed rungs in the VE group compared to that in S/oil group. At week 16, there was a significant decrease (P<0.01) in the number of missed rungs in the CPF group compared to the VE group but no significant change (P>0.05) when compared to that recorded in S/oil or VE+CPF group. There was no significant change (P>0.05) in the VE+CPF group compared to that in S/oil or VE group.

in week 8 or 16 but no significant change at week 8 compared to that of week 16.

**0**

dynamic of grip time in Wistar rats.

**20**

**40**

**60**

**Grip time (secs)**

**80**

**100**

**120**

Similarly, there was no significant change (P>0.05) in the number of missed rungs in the VE group compared to that in the S/oil group (Fig. 4).

Fig. 3. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the dynamics of locomotion efficiency in Wistar rats.

#### **3.6 Effect of treatments on learning acquisition**

There was a significant increase (P<0.01) in the number of footshocks applied to the CPF group relative to that recorded in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the number of footshocks in the VE+CPF group relative to that in S/oil or VE group (Fig. 5).

Fig. 4. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the dynamics of incline plane performance in Wistar rats.

Ameliorative Effect of Vitamin E on Sensorimotor and

**0**

**0**

groups, respectively.

**0.05**

**0.1**

**0.15**

**0.2**

**0.25**

**Brain malonaldehyde concentration (mMol/mg protein)**

**0.3**

**0.35**

**0.4**

**0.45**

**0.5**

**20**

**40**

**60**

**80**

**Latency on platform (Secs)**

**100**

**120**

**140**

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 217

**S/oil VE CPF VE+CPF**

**S/oil VE CPF VE+CPF**

Fig. 7. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the brain malonaldehyde concentration in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF

Fig. 6. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on shortterm memory in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

**abc**

 **abc**

Fig. 5. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the learning task in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

#### **3.7 Effect of treatments on short-term memory**

A significant decrease (P<0.01) in the duration of stay on platform (latency on platform) was recorded in the CPF group compared to that in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the duration of stay on the platform in the VE+CPF group compared to that in the S/oil or VE group (Fig. 6).

#### **3.8 Effect of treatments on brain malonaldehyde concentration**

A significant increase (P<0.01) in MDA concentration was recorded in the CPF group relative to that in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the brain MDA concentration in the VE+CPF group compared to that in S/oil or VE group, nor between VE and S/oil groups (Fig. 7).

#### **3.9 Effect of treatments on brain superoxide dismutase activity**

There was a significant decrease (P<0.01) in SOD activity in the CPF group relative to the S/oil, VE or VE+CPF group. No significant change (P>0.05) was recorded in SOD activity in the VE+CPF group relative to that in S/oil or VE group, nor between VE and that recorded in the S/oil group (Fig. 8).

#### **3.10 Effect of treatments on brain catalase activity**

A significant decrease (P<0.01) in brain CAT activity was recorded in the CPF group relative that in the S/oil, VE or VE+CPF group. The CAT activity in the VE+CPF group did not

**abc**

**S/oil VE CPF VE+CPF**

Fig. 5. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the learning task in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

A significant decrease (P<0.01) in the duration of stay on platform (latency on platform) was recorded in the CPF group compared to that in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the duration of stay on the platform in the VE+CPF group

A significant increase (P<0.01) in MDA concentration was recorded in the CPF group relative to that in the S/oil, VE or VE+CPF group. There was no significant change (P>0.05) in the brain MDA concentration in the VE+CPF group compared to that in S/oil or VE

There was a significant decrease (P<0.01) in SOD activity in the CPF group relative to the S/oil, VE or VE+CPF group. No significant change (P>0.05) was recorded in SOD activity in the VE+CPF group relative to that in S/oil or VE group, nor between VE and that recorded

A significant decrease (P<0.01) in brain CAT activity was recorded in the CPF group relative that in the S/oil, VE or VE+CPF group. The CAT activity in the VE+CPF group did not

**3.7 Effect of treatments on short-term memory** 

compared to that in the S/oil or VE group (Fig. 6).

group, nor between VE and S/oil groups (Fig. 7).

**3.10 Effect of treatments on brain catalase activity** 

in the S/oil group (Fig. 8).

**3.8 Effect of treatments on brain malonaldehyde concentration** 

**3.9 Effect of treatments on brain superoxide dismutase activity** 

**0**

**1**

**2**

**3**

**4**

**Number of footshocks applied**

**5**

**6**

**7**

Fig. 6. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on shortterm memory in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

Fig. 7. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the brain malonaldehyde concentration in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

Ameliorative Effect of Vitamin E on Sensorimotor and

**0**

**4. Discussion** 

respectively; cP<0.05 versus VE group.

**100**

**200**

**300**

**Acetylcholinesterase activity (IU/g tissue)**

**400**

**500**

**600**

**3.11 Effect of treatments on brain acetylcholinesterase activity** 

groups, respectively, or between VE and S/oil groups (Fig. 10).

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 219

There was a significant decrease in brain AChE activity in the CPF group compared to that in the S/oil (P<0.01), VE (P<0.01) or VE+CPF (P<0.05) group. There was no significant change (P>0.05) recorded in CAT activity in the VE+CPF relative to that in the S/oil and VE

**S/oil VE CPF VE+CPF**

Fig. 10. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the

The increase in brain MDA concentration and low SOD and CAT activities in the CPF group is an indication of the ability of this pesticide to elevate lipoperoxidative changes and thereby induce oxidative stress. This was in agreement with the findings from our previous studies (Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). The brain due to its biochemical and physiological properties is especially sensitive to free radicals, which destroy its functions and structure (Drewa et al., 1998). The brain is highly vulnerable to oxidative stress because in addition to harboring large amount of oxygen in a relatively small mass, it contains a significant quantity of metals (Fe), and has fewer antioxidant molecules than other organs (Halliwell and Gutteridge, 1999; Naffa-Mazzacoratt et al., 2001). For instance, the CNS is relatively poorly endowed with SOD, CAT, and glutathione peroxidase, and is also relatively lacking in vitamin E (Halliwell & Gutteridge, 1985). CPF is lipophilic and may enhance lipid peroxidation by directly interacting with cellular plasma membrane (Hazarika et al., 2003). The increased MDA concentration which is due to induction of free radical has been shown to alter the composition of membrane lipids, proteins, carbohydrates and DNA. Membrane lipids are vital for the maintenance of cellular integrity and survival (Jain, 1989). Peroxidation of membrane lipids results in the

acetylcholinesterase activity in Wistar rats. abcP<0.01versus S/oil and VE groups,

**ab**

**c**

differ significantly (P>0.05) when compared to that in the S/oil or VE group, and between VE and that recorded in the S/oil group (Fig. 9).

Fig. 8. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the superoxide dismutase activity in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively.

Fig. 9. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the catalase activity in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively

#### **3.11 Effect of treatments on brain acetylcholinesterase activity**

There was a significant decrease in brain AChE activity in the CPF group compared to that in the S/oil (P<0.01), VE (P<0.01) or VE+CPF (P<0.05) group. There was no significant change (P>0.05) recorded in CAT activity in the VE+CPF relative to that in the S/oil and VE groups, respectively, or between VE and S/oil groups (Fig. 10).

Fig. 10. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the acetylcholinesterase activity in Wistar rats. abcP<0.01versus S/oil and VE groups, respectively; cP<0.05 versus VE group.

#### **4. Discussion**

218 Insecticides – Basic and Other Applications

differ significantly (P>0.05) when compared to that in the S/oil or VE group, and between

**S/oil VE CPF VE+CPF**

**S/oil VE CPF VE+CPF**

Fig. 9. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the catalase activity in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups, respectively

Fig. 8. Effect of chronic administration of soya oil, vitamin E and/or chlorpyrifos on the superoxide dismutase activity in Wistar rats. abcP<0.01versus S/oil, VE and VE+CPF groups,

 **abc**

**abc**

VE and that recorded in the S/oil group (Fig. 9).

**0**

respectively.

**0**

**10**

**20**

**30**

**Catalase activity (mMol/mg protein)**

**40**

**50**

**60**

**70**

**80**

**0.5**

**1**

**1.5**

**Superoxide dismuatse activity (mMol/mg protein)**

**2**

**2.5**

**3**

The increase in brain MDA concentration and low SOD and CAT activities in the CPF group is an indication of the ability of this pesticide to elevate lipoperoxidative changes and thereby induce oxidative stress. This was in agreement with the findings from our previous studies (Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). The brain due to its biochemical and physiological properties is especially sensitive to free radicals, which destroy its functions and structure (Drewa et al., 1998). The brain is highly vulnerable to oxidative stress because in addition to harboring large amount of oxygen in a relatively small mass, it contains a significant quantity of metals (Fe), and has fewer antioxidant molecules than other organs (Halliwell and Gutteridge, 1999; Naffa-Mazzacoratt et al., 2001). For instance, the CNS is relatively poorly endowed with SOD, CAT, and glutathione peroxidase, and is also relatively lacking in vitamin E (Halliwell & Gutteridge, 1985). CPF is lipophilic and may enhance lipid peroxidation by directly interacting with cellular plasma membrane (Hazarika et al., 2003). The increased MDA concentration which is due to induction of free radical has been shown to alter the composition of membrane lipids, proteins, carbohydrates and DNA. Membrane lipids are vital for the maintenance of cellular integrity and survival (Jain, 1989). Peroxidation of membrane lipids results in the

Ameliorative Effect of Vitamin E on Sensorimotor and

improves neuronal transmission.

increases the detoxification of OP compounds (Shih et al., 1998).

involved in motor coordination deficits induced by chronic CPF exposure.

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 221

and nicotinic cholinergic receptors. The ability of vitamin E to remedy the CPF-induced cholinergic signs may be attributed its AChE restoration activity. Furthermore, vitamin E has been shown to increase the activity of paraoxonase 1 (Jarvik et al., 2002), an enzyme that

Beam walking across bridges of different cross-sections provides a well-established method of monitoring motor coordination and balance in rodents. The progressive increase in the width at which rats in the CPF group slipped off the beam which indicates impairment of motor coordination has been reported in previous studies (Ambali et al., 2010a; Ambali & Aliyu, 2012). Abou-Donia et al. (2002) observed similar results following repeated exposure of rats to sarin. Beam-walking performance is an integrated form of behavior requiring pertinent level of consciousness, memory, sensorimotor and cortical functions mediated by the cortical area (Abou-Donia et al., 2001). Cortical injury may therefore have been responsible for the deficit in beam-walk performance in the CPF group (Abou-Donia et al., 2001) partly due to oxidative damage. Indeed, CPF and CPF-oxon have been shown to induce apoptosis in rat cortical neuron independent of AChE inhibition (Caughlan et al., 2004). Pretreatment with vitamin E mitigated but did not completely abolish the motor coordination deficits induced by chronic CPF exposure. This is because there was a significant increase in the width at which the VE+CPF group slipped off the beam at week 16 compared to day 0. This shows that oxidative stress may not be the only mechanism

The present study has also shown a significant reduction in forepaw grip time, reflecting deficit in forepaw motor strength following chronic CPF exposure in rats. The result agreed with the finding obtained in an earlier study which showed reduction in hind limb grip strength following repeated CPF administration in rats (Terry et al., 2003). The impairment of motor strength by CPF may have also been due to the decrease in anterograde axonal transport (Terry et al., 2007) or reduced neuronal viability associated with impaired microtubule synthesis and/or function (Prendergast et al., 2007). It has also been postulated that disruption of kinesin-dependent intracellular transport may account for some of the long-term effects of OPs on the peripheral and central nervous system (Gearharta et al., 2007). Reduced hand strength (Miranda et al., 2004) and loss of muscle strength (Steenland et al*.*, 2000) have been observed in humans following prolonged exposure to OPs. Relationship has also been established between higher OP exposure and the development of chronic fatigue syndrome (Tahmaz et al., 2003). Furthermore, the role of muscle (Ambali and Ayo, 2011b) and brain oxidative damage induced by CPF which causes impairment of neuronal viability (Ambali & Ayo, 2011a) hence reduction of motor strength cannot be over emphasized. Although there was a significant deficit in motor strength in the VE+CPF group at weeks 16 and 8 when respectively compared to day 0, the fact that there was no significant change especially at week 16 compared to S/oil and VE groups reflect improvement in motor strength in this group. This may be partly due to reduced brain and perhaps muscle oxidative damage complemented by improvement in AChE activity which

Chronic CPF exposure has been shown in the present study to interfere with neuromuscular coordination as shown by the decline in the incline plane performance at weeks 8 and 16. The inclined plane test has been used to evaluate integrated muscle function and strength in rodents by evaluating their ability to maintain body position on a board as its angle of inclination is increased. We have earlier demonstrated the ability of acute CPF exposure to impair short-term neuromuscular coordination (Ambali et al., 2010a; Ambali & Aliyu, 2012).

inactivation of enzymes and cross-linking of membrane lipids and proteins and in cell death (Pfafferott et al., 1982; Jain et al., 1983; Jain, 1984). Furthermore, by-products of lipid peroxidation have been shown to cause profound alterations in the structural organization and functions of the cell membrane including decreased membrane fluidity, increased membrane permeability, inactivation of membrane-bound enzymes and loss of essential fatty acids (Van Ginkel & Sevanian, 1994). This lipoperoxidative changes may cause alterations in the structural and functional components of the brain neuronal cells.

The decrease in the SOD and CAT activities in the CPF group has been reported in previous studies (Tuzmen et al., 2007, 2008; Aly et al., 2010; Ambali & Ayo, 2011a) and may reflect the level of oxidative damage caused by the pesticide. SOD is involved in dismutation of the O2•− to H2O2 and oxygen. The significant reduction recorded in the CPF group may be due to either reduction in its synthesis or elevated degradation or inactivation of the enzyme. CAT, on the other hand is known to neutralize H2O2 and covert it to H2O and O2. The significant decline in the CAT activity observed in group exposed to CPF only may be due to the reduced conversion of O2 •− to H2O2 by SOD thereby resulting in the accumulation of O2•−. This accumulated O2•− inhibits the activity of CAT (Kono & Fridovich, 1982). The decline in the activity of the antioxidant enzymes following chronic CPF exposure in the present study may be due to downregulation in the synthesis of antioxidant enzymes due to persistent toxicant insult (Irshad & Chaudhuri, 2002). Furthermore, O2 •− converts ferroxy state of CAT to ferryl state, which is an inactive form of the enzyme (Freeman & Crapo, 1982), thereby exacerbating the free radical-induced damage to the body tissue.

Pretreatment with vitamin E was shown by the present study to reduce the brain MDA concentration and increase the activities of the antioxidant enzymes, SOD and CAT reflecting its antioxidant properties. α-tocopherol prevents the peroxidation of membrane phospholipids and prevent cell membrane damage through its antioxidant action. The lipophilic character of tocopherol makes it easier to locate the interior of the cell membrane bilayer to exert its antioxidant action. Tocopherol-OH transfers a hydrogen atom with a single electron to a free radical, thus removing the radical before it can interact with the cell membrane (Krishnamoorty et al., 2007). The decreased lipoperoxidation of the membrane due to free radical scavenging effect of vitamin E may have been responsible for the restoration of SOD and CAT activities, since the vitamin may have prevent their full participation in free radical neutralization, hence preserving their activities.

The result also revealed that chronic CPF exposure caused reduction in the brain AChE activity similar to what has been reported in previous studies (Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). The ability of CPF to phosphorylate AChE results in impairment of its activity, hence the cholinergic crisis. Apart from this, the induction of lipoperoxidation may have partly contributed to the impaired AChE activity recorded in the CPF group. Oxidative stress affects the activities of various membranebound enzymes, including AChE (Mehta et al., 2005) via their direct attack by free radicals or peroxidation of the membrane lipids in which they are embedded (Souza et al., 2010). Besides, OH has been shown to cause significant reduction in AChE activity in the rat brain (Tsakiris et al., 2000). Vitamin E was shown in the present study to restore the activity of AChE probably due to its antioxidant activity. Vitamin E has been shown in previous studies to restore AChE activity impaired by CPF (Yavuz et al., 2004; Ambali & Aliyu, 2012). The lacrimation and intermittent tremors observed in the CPF group is part of the cholinergic syndrome typical of OP insecticides (Eaton et al., 2008*)*. These cholinergic signs were due to inhibition of AChE by CPF, resulting in accumulation of ACh in the muscarinic

inactivation of enzymes and cross-linking of membrane lipids and proteins and in cell death (Pfafferott et al., 1982; Jain et al., 1983; Jain, 1984). Furthermore, by-products of lipid peroxidation have been shown to cause profound alterations in the structural organization and functions of the cell membrane including decreased membrane fluidity, increased membrane permeability, inactivation of membrane-bound enzymes and loss of essential fatty acids (Van Ginkel & Sevanian, 1994). This lipoperoxidative changes may cause

The decrease in the SOD and CAT activities in the CPF group has been reported in previous studies (Tuzmen et al., 2007, 2008; Aly et al., 2010; Ambali & Ayo, 2011a) and may reflect the level of oxidative damage caused by the pesticide. SOD is involved in dismutation of the O2•− to H2O2 and oxygen. The significant reduction recorded in the CPF group may be due to either reduction in its synthesis or elevated degradation or inactivation of the enzyme. CAT, on the other hand is known to neutralize H2O2 and covert it to H2O and O2. The significant decline in the CAT activity observed in group exposed to CPF only may be due

decline in the activity of the antioxidant enzymes following chronic CPF exposure in the present study may be due to downregulation in the synthesis of antioxidant enzymes due to persistent toxicant insult (Irshad & Chaudhuri, 2002). Furthermore, O2•− converts ferroxy state of CAT to ferryl state, which is an inactive form of the enzyme (Freeman & Crapo,

Pretreatment with vitamin E was shown by the present study to reduce the brain MDA concentration and increase the activities of the antioxidant enzymes, SOD and CAT reflecting its antioxidant properties. α-tocopherol prevents the peroxidation of membrane phospholipids and prevent cell membrane damage through its antioxidant action. The lipophilic character of tocopherol makes it easier to locate the interior of the cell membrane bilayer to exert its antioxidant action. Tocopherol-OH transfers a hydrogen atom with a single electron to a free radical, thus removing the radical before it can interact with the cell membrane (Krishnamoorty et al., 2007). The decreased lipoperoxidation of the membrane due to free radical scavenging effect of vitamin E may have been responsible for the restoration of SOD and CAT activities, since the vitamin may have prevent their full

The result also revealed that chronic CPF exposure caused reduction in the brain AChE activity similar to what has been reported in previous studies (Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). The ability of CPF to phosphorylate AChE results in impairment of its activity, hence the cholinergic crisis. Apart from this, the induction of lipoperoxidation may have partly contributed to the impaired AChE activity recorded in the CPF group. Oxidative stress affects the activities of various membranebound enzymes, including AChE (Mehta et al., 2005) via their direct attack by free radicals or peroxidation of the membrane lipids in which they are embedded (Souza et al., 2010). Besides, OH has been shown to cause significant reduction in AChE activity in the rat brain (Tsakiris et al., 2000). Vitamin E was shown in the present study to restore the activity of AChE probably due to its antioxidant activity. Vitamin E has been shown in previous studies to restore AChE activity impaired by CPF (Yavuz et al., 2004; Ambali & Aliyu, 2012). The lacrimation and intermittent tremors observed in the CPF group is part of the cholinergic syndrome typical of OP insecticides (Eaton et al., 2008*)*. These cholinergic signs were due to inhibition of AChE by CPF, resulting in accumulation of ACh in the muscarinic

1982), thereby exacerbating the free radical-induced damage to the body tissue.

participation in free radical neutralization, hence preserving their activities.

•− to H2O2 by SOD thereby resulting in the accumulation of

•− inhibits the activity of CAT (Kono & Fridovich, 1982). The

alterations in the structural and functional components of the brain neuronal cells.

to the reduced conversion of O2

O2•−. This accumulated O2

and nicotinic cholinergic receptors. The ability of vitamin E to remedy the CPF-induced cholinergic signs may be attributed its AChE restoration activity. Furthermore, vitamin E has been shown to increase the activity of paraoxonase 1 (Jarvik et al., 2002), an enzyme that increases the detoxification of OP compounds (Shih et al., 1998).

Beam walking across bridges of different cross-sections provides a well-established method of monitoring motor coordination and balance in rodents. The progressive increase in the width at which rats in the CPF group slipped off the beam which indicates impairment of motor coordination has been reported in previous studies (Ambali et al., 2010a; Ambali & Aliyu, 2012). Abou-Donia et al. (2002) observed similar results following repeated exposure of rats to sarin. Beam-walking performance is an integrated form of behavior requiring pertinent level of consciousness, memory, sensorimotor and cortical functions mediated by the cortical area (Abou-Donia et al., 2001). Cortical injury may therefore have been responsible for the deficit in beam-walk performance in the CPF group (Abou-Donia et al., 2001) partly due to oxidative damage. Indeed, CPF and CPF-oxon have been shown to induce apoptosis in rat cortical neuron independent of AChE inhibition (Caughlan et al., 2004). Pretreatment with vitamin E mitigated but did not completely abolish the motor coordination deficits induced by chronic CPF exposure. This is because there was a significant increase in the width at which the VE+CPF group slipped off the beam at week 16 compared to day 0. This shows that oxidative stress may not be the only mechanism involved in motor coordination deficits induced by chronic CPF exposure.

The present study has also shown a significant reduction in forepaw grip time, reflecting deficit in forepaw motor strength following chronic CPF exposure in rats. The result agreed with the finding obtained in an earlier study which showed reduction in hind limb grip strength following repeated CPF administration in rats (Terry et al., 2003). The impairment of motor strength by CPF may have also been due to the decrease in anterograde axonal transport (Terry et al., 2007) or reduced neuronal viability associated with impaired microtubule synthesis and/or function (Prendergast et al., 2007). It has also been postulated that disruption of kinesin-dependent intracellular transport may account for some of the long-term effects of OPs on the peripheral and central nervous system (Gearharta et al., 2007). Reduced hand strength (Miranda et al., 2004) and loss of muscle strength (Steenland et al*.*, 2000) have been observed in humans following prolonged exposure to OPs. Relationship has also been established between higher OP exposure and the development of chronic fatigue syndrome (Tahmaz et al., 2003). Furthermore, the role of muscle (Ambali and Ayo, 2011b) and brain oxidative damage induced by CPF which causes impairment of neuronal viability (Ambali & Ayo, 2011a) hence reduction of motor strength cannot be over emphasized. Although there was a significant deficit in motor strength in the VE+CPF group at weeks 16 and 8 when respectively compared to day 0, the fact that there was no significant change especially at week 16 compared to S/oil and VE groups reflect improvement in motor strength in this group. This may be partly due to reduced brain and perhaps muscle oxidative damage complemented by improvement in AChE activity which improves neuronal transmission.

Chronic CPF exposure has been shown in the present study to interfere with neuromuscular coordination as shown by the decline in the incline plane performance at weeks 8 and 16. The inclined plane test has been used to evaluate integrated muscle function and strength in rodents by evaluating their ability to maintain body position on a board as its angle of inclination is increased. We have earlier demonstrated the ability of acute CPF exposure to impair short-term neuromuscular coordination (Ambali et al., 2010a; Ambali & Aliyu, 2012).

Ameliorative Effect of Vitamin E on Sensorimotor and

neurotransmission and other brain activities.

1991).

present study.

**5. Conclusion** 

**6. References** 

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 223

et al., 2010a; Ambali & Aliyu, 2012). In addition, studies in humans have shown persistent cognitive deficits in farmers and pesticide applicators repeatedly exposed to OPs but are symptom-free (Steenland et al*.*, 2000; Dick et al., 2001). The impairment of cognition observed in the CPF group may be due to alteration in ACh metabolism due to reduction of AChE activity. Since ACh has been demonstrated to be involved in cognition, agents such as OPs which alter ACh metabolism may interfere with this role. Many studies have linked central cholinergic system to synaptic plasticity, learning and memory processes (Baskerville et al., 1997; Sachdev et al., 1998). It is believed that OP compounds play a role in memory loss by producing cholinergic dysfunction at the level of the synapse (Carr & Chambers,

Furthermore, CPF has been shown to induce cytotoxicity directly on the hippocampal cells via the induction of apoptosis, irrespective of its effect on AChE (Terry et al., 2003). Induction of apoptosis has been described as the toxic end-point of CPF neurotoxicity in the brain as it induces structural changes in the brain that may cause functional deficits, including those involved in memory and learning (Caughlan et al., 2004). Apoptosis probably resulting from oxidative damage to cellular macromolecules may have been responsible for the massive degenerative changes in the brain neurons and glial cells of rats chronically exposed to CPF that we reported in an earlier study (Ambali & Ayo, 2011a). CPF-induced oxidative stress may be central to apoptosis, since free radicals have been implicated in apoptotic death of cells (Corcoran et al., 1994; McConkey et al., 1994). Degenerative changes in the neurons leads to functional deficits as it relates to

Vitamin E has been shown in the present study to improve learning and short-term memory impaired by chronic CPF exposure. We have earlier demonstrated the ability of either vitamin C or E to mitigate short-term cognitive changes induced by acute CPF exposure in rats (Ambali et al., 2010a; Ambali and Aliyu, 2012). The improved learning and short-term memory recorded following pretreatment with vitamin E may be due to its antioxidant and AChE restoration properties. Apart from its antioxidant function, vitamin E influences the cellular response to oxidative stress through modulation of signal-transduction pathways (Azzi et al., 1992), which may have further enhanced the neuronal function. Similarly, neuroprotective effect of vitamin E has been established in several studies (Frantseva et al., 2000a, 2000b; Pace et al., 2003; El-Hossary et al., 2009) and may have contributed in mitigating the behavioural changes induced by CPF in the

The present study has shown that the impaired sensorimotor and cognitive changes induced by chronic CPF exposure mitigated by pretreatment with vitamin E are partly due to its

Abou-Donia, M.B. (1992). Introduction. *In*: *Neurotoxicology,* M.B. Abou-Donia (Ed.), 3-24

antioxidant, neuroprotective and AChE restoration properties.

CRC Press, Boca Raton, FL.

Abou-Donia et al. (2002) similarly showed the ability of the OP warfare agent, sarin to impair incline plane performance in rats. The impairment of neuromuscular coordination may be due to increase in brain oxidative changes induced by CPF, which alters the morphological and functional capacity of the brain region involved in neuromuscular coordination. Oxidative damage to the brain following CPF exposure has been reported in previous studies (Verma, 2001; Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). Furthermore, the reduction of AChE activity may have been partly involved in the impaired neuromuscular coordination recorded in the CPF group, since alterations in ACh metabolism may alter neuronal activity.

Although the incline plane performance in the group pretreated with vitamin E at week 16 was significantly lower than that obtained at day 0, the study generally showed that performance in weeks 16 and 8 in the VE+CPF group was not significantly different from that of S/oil or VE group. This shows that the vitamin mitigated the CPF-evoked deficit in neuromuscular coordination. The fact that vitamin E did not completely abolish the CPFinduced impaired incline plane performance shows that oxidative stress and restoration of AChE activity may not be the only factor responsible for the sensorimotor deficit.

The lower ladder score characterized by lower number of missed rungs observed in rats chronically exposed to CPF indicates that the legs of the rats were frequently being held stationary above the rungs for a relatively longer period. This observation demonstrated difficulty in the ability of CPF group to move fast through the obstacles, and hence a deficit in locomotor activity. The deficit in locomotor efficiency observed in the CPF group was dependent on the duration of exposure, with much more impairment recorded at week 16 compared to week 8. The results agreed with the previous findings that slowness of movement is one of the extrapyramidal symptoms (Parkinsonism) observed in humans exposed to non-specific agricultural pesticides, which increased with the duration of exposure (Ritz & Yu, 2000; Alavanja et al., 2004). Thus, the locomotion deficit in the CPF group observed in the present study is part of the sensorimotor deficits occurring in animals chronically exposed to CPF. This impaired mobility may be due to oxidative stress as oxidative damage to the muscle induced by CPF (Ambali & Ayo, 2011b) may have probably caused necrosis thereby impairing locomotion efficiency. Carr et al. (2001) attributed reduced mobility observed in OP poisoning partly to damage in the peripheral musculature, probably due to necrosis of skeletal muscle fibre. Muscle necrosis has been observed following exposure to the OP insecticide, isofenphos and the insecticide metabolite, paraoxon (Dettbarn, 1984; Calore et al., 1999). Similarly, the impaired mobility may be due to inhibition of AChE activity and the subsequent cholinergic paralysis induced by CPF. The severity of the muscle necrosis may be dependent on the level and duration of AChE inhibition (Carr et al., 2001). The amelioration of the locomotor deficits manifested in the improvement of ladder walk and characterized by increase in the number of missed rungs in rats pretreated with vitamin E demonstrated the important role played by oxidative stress and AChE inhibition in the locomotor deficit induced by CPF.

The significant increase in the number of footshocks received by the CPF group relative to the other groups indicates learning impairment. Similarly, the significant reduction in the duration the animal in the CPF group stayed on the platform indicates deficit in memory. This shows that CPF exposure even at low dose is capable of cognitive impairment. CPFinduced cognitive impairment have been reported in several studies in rats (Bushnell et al. 1991; 1994; Prendergast et al., 1997, 1998, 2007; Stone et al., 2000, Moser et al., 2005; Ambali et al., 2010a; Ambali & Aliyu, 2012). In addition, studies in humans have shown persistent cognitive deficits in farmers and pesticide applicators repeatedly exposed to OPs but are symptom-free (Steenland et al*.*, 2000; Dick et al., 2001). The impairment of cognition observed in the CPF group may be due to alteration in ACh metabolism due to reduction of AChE activity. Since ACh has been demonstrated to be involved in cognition, agents such as OPs which alter ACh metabolism may interfere with this role. Many studies have linked central cholinergic system to synaptic plasticity, learning and memory processes (Baskerville et al., 1997; Sachdev et al., 1998). It is believed that OP compounds play a role in memory loss by producing cholinergic dysfunction at the level of the synapse (Carr & Chambers, 1991).

Furthermore, CPF has been shown to induce cytotoxicity directly on the hippocampal cells via the induction of apoptosis, irrespective of its effect on AChE (Terry et al., 2003). Induction of apoptosis has been described as the toxic end-point of CPF neurotoxicity in the brain as it induces structural changes in the brain that may cause functional deficits, including those involved in memory and learning (Caughlan et al., 2004). Apoptosis probably resulting from oxidative damage to cellular macromolecules may have been responsible for the massive degenerative changes in the brain neurons and glial cells of rats chronically exposed to CPF that we reported in an earlier study (Ambali & Ayo, 2011a). CPF-induced oxidative stress may be central to apoptosis, since free radicals have been implicated in apoptotic death of cells (Corcoran et al., 1994; McConkey et al., 1994). Degenerative changes in the neurons leads to functional deficits as it relates to neurotransmission and other brain activities.

Vitamin E has been shown in the present study to improve learning and short-term memory impaired by chronic CPF exposure. We have earlier demonstrated the ability of either vitamin C or E to mitigate short-term cognitive changes induced by acute CPF exposure in rats (Ambali et al., 2010a; Ambali and Aliyu, 2012). The improved learning and short-term memory recorded following pretreatment with vitamin E may be due to its antioxidant and AChE restoration properties. Apart from its antioxidant function, vitamin E influences the cellular response to oxidative stress through modulation of signal-transduction pathways (Azzi et al., 1992), which may have further enhanced the neuronal function. Similarly, neuroprotective effect of vitamin E has been established in several studies (Frantseva et al., 2000a, 2000b; Pace et al., 2003; El-Hossary et al., 2009) and may have contributed in mitigating the behavioural changes induced by CPF in the present study.

#### **5. Conclusion**

222 Insecticides – Basic and Other Applications

Abou-Donia et al. (2002) similarly showed the ability of the OP warfare agent, sarin to impair incline plane performance in rats. The impairment of neuromuscular coordination may be due to increase in brain oxidative changes induced by CPF, which alters the morphological and functional capacity of the brain region involved in neuromuscular coordination. Oxidative damage to the brain following CPF exposure has been reported in previous studies (Verma, 2001; Ambali et al., 2010a; Ambali & Ayo, 2011a, 2011b; Ambali & Aliyu, 2012). Furthermore, the reduction of AChE activity may have been partly involved in the impaired neuromuscular coordination recorded in the CPF group, since alterations in

Although the incline plane performance in the group pretreated with vitamin E at week 16 was significantly lower than that obtained at day 0, the study generally showed that performance in weeks 16 and 8 in the VE+CPF group was not significantly different from that of S/oil or VE group. This shows that the vitamin mitigated the CPF-evoked deficit in neuromuscular coordination. The fact that vitamin E did not completely abolish the CPFinduced impaired incline plane performance shows that oxidative stress and restoration of

The lower ladder score characterized by lower number of missed rungs observed in rats chronically exposed to CPF indicates that the legs of the rats were frequently being held stationary above the rungs for a relatively longer period. This observation demonstrated difficulty in the ability of CPF group to move fast through the obstacles, and hence a deficit in locomotor activity. The deficit in locomotor efficiency observed in the CPF group was dependent on the duration of exposure, with much more impairment recorded at week 16 compared to week 8. The results agreed with the previous findings that slowness of movement is one of the extrapyramidal symptoms (Parkinsonism) observed in humans exposed to non-specific agricultural pesticides, which increased with the duration of exposure (Ritz & Yu, 2000; Alavanja et al., 2004). Thus, the locomotion deficit in the CPF group observed in the present study is part of the sensorimotor deficits occurring in animals chronically exposed to CPF. This impaired mobility may be due to oxidative stress as oxidative damage to the muscle induced by CPF (Ambali & Ayo, 2011b) may have probably caused necrosis thereby impairing locomotion efficiency. Carr et al. (2001) attributed reduced mobility observed in OP poisoning partly to damage in the peripheral musculature, probably due to necrosis of skeletal muscle fibre. Muscle necrosis has been observed following exposure to the OP insecticide, isofenphos and the insecticide metabolite, paraoxon (Dettbarn, 1984; Calore et al., 1999). Similarly, the impaired mobility may be due to inhibition of AChE activity and the subsequent cholinergic paralysis induced by CPF. The severity of the muscle necrosis may be dependent on the level and duration of AChE inhibition (Carr et al., 2001). The amelioration of the locomotor deficits manifested in the improvement of ladder walk and characterized by increase in the number of missed rungs in rats pretreated with vitamin E demonstrated the important role played by oxidative stress and AChE inhibition in the

The significant increase in the number of footshocks received by the CPF group relative to the other groups indicates learning impairment. Similarly, the significant reduction in the duration the animal in the CPF group stayed on the platform indicates deficit in memory. This shows that CPF exposure even at low dose is capable of cognitive impairment. CPFinduced cognitive impairment have been reported in several studies in rats (Bushnell et al. 1991; 1994; Prendergast et al., 1997, 1998, 2007; Stone et al., 2000, Moser et al., 2005; Ambali

AChE activity may not be the only factor responsible for the sensorimotor deficit.

ACh metabolism may alter neuronal activity.

locomotor deficit induced by CPF.

The present study has shown that the impaired sensorimotor and cognitive changes induced by chronic CPF exposure mitigated by pretreatment with vitamin E are partly due to its antioxidant, neuroprotective and AChE restoration properties.

#### **6. References**

Abou-Donia, M.B. (1992). Introduction. *In*: *Neurotoxicology,* M.B. Abou-Donia (Ed.), 3-24 CRC Press, Boca Raton, FL.

Ameliorative Effect of Vitamin E on Sensorimotor and

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 225

Bazylewicz-Walczak, B.; Majczakowa, W. & Szymczak, M. (1992). Behavioural effects of

Bushnell, P. J.; Padilla, S. S.; Ward, T.; Pope, C. N. & Olszyk, V. B. (1991). Behavioural and

*Journal of Pharmacology and Experimental Therapeutics,* Vol. 256, pp. 741-750. Bushnell, P.J.; Kelly, K.C. & Ward, T.R. (1994). Repeated inhibition of cholinesterase by

Caňadas, F.; Cardona, D.; Dávila, E.; Sánchez-Santed, F. (2005). Long-term neurotoxicity of

Carlock, L.L.; Chen, W.L.; Gordon, E.B.; Killeen, J. C.; Manley, A.; Meyer, L.S.; Mullin, L.S.;

relationships. *Pharmacology Biochemistry and Behaviour,* Vol. 40, pp. 929-936. Carr, R.L.; Chambers, H.W.; Guansco, J.A.; Richardson, J.R.; Tang, J. & Chambers, J.E. (2001).

Casida, J.E. and Quistad, G.B. (2004). Organophosphate toxicology: Safety aspects of non-

Chakraborti, T.K.; Farrar, J.D. & Pope, C.N. (1993). Comparative neurochemical and

Clegg, D. J. & van Gemert, M. (1999). Expert panel report of human studies on chlorpyrifos

Colborn, T. (2006). A case for revisiting the safety of pesticides: A closer look at neurodevelopment. *Environmental Health Perspectives* Vol. 114, pp. 10–17. Corcoran, G.B.; Fix, L.; Jones, D.P.; Moslen, M.T.; Nicotera, P.; Oberhammer, F.A. & Buttyan,

Costa, L.G.; Giordano, G.; Guizzetti M. & Vitalone A. (2008). Neurotoxicity of pesticides: a

MAP kinases. *Toxicological Sciences,* Vol. 78, pp. 125-134.

*Pharmacology Biochemistry and Behaviour,* Vol. 46, pp. 219-224.

planting workers. *Neurotoxicology,* Vol. 20, pp. 819-826.

*Ecotoxicology and Environmental Safety*,Vol. 43, pp. 187-194.

*Toxicological Sciences,* Vol. 85, pp.944-951.

260-267.

*Health B* 2, pp. 257–279.

*Pharmacology,* Vol. 128, pp. 169-181.

brief review. Frontiers in Bioscience, 13:1240–1249.

occupational exposure to organophosphorous pesticides in female greenhouse

neurochemical changes in rats dosed repeatedly with diisopropylfluorophos-phate.

chlorpyrifos in rats: behavioural, neurochemical and pharmacological indices of tolerance. *Journal of Pharmacology and Experimental Therapeutics,* Vol. 270, pp. 15-25. Calore, E.E.; Sesso, A.; Puga, F.R.; Cavaliere, M.J.; Calore, N.M. & Weg, R. (1999). Early

expression of ubiquitin in myofibres of rats in organophosphate intoxication.

chlorpyrifos: spatial learning impairment on repeated acquisition in a water maze.

Pendino, K.J.; Percy, A.; Sargent, D.E.; & Seaman, L.R. (1999). Regulating and assessing risks of cholinesterase-inhibiting pesticides: Divergent approaches and interpretations. *Journal of Toxicology and Environmental. Health B* 2, pp. 105–160. Carr, R.L. & Chambers, J.E. (1991). Acute effects of the organophosphate paraoxon on

schedule-controlled behaviour and esterase activity in rats: Dose-response

Effect of repeated open-field behaviour in juvenile rats. *Toxicological Sciences,* 59:

acetylcholinesterase secondary targets*. Chemical Research in Toxicology,* 17: 983-898. Caughlan, A.; Newhouse, K.; Namgung, U. & Xia, Z. (2004). Chlorpyrifos induces apoptosis

in rat cortical neurons that is regulated by a balance between p38 and ERK/JNK

neurobehavioural effects of repeated chlorpyrifos exposures in young rats.

and/or other organophosphate exposures. *Journal of Toxicology and Environmental* 

R. (1994). Apoptosis: Molecular control point in toxicity. *Toxicology and Applied* 


Abou-Donia, M.B.; Dechkovskaia, A.M; Goldstein, L.B.; Bullman S.L. & Khan, W.A. (2002).

Abou-Donia, M.B.; Goldstein, L.B.; Jones, K.H.; Abdel-Rahaman, A.A.; Damodaran, T.;

Alavanja, M.C.; Hoppin, J.A.; & Kamel, F. (2004). Health effects of chronic pesticide

Aly, N.; EL-Gendy, K.; Mahmoud F.; & El-Sebae, A.K. (2010). Protective effect of vitamin C

Ambali, S.F. (2009). Ameliorative effect of vitamin C and E on neurotoxicological,

Ambali, S.F. & Aliyu, M.B. (2012). Short-term sensorimotor and cognitive changes induce by

Ambali, S.F.& Ayo, J.O. (2011a) Sensorimotor performance deficits induced by chronic

Ambali, S.F. & Ayo, J.O. (2011b). Vitamin C attenuates chronic chlorpyrifos-induced

Ambali, S.F.; Ayo, J.O.; Ojo, S.A. & Esievo, K.A.N. (2010b). Vitamin E protects rats from

Ambali, S.F.; Idris, S.B.; Onukak, C.; Shittu, M. & Ayo, J.O. (2010a). Ameliorative effects of

Azzi, A.; Boscobonik, D. & Hensey, C. (1992). The protein kinase C family. *European Journal* 

Bagchi, D.; Bagchi, M.; Hassoun, E.A. & Stohs, S.J. (1995). *In vitro* and *in vivo* generation of

Baskerville, K.A.; Schweitzer, J.B. & Herron, P. (1997). Effects of cholinergic depletion on

*Environmental Chemistry,* Vol. 93, No 6, pp. 1212–1226.

*and Chemical Toxicology,* Vol. 48, pp. 3477-3480.

selected pesticides. *Toxicology,* Vol. 104, pp. 129-140..

*of Biochemistry,* Vol. 208, pp. 547-557.

158.

197.

Vol. 60, pp. 305-314.

Nigeria, 355pp.

No 2, pp. 31-38.

pp. 547-558.

1159-1169.

(Accepted manuscript).

*Physiology,* Vol. 97, pp. 7–12.

Sensorimotor deficit and cholinergic changes following coexposure with pyridostigmine bromide and sarin in rats. *Toxicological Sc*i*ences,* Vol. 66, pp. 148–

Dechkovskaia, A.M.; Bullman, S.L.; Amir, B.E. & Khan, W.A. (2001). Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET and permethrin alone and in combination. *Toxicological Sciences,* 

exposure: cancer and neurotoxicity. *Annual Review of Public Health,* Vol. 25, pp. 155-

against chlorpyrifos oxidative stress in male mice. *Pesticide Biochemistry and* 

hematological and biochemical changes induced by chronic chlorpyrifos administration in Wistar rats. *PhD Dissertation*, Ahmadu Bello University, Zaria,

acute chlorpyrifos exposure: Ameliorative effect of vitamin E. *Pharmacologia,* Vol 3,

chlorpyrifos exposure in Wistar rats: mitigative effect of vitamin C. *Toxicological and* 

alteration of neurobehavioural parameters in Wistar rats. *Toxicology International*

chlorpyrifos-induced increased erythrocyte osmotic fragility in Wistar rats. *Food* 

vitamin C on short-term sensorimotor and cognitive changes induced by acute chlorpyrifos exposure in Wistar rats. *Toxicology and Industrial Health,* Vol. 26, No. 9,

reactive oxygen species, DNA damage and lactate dehydrogenase leakage by

experience dependent plasticity in the cortex of the rat. *Neuroscience* Vol. 80, pp.


Ameliorative Effect of Vitamin E on Sensorimotor and

*Archives of Toxicology,* Vol. 74, pp. 533- 538.

consequence? *Lancet,* Vol. 344, pp. 721-724.

study. *Toxicology,* Vol. 185, No. 1–2, pp. 1-8.

*Neurotoxicology,* Vol. 21, pp. 829–835.

University Press, London, England.

*Neuroscience,* Vol. 8, pp. 22-26.

*Toxicology*, Vol. 75, No. 2, pp. 88-96.

20892.

No. 1, pp. 47-62.

pp. 33 – 44.

pp. 21340-21345.

3394.

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 227

Guide for the care and use of laboratory animals, DHEW Publication No. (NIH) 85-23,

Gultekin F.; Ozturk, M. & Akdogan, M. (2000). The effect of organophosphate insecticide

Gultekin, F.; Delibas, N.; Yasar, S. & Kilinc, I. (2001). *In vivo* changes in antioxidant systems

Gultekin, F.; Karakoyun, I.; Sutcu, R.; Savik, E.; Cesur, G.; Orhan, H. & Delibas, N. (2007).

Halliwell, B. (1994). Free radicals, antioxidants and human disease: curiosity, cause or

Halliwell, B. & Gutteridge, J. C. (1999). *Free Radicals in Biology and Medicine*, 3rd ed., Oxford

Halliwell, B. & Gutteridge, J.M.C. (1985). Oxygen radicals and the nervous system. *Trends in* 

Hazarika, A.; Sarkar, S.N.; Hajare, S.; Kataria, M. & Malik, J.K. (2003). Influence of malathion

He, F. (2000). Neurotoxic effects of insecticides—Current and future research: A review.

Hill, R.; Head, S.; Baker, S.; Gregg, M.; Shealy, D.; Bailey, S.; Williams, C.; Sampson, E. &

reference range concentrations. *Environmental Research,* Vol. 71, pp. 88-108. Hsu, P. C. & Guo, Y. L. (2002): Antioxidant nutrients and lead toxicity. *Toxicology,* Vol. 180,

Irshad, M. & Chaudhuri, B.S. (2002). Oxidant-antioxidant system: role and significance in human body. *Indian Journal of Experimental Biology,* Vol. 40, pp. 1233–1239. Jain, S.K. (1989). Hyperglycaemia can cause membrane lipid peroxidation and osmotic

Jain, S.K. (1984). The accumulation of malonyldialdehyde, a product of fatty acid

Jain, S.K.; Mohandas, N.; Clark, M.R. & Shohet, S.B. (1983). The effect of malonyldialdehyde,

survival of erythrocytes. *British Journal of Haematology,* Vol. 53, pp. 247-255. Jarvik, G.P.; Tsai, T.N.; McKinstry, L.A.; Wani, R.; Brophy, V.; Richter, R.J.; Schellenberg,

pretreatment on the toxicity of anilofos in male rats: a biochemical interaction

Needham, L. (1995). Pesticide residues in urine of adults living in the United States:

fragility in human red blood cells. *Journal of Biological Chemistry,* Vol. 264, No. 35,

peroxidation, can disturb aminophospholipid organization in the membrane bilayer of human erythrocytes. *Journal of Biological Chemistry*, Vol. 259, pp. 3391-

a product of lipid peroxidation, on the deformability, dehydration and 51Cr-

G.D.; Heagerty, P.J.; Hatsukami, T. & Furlong, C.E. (2002). Vitamin C and E intake

Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD

chlorpyrifos–ethyl on lipid peroxidation and antioxidant enzymes (*in-vitro*).

and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats. *Archives of* 

Chlorpyrifos increases the levels of hippocampal NMDA receptor subunits NR2A and NR2B in juvenile and adult rats. *International Journal of Neuroscience,* Vol. 117,


Dettbarn, W.D. (1984). Pesticide-induced muscle necrosis: mechanisms and prevention.

Dick, R.B.; Steenland, K.; Krieg, E.F. & Hines, C.J. (2001). Evaluation of acute sensory-motor

Dietrich, K.N.; Eskenazi, B.; Schantz, S.; Yolton, K.; Rauh, V. A.; Johnson, C. B.; Alkon, A.;

Draper, H.H. & Hadley, M. (1990). Malondialdehyde determination as index of lipid

Drewa, G.; Jakbczyk, M. & Araszkiewicz, A. (1998). Role of free 1 radicals in schizophrenia.

Eaton, D.L.; Daroff, R.B.; Autrup, H.; Buffler, P.; Costa, L.G.; Coyle, J.; Mckhann, G.; Mobley,

El-Hossary, G.G.; Mansour, S.M. & Mohamed, A.S. (2009). Neurotoxic effects of

Farag, A.T.; Radwana, A.H.; Sorourb, F.; El Okazyc A.; El-Agamyd, E. & El-Sebae, A. (2010).

Fiedler, N.; Kipen, H.; Kelly-McNeil, K. & Fenske, R. (1997). Long-term use of

Frantseva, M.V.; Valazquez, J.L.; Hwang, P.A. & Carlen, P.L. (2000a). Free radicals

Frantseva, M.V.; Valazquez, J.L.; Tsoraklidis, G.; Mendonca, A.J.; Adamchik, Y.; Mills, L.R.;

Freeman, B.A. & Crapo, J.D. (1982). Biology of disease: Free radicals and tissue injury.

Freitas, R.M.; Vasconcelos, S.M.M.; de Souza, F.G.F.; Viana, G.S.B. & Fonteles, M.M.F. (2005).

Gearharta, D.A.; Sicklesb, D.W.; Buccafuscoa, J.J.; Prendergast, M.A. & Terry, Jr, A.V. (2007).

W.C.; Nadel, L.; Neubert, D.; Schukte-Hermann, R.; Peter, S. & Spencer, P.S. (2008). Review of the toxicology of chlorpyrifos with an emphasis on human exposure and

chlorpyrifos and the possible protective role of antioxidant supplements: an experimental study. *Journal of Applied Science Research,* Vol. 5, No. 9, pp. 1218-

Chlorpyrifos induced reproductive toxicity in male mice. *Reproductive Toxicology,*

organophosphates and neuropsychological performance. *American Journal of* 

production correlates with cell death in an *in vitro* model of epilepsy. *European*

Carlen, P.L. & Burnham, M.V. (2000b). Oxidative stress in involved in seizureinduced neurodegeneration in the kindling model of epilepsy. *Neuroscience,* Vol. 97,

Oxidative stress in the hippocampus after pilocarpine induced status epilepticus in

Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesindependent microtubule motility. *Toxicology and Applied Pharmacology,* Vol. 218,

effects and test sensitivity using termiticide workers exposed to chlorpyrifos.

Canfield, R.L.; Pessah, I.N. & Berman, R.F. (2005). Principles and practices of neurodevelopmental assessment in children: Lessons learned from the centers for children's environmental health and disease prevention research. *Environmental* 

*Fundamental and Applied Toxicology,* Vol. 4, pp. S18-S26.

peroxidation. *Methods in Enzymology,* Vol. 186, pp. 421-431.

2 *Medical Science Monitoring,* Vol. 4, No. 6, pp. 1111-1115..

neurodevelopment. *Critical Reviews in Toxicology* S2, pp. 1-125.

*Neurotoxicology and Teratology,* Vol. 23, pp. 381-393.

*Health Perspectives,* Vol. 113; pp. 1437–1446.

*Industrial Medicine,* Vol. 32, pp. 487–496.

*Journal of Neuroscience,* Vol. 12, pp. 1413-1419.

*Laboratory Investigations,* Vol. 47, pp. 412-426.

Wistar rats. *FEBS Journal,* Vol. 272, pp. 1307-1312.

1222.

Vol. 29, pp. 80–85.

pp. 431-435.

No.1, pp. 20-29.


Ameliorative Effect of Vitamin E on Sensorimotor and

*Environmental Medicine*, Vol. 61, No. 1, pp. e4.

*Epilepsy Research,* Vol. 46, pp. 121-128.

*of Clinical Oncology,* Vol. 21, pp. 927-931.

erythrocyte deformability. *Blood,* Vol. 59, pp. 12-15.

*Pharmacology, Biochemistry and Behaviour*, Vol. 42, pp. 251-256.

learning. *Psychopharmacology* (Berl), Vol. 130, pp. 276-284.

*Health Perspectives*, Vol. 11, pp. 536–544.

6-63.

630.

1, pp. 330-339.

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 229

Moser, V.C.; Phillips, P.M.; McDaniel, K.L.; Marshall, R.S.; Hunter, D.L. & Padilla, S. (2005).

Naffah-Mazzacoratti, M.G.; Cavalheiro, E.A.; Ferreira, E.C.; Abdalla, D.S.P.; Amado, D. &

Osfor, M.M.H.; Ibrahim, H.S.; Mohamed, Y.A.; Ahmed, S.M.; Abd El Azeem, A.S. & Hegazy,

Pace, A.; Savarese, A.; Picardo, M.; Maresca V.; Pacetti, U.; Del Monte, G.; Biroccio, A.;

Pancetti, F.; Olmos, C.; Dagnino-Subiabre, A.; Rozas, C. & Morales, B. (2007).

Pfafferott, C.; Meiselman, H.J. and Hochstein, P. (1982). The effect of malonyldialdehyde on

Pope, C.N. (1999). Organophosphorus pesticides: do they all have the same mechanism of toxicity? *Journal of Toxicology and Environmental Health*, Vol. 2, pp. 161-181. Pope, C.N.; Chakraborti, T.K.; Chapman, M.L. & Farrar J.D. (1992). Long-term

Prendergast, M.A.; Self, S.L.; Smith, K.J.; Ghayoumi, L.; Mullins, M.M.; Butler, T.R.;

Prendergast, M.A.; Terry, A.V. Jr. & Buccafusco, J.J. (1997). Chronic, low-level exposure to

Prendergast, M.A.; Terry, A.V. Jr. & Buccafusco, J.J. (1998). Effects of chronic low-level

monkeys and rats. *Neurotoxicology and Teratolology,* Vol. 20, pp. 115-122. Qiao, D.; Seidler, F.J.; Tate, C. A.; Cousins, M. M. & Slotkin, T. A. (2003). Fetal chlorpyrifos

to chlorpyrifos in rats. *Toxicological Sciences,* Vol. 86, pp. 375-386.

after acute poisoning with organophosphate insecticides. *Occupational and* 

Neurobehavioural effects of chronic dietary and repeated highlevel spike exposure

Bellissimo, M.I. (2001). Superoxide dismutase, glutathione peroxidase activities and the hydroperoxide concentration are modified in the hippocampus of epileptic rats.

A.M. (2010). Effect of Alpha Lipoic Acid and Vitamin E on Heavy Metals Intoxication in Male Albino Rats. *Journal of American Science,* Vol. 6, No. 8, pp.

Leonetti, C.; Jandolo, B.; Cognetti, F. & Bove, L. (2003). Neuroprotective effect of vitamin e supplementation in patients treated with cisplatin chemotherapy. *Journal* 

Noncholinesterase effects induced by organophosphate pesticides and their relationship to cognitive processes: implication for the action of acylpeptide hydrolase. *Journal of Toxicology and Environmental Health, Part B*, Vol. 10, pp. 623–

neurochemical and behavioural effects induced by acute chlorpyrifos treatment.

Buccafusco, J.J.; Gearhart, D.A. & Terry, A.V. Jr. (2007). Microtubule-associated targets in chlorpyrifos oxon hippocampal neurotoxicity. *Neuroscience,* Vol. 146, No.

diisopropyl fluorophosphates causes protracted impairment of spatial navigation

organophosphate exposure on delayed recall, discrimination and spatial learning in

exposure: Adverse effects on brain cell development and cholinergic biomarkers emerge postnatally and continue into adolescence and adulthood. *Environmental* 

is associated with increase paraoxonase activity. *Arterioscleriosis, Thrombosis and Vascular Biology,* Vol. 22, pp. 1329 -1333.


Kamel, F.; Engel, L.S.; Gladen, B.C.; Hoppin, J.A.; Alavanja, M.C.R. & Sandler, S.P. (2007).

Kamel, F. and Hoppin, J.A. (2004). Association of pesticide exposure with neurologic

Kamel, F.; Rowland, A.S.; Park, L.P.; Anger, W.K.; Baird, D.D.; Gladen, B.C.; Moreno, T.;

Kehrer, J.P. (1993). Free radicals as mediators of tissue injury and disease. *Critical Reviews in* 

Kingston, R.L.; Chen, W.L.; Borron, S.W.; Sioris, L.J.; Harris, C.R. & Engebretsen, K.M.

Kono, Y. & Fridovich I. (1982). Superoxide radical inhibits catalase. *Biological Chemistry*, Vol.

Krishnamoorthy, G.; Ventaraman, P.; Arunkumar, A.; Vignesh, R. C.; Aruldhas, M. M. &

Iwasaki, M.; Sato, I.; Jin, Y.; Saito, N. & Tsoda, S. (2007). Problems of positive list system

Lizardi, P.S.; O'Rourke, M.K. & Morris, R.J. (2008). The effects of organophosphate pesticide

Lotti M. (2000). *Experimental and Clinical Neurotoxicology*. 2nd Ed., Oxford University Press,

Lowry, H.; Rosebrough, N.J.; Farr, A.L. & Randall, R.J. (1951). Protein measurements with the folin phenol reagent. *Journal of Biological Chemistry*, Vol. 193, pp. 265–275. Maxwell, S.R. (1995): Prospects for the use of antioxidants therapies. *Drugs*, Vol. 49, pp.

McConkey, D.J., Jondal M.B. and Orrenius, S.G. (1994). Chemical-induced Apoptosis in the

Mehta, A.; Verma, R.S. & Vasthava S. (2005). Chlorpyrifos-induced alterations in rat brain

Miranda, J.; McConnell, R.; Wesseling, C.; Cuadra, R.; Delgado, E.; Torres, E.; Keifer, M. &

Immune System. In*: Immunotoxicology and Immunopharmacology,* (J.H., Dean, M.I., Luster, A.E., Munson & I. Kimber, (Eds.), 473-485, 2nd Edition, Raven Press Ltd.

acetylcholine esterase, lipid peroxidation and ATPase. *Indian Journal of Biochemistry* 

Lundberg, I. (2004). Muscular strength and vibration thresholds during two years

study. *Human and Experimental Toxicology,* Vol. 26, pp. 243-250.

*Vascular Biology,* Vol. 22, pp. 1329 -1333.

*Toxicology,* Vol. 23, No 1, pp. 21-48.

*and Human Toxicology,* Vol. 41, pp. 87-92.

*Reproductive Toxicology*, Vol. 23, pp. 239-245.

*Pediatric Psychology*, Vol. 33, No. 1, pp. 91–101.

958.

765-772.

257, pp. 5751-5754.

32, No. 2, pp. 179-184.

New York.

NewYork.

*and Biophysics*, Vol. 42, pp. 54-58.

345.

is associated with increase paraoxonase activity. *Arterioscleriosis, Thrombosis and* 

Neurologic symptoms in licensed pesticide applicators in the agricultural health

dysfunction and disease. *Environmental Health Perspectives,* Vol. 112, No. 9, pp. 950-

Stallone, L. & Sandler, D.P. (2003). Neurobehavioural performance and work experience in Florida farmworkers*. Environmental Health Perspectives,* Vol. 111, pp.

(1999). Chlorpyrifos: a ten-year U.S. poison center exposure experience. *Veterinary* 

Arunakaran, J. (2007). Ameliorative effect of vitamins (α–tocopherol and ascorbic acid) on PCB (Aroclor 1254)-induced oxidative stress in rat epididymal sperm.

revealed by survey of pesticide residue in food. *Journal of Toxicological Sciences*, Vol.

exposure on hispanic children's cognitive and behavioral functioning. *Journal of* 

after acute poisoning with organophosphate insecticides. *Occupational and Environmental Medicine*, Vol. 61, No. 1, pp. e4.


Ameliorative Effect of Vitamin E on Sensorimotor and

sheep dip. *Lancet,* Vol. 315, pp. 1135-1139.

rats. *Brain Research,* Vol. 882, pp. 9–18.

261-267.

1117-1128.

No. 535–540.

39, pp. 174-177.

Vol. 88 pp. 191–196.

124.

1036.

Cognitive Changes Induced by Chronic Chlorpyrifos Exposure in Wistar Rats 231

Stephens, R.; Spurgeon, A.; Calvert, I.A.; Beach, J.; Levy, L.S.; Berry, H. & Harrington, J.

Stone, J.D.; Terry, A.V. Jr.; Pauly, J.R.; Prendergast, M.A. & Buccafusco, J.J. (2000).

Tahmaz, N.; Soutar, A. & Cherrie, J.W. (2003). Chronic fatigue and organophosphate

Terry, A. V. Jr.; Stone, J. D.; Buccafusco, J.J.; Sicles, D. W.; Sood, A. & Prendergast, M.A.

*Journal of Pharmacology and Experimental Therapeutics,* Vol. 305, pp. 375-384. Terry, A.V. Jr; Gearhart, D.A.; Beck, W.D. Jr.; Truan, J.N.; Middlemore, M.; Williamson,

Tsakiris, S.; Angelogianni, P.; Schulpis, K.H. & Stavridis, J.C. (2000). Protective effect of L-

Tuzmen, N.; Candan, N. & Kaya, E. (2007). The evaluation of altered antioxidative defense

Tuzmen, N.; Candan, N.; Kaya, E. & Demiryas, N. (2008). Biochemical effects of chlorpyrifos

Van Ginkel, G. & Sevanian, A., (1994). Lipid peroxidation induced membrane structural

Verma, R.S. (2001). Chlorpyrifos-induced alterations in levels of thiobarbitunc acid reactive

Verma, R.S.; Mnugya, A. & Srivastava, N. (2007). *In vivo* chlorpyrifos induced oxidative

Weiss, B.; Amler, S. & Amler, R.W. (2004). Pesticides. *Pediatrics,* Vol. 113, pp. 1030-

Yavuz, T.; Delibao, N.; YÂldÂrÂm, B.; Altuntao, I.; CandÂr, O.; Cora, A.; Karahan, N.;

alterations. *Methods in Enzymology*, Vol. 233, pp. 273-288.

*Clinical Biochemistry,* Vol. 33, No. 2, pp. 103-106.

(1995). Neuropsychological effects of long-term exposure to organophosphates in

Protractive effects of chronic treatment with an acutely sub-toxic regimen of diisopropylflurophosphate on the expression of cholinergic receptor densities in

pesticides in sheep farming: A retrospective study amongst people reporting to a UK pharmacovigilance scheme. *Annals of Occupational Hygiene,* Vol. 47, No. 4, pp.

(2003). Repeated exposures to subthreshold doses of chlorpyrifos in rats: Hippocampal damage, impaired axonal transport, and deficits in spatial learning.

L.N.; Bartlett, M.G.; Prendergast, M.A.; Sickles, D.W. & Buccafusco, J.J. (2007). Chronic intermittent exposure to chlorpyrifos in rats: Protracted effects on axonal transport, neurotrophin receptors, cholinergic markers, and information processing. *Journal of Pharmacology and Experimental Therapeutics,* Vol. 322, pp.

phenylalanine on rat brain acetylcholinesterase inhibition induced by free radicals.

mechanism and acetylcholinesterase activity in rat brain exposed to chlorpyrifos, deltamethrin, and their combination. *Toxicology Mechanisms and Methods*, Vol. 17,

and deltamethrin on altered antioxidative defense mechanisms and lipid peroxidation in rat liver. *Cell Biochemistry and Function*, Vol. 26, pp. 119-

substances and glutathione in rat brain. *Indian Journal of Experimental Biology,* Vol.

stress: attenuation by antioxidant vitamins. *Pesticides Biochemistry and Physiology*,

Ãbrioim, E. & Kutsal, A. (2004). Vascular wall damage in rats induced by


Rack, K.D. (1993). Environmental fate of chlorpyrifos. *Review of Environmental Contamination* 

Ritz, B. & Yu, F. (2000). Parkinson's disease mortality and pesticide exposure in California

Sachdev, R.; Lu, S.; Wiley, R. & Ebner, F. (1998). Role of the basal forebrain cholinergic

Sally, A.M.; Sharee, A.W., & Janet, D. (2003). What advanced practice Nurses Need to know

Sánchez-Santed, F.; Canâdas, F.; Flores, P.; Lo´pez-Grancha, M. & Cardona, D. 2004. Long-

Saulsbury, M.D.; Heyliger, S.O.; Wang, K. & Johnson, D.J. (2009). Chlorpyrifos induces

Shih, D.M.; Gu, L.; Xia, Y.R.; Navab, M.; Li, W.F.; Hama, S.; Castellani, L.W.; Furlong, C.E.;

Sies, H. (1991). Oxidative stress: Introduction. *In* Sies, H. (Ed.), *Oxidative Stress: Oxidants and* 

Slotkin, T.A. (2004). Cholinergic systems in brain development and disruption by

Slotkin, T.A. (2005). Developmental neurotoxicity of organophosphates: a case study of

Slotkin, T.A.; Levin, E. D. & Seidler, F. J. (2006). Comparative developmental neurotoxicity

systemic toxicity. *Environmental Health Perspectives*, Vol. 114, pp. 746–751. Souza, G.F.; Saldanha G.B. & Freitas, R.M. (2010). Lipoic acid increases glutathione

Stamper, C.R.; Balduini, W.; Murphy, S.D. & Costa, L.G. (1988). Behavioral and biochemical

Steenland, K.; Dick, R.B.; Howell, R.J.; Chrislip, D.W.; Hines, C.J.; Reid, T.M.; Lehman, E.;

*Antioxidants,* Vol 23., 21-48, Academic Press, San Diego, CA, USA.

*Toxicology and Applied Pharmacology,* Vol. 198, pp. 132-151.

Ed), , 293-314, Elsevier Academic Press, San Diego.

projection in somatosensory cortical plasticity. *Journal of Neurophysiology,* Vol. 79,

about free radicals? *International Journal of Advanced Nursing Practice,* Vol. 6,

term functional neurotoxicity of paraoxon, and chlorpyrifos: Behavioural and pharmacological evidence. *Neurotoxicology and Teratology*, Vol. 26, pp. 305-

oxidative stress in oligodendrocyte progenitor cells. *Toxicology*, Vol. 259, pp.

Costa, L.G.; Fogelman, A.M. & Lusis, A.J. (1998). Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. *Nature*, Vol. 394,

neurotoxicants: nicotine, environmental tobacco smoke, organophosphates.

chlorpyrifos. In: *Toxicity of Organophosphate and Carbamate Pesticides,* (R.C. Gupta,

of organophosphate insecticides: Effects on brain development are separable from

peroxidase, Na+, K+-ATPase and acetylcholinesterase activities in rat hippocampus after pilocarpine-induced seizures? *Arquivos de Neuro Psiquiatria*, Vol, 68, pp. 586–

effects of postnatal parathion exposure in the rat. *Neurotoxicology and Teratology*,

Laber, P.; Krieg, E.F. & Knott, C. (2000). Neurologic function among termiticide applicators exposed to chlorpyrifos. *Environmental Health Perspectives,* Vol. 108, No.

1984-1994. *International Journal of Epidemiology,* Vol. 29, pp. 323-329.

*and Toxicology*, Vol. 131, pp. 1–150.

pp. 3216-3228.

pp. 1.

317.

1–9.

591.

Vol. 10, pp. 261-266.

4, pp. 293-300.

No. 6690, 284-287.


**12** 

*USA* 

**Non-Chemical Disinfestation of Food** 

The presence of microbial and insect/mite pests in foods and agricultural commodities, particularly in fresh produce, dried foods, nuts, grains, seeds, nursery plants, ornamental flowers and in wood products (i.e. pallets), continues to be a major factor affecting their condition for safe distribution and use in local, regional and international markets. As a mean to reduce the potential of propagating non-indigenous pests, postharvest (mandatory) treatment modalities and quarantine barriers have been imposed to regulate transportation and distribution of many of these products worldwide. These regulations define strategies for the detection, control, or eradication techniques for controlling quarantine insect and

Today, more than 6,500 nonnative species are already established in the United Sates and approximately 15% of these species are either economically or environmentally harmful (Pimentel, Lach, Zuñiga et al., 1999). Control or eradication practices for arthropod pests are mostly based on chemical pesticides, although host removal, adequate agricultural production practices, biological control agents, and sterile insect release are often techniques

Among the most important quarantine plant pests, various exotic fruit flies have been identified in the USA as threats to more than 250 crops. On the other hand, the presence of moths in stored products represents important and unacceptable risks to many growing and expanding agricultural regions worldwide. If detected, affected commodities must be processed with effective control or eradication techniques. If unattended, losses in product's

Chemical pesticides, waxes, coatings, thermal treatments (heated air; hot water immersion), modified atmospheres, cold storage (refrigeration), and irradiation are some of the processes that have helped industry meet current challenges and demands. Lately, however, new consumer preferences, trends and regulatory interventions have increased the needs for minimally processed foods with low or no residual chemicals. This new trend requires that less invasive or chemical-free alternatives become available to replace or minimize the use of pesticides. Furthermore, recent concerns associated with potential terrorist threats using microbial contaminants or other pests, have increased the need to develop alternatives to

applied in place off or in conjunction with pesticides.

quality represent unacceptable economic losses.

**1. Introduction** 

mite pests.

**and Agricultural Commodities** 

**with Radiofrequency Power** 

Manuel C. Lagunas-Solar *University of California, Davis* 

*RF Biocidics Inc., Vacaville, California* 

methidathion and ameliorating effect of vitamins E and C. *Archives of Toxicology,* Vol. 78, pp. 655-659.

Zhu, H.; Robin, W.; Rockhold, R.W.; Baker, R.C.; Kramer, R.E. & Ho, I.K. (2001). Effects of single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats. *Journal of Biomedical Sciences,* Vol. 8, pp. 467- 474.

### **Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power**

Manuel C. Lagunas-Solar *University of California, Davis RF Biocidics Inc., Vacaville, California USA* 

#### **1. Introduction**

232 Insecticides – Basic and Other Applications

Zhu, H.; Robin, W.; Rockhold, R.W.; Baker, R.C.; Kramer, R.E. & Ho, I.K. (2001). Effects of

Vol. 78, pp. 655-659.

474.

methidathion and ameliorating effect of vitamins E and C. *Archives of Toxicology,*

single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats. *Journal of Biomedical Sciences,* Vol. 8, pp. 467-

> The presence of microbial and insect/mite pests in foods and agricultural commodities, particularly in fresh produce, dried foods, nuts, grains, seeds, nursery plants, ornamental flowers and in wood products (i.e. pallets), continues to be a major factor affecting their condition for safe distribution and use in local, regional and international markets. As a mean to reduce the potential of propagating non-indigenous pests, postharvest (mandatory) treatment modalities and quarantine barriers have been imposed to regulate transportation and distribution of many of these products worldwide. These regulations define strategies for the detection, control, or eradication techniques for controlling quarantine insect and mite pests.

> Today, more than 6,500 nonnative species are already established in the United Sates and approximately 15% of these species are either economically or environmentally harmful (Pimentel, Lach, Zuñiga et al., 1999). Control or eradication practices for arthropod pests are mostly based on chemical pesticides, although host removal, adequate agricultural production practices, biological control agents, and sterile insect release are often techniques applied in place off or in conjunction with pesticides.

> Among the most important quarantine plant pests, various exotic fruit flies have been identified in the USA as threats to more than 250 crops. On the other hand, the presence of moths in stored products represents important and unacceptable risks to many growing and expanding agricultural regions worldwide. If detected, affected commodities must be processed with effective control or eradication techniques. If unattended, losses in product's quality represent unacceptable economic losses.

> Chemical pesticides, waxes, coatings, thermal treatments (heated air; hot water immersion), modified atmospheres, cold storage (refrigeration), and irradiation are some of the processes that have helped industry meet current challenges and demands. Lately, however, new consumer preferences, trends and regulatory interventions have increased the needs for minimally processed foods with low or no residual chemicals. This new trend requires that less invasive or chemical-free alternatives become available to replace or minimize the use of pesticides. Furthermore, recent concerns associated with potential terrorist threats using microbial contaminants or other pests, have increased the need to develop alternatives to

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 235

solely based on the use of energy, they are naturally free of residues and therefore can serve

Since 2002, research at the University of California, Davis established the use of RF power for disinfestation as well as for many novel sanitation and preservation purposes for a variety of food, non-food and agricultural commodities. Since then, RF processing has been established as a novel methodology able to provide new alternatives for chemical-free disinfestation, disinfection and enzyme deactivation effects on various commodities (Lagunas-Solar, 2003; Lagunas-Solar, Zeng & Essert, 2003; Lagunas-Solar, Zeng, Essert et al. 2005a; Lagunas-Solar, Cullor, Zeng, et al. 2005b; Lagunas-Solar, Zeng, Essert et al. 2006a). RF disinfestation, in particular, was proven as an effective, rapid, and a reliable chemical-free

Radiofrequency waves using designated, single frequencies are approved for industrial, scientific and medical uses by national (US Federal Communication Commission, FCC) and international organizations. Currently, limited but increasing commercial use in all these areas to heat-treat and dry a variety of commodities is underway. Radiofrequency power provides well-controlled, volumetric (internal) and rapid heating of a diverse variety of food and non-food commodities. Appropriate food and non-food products to be processed and heated with RF power are generally known as dielectrics (poor electric conductors) and include pests, microbes, foods and non-food agricultural commodities such as soil,

Dielectric properties are directly related to the material's chemical (molecular) composition and due to the presence and relative abundance of dipoles like water and/or induced dipoles like proteins, lipids, and carbohydrates. Therefore, the material's ability to absorb RF power and convert it to thermal power resides at the molecular level. Because molecules are well distributed and organized within and on the surface of dielectric materials, the effect of absorbing RF power occurs throughout its volume and to a lesser extent on its surface (lower concentrations) where temperatures are slightly lower than its internal volume (< 1oC). For this reason, RF processing is said to be a volumetric process, comparable to microwave heating, but in contrast with any other conventional surface thermal process known today. By comparison, the volumetric nature of RF processing provides with unique opportunities to reduce the needed thermal load (i.e. temperature over time) required for an intended effect as heat losses by radiation are larger at the surface. This volumetric property applies equally to arthropod and microbial pests as well as to the host commodity and its

The RF disinfestation process is rapid (seconds to minutes) and proven effective when reaches lethal thermal levels (50-60oC). These levels are sufficient to provide thermal loads able to irreversibly disrupt essential and common metabolic pathways and to affect all biological stages of arthropod (and other) pests. Furthermore, as the interaction of RF photons with molecules is frequency dependent, at specific frequencies insect pests exhibit a higher heating rate than the host commodity allowing a somewhat selective heating process to be realized. This selective process minimizes processing time and lowers the overall thermal load applied to the commodity thus decreasing the potential for any adverse effects

The fundamental physical concepts and the rationale behind the RF disinfestation process, including the interactive energy-transfer and conversion mechanisms (RF to thermal power)

the needs of both conventional and organic agriculture.

alternative to pesticides and capable of large-scale processing.

packaging and wood (pallets) products.

package.

on its quality attributes.

with arthropod pests are explained below.

assure the safety of the food supply while minimizing economical risks associated with production and export agriculture. These combined challenges are now familiar to affected governments as well as to industry and regulators worldwide.

Historically, and with a few exceptions, pesticides have provided an ample spectrum of effective techniques to control pests and there is a continual industry trend to maintain and improve their use. However, this practice and its effects and limitations have partially fueled the emergence of organic agriculture. This in turn has prompted conventional agriculture to review its practices, its traditional processes, and to investigate new types of pesticides as well as to develop new disinfestation techniques. The incorporation of fluorine in agrochemicals to enhance stability and bioavailability is the latest attempt to increase their effectiveness while reducing their secondary impact (Jeschke, 2004). Nevertheless, their invasiveness and persistence in all environs surrounding agricultural practices continues to be resisted by consumers and by increased limiting regulations.

Past and even present industry reliance on methyl bromide fumigation for quarantine pest controls is the best and most recent example of the changing attitude that exists today with respect to invasive chemical processes. The existing ban and the new restrictions on production levels have forced agriculture to look for new and better alternatives. Fumigation, vacuum techniques and controlled atmospheres (CA) for insect (quarantine) control are marginally successful and restricted to long-storage commodities (i.e. grains, nut products, raisins) (Bond, 2007; Calderon, 1990). For perishable fresh commodities, these techniques have failed to provide the required and timely disinfestation level. Nevertheless, while somewhat successful, the needed long processing times (days or weeks) increases cost and is inadequate to fit with the logistics of marketing fresh agricultural products.

The use of low-level doses of ionizing radiation (i.e. food irradiation) is another effective and approved technique providing an alternative to disinfestation and disinfection of many commodities (Urbain, 1986). However, while technically useful and approved for certain applications, this approach prompts many public concerns and is usually and effectively resisted. Furthermore, because irradiation facilities require a high capital investment to install and operate in order to remain economically viable, it also forces the irradiation industry to operate as major centralized facilities located near high productivity agricultural areas. The seasonal nature of agriculture, however, forces the irradiation industry to meet the peak demands with excess processing capacity and to broaden off-season applications (i.e. disinfection of medical supplies) to remain viable. Consequently, the handling and distribution of to-be-treated food and agricultural commodities imposes new and severe logistical and cost adjustments to the user community. As a result, few if any agricultural export areas rely on irradiation facilities and those operating represent a small and stagnant resource for insect control.

Despite the above limitations, ionizing radiation also provide means to sterilize insects that once released in specific areas can reduce the impact of local/regional infestations.

As of today, with the exception of food irradiation, few attempts to fulfill the need for new alternatives to pesticides have been investigated using single or combined physical processes. If effective, these processes are inherently safer, eliminating the risks associated with the presence of pesticides in products and ultimately easing the current concerns with disposal issues, worker safety, and environmental impacts. Non-chemical or residue-free alternatives also provide opportunities to yield products with attributes closer to their natural sensory and nutritional properties. Furthermore, because physical processes are

assure the safety of the food supply while minimizing economical risks associated with production and export agriculture. These combined challenges are now familiar to affected

Historically, and with a few exceptions, pesticides have provided an ample spectrum of effective techniques to control pests and there is a continual industry trend to maintain and improve their use. However, this practice and its effects and limitations have partially fueled the emergence of organic agriculture. This in turn has prompted conventional agriculture to review its practices, its traditional processes, and to investigate new types of pesticides as well as to develop new disinfestation techniques. The incorporation of fluorine in agrochemicals to enhance stability and bioavailability is the latest attempt to increase their effectiveness while reducing their secondary impact (Jeschke, 2004). Nevertheless, their invasiveness and persistence in all environs surrounding agricultural practices continues to

Past and even present industry reliance on methyl bromide fumigation for quarantine pest controls is the best and most recent example of the changing attitude that exists today with respect to invasive chemical processes. The existing ban and the new restrictions on production levels have forced agriculture to look for new and better alternatives. Fumigation, vacuum techniques and controlled atmospheres (CA) for insect (quarantine) control are marginally successful and restricted to long-storage commodities (i.e. grains, nut products, raisins) (Bond, 2007; Calderon, 1990). For perishable fresh commodities, these techniques have failed to provide the required and timely disinfestation level. Nevertheless, while somewhat successful, the needed long processing times (days or weeks) increases cost

The use of low-level doses of ionizing radiation (i.e. food irradiation) is another effective and approved technique providing an alternative to disinfestation and disinfection of many commodities (Urbain, 1986). However, while technically useful and approved for certain applications, this approach prompts many public concerns and is usually and effectively resisted. Furthermore, because irradiation facilities require a high capital investment to install and operate in order to remain economically viable, it also forces the irradiation industry to operate as major centralized facilities located near high productivity agricultural areas. The seasonal nature of agriculture, however, forces the irradiation industry to meet the peak demands with excess processing capacity and to broaden off-season applications (i.e. disinfection of medical supplies) to remain viable. Consequently, the handling and distribution of to-be-treated food and agricultural commodities imposes new and severe logistical and cost adjustments to the user community. As a result, few if any agricultural export areas rely on irradiation facilities and those operating represent a small and stagnant

Despite the above limitations, ionizing radiation also provide means to sterilize insects that

As of today, with the exception of food irradiation, few attempts to fulfill the need for new alternatives to pesticides have been investigated using single or combined physical processes. If effective, these processes are inherently safer, eliminating the risks associated with the presence of pesticides in products and ultimately easing the current concerns with disposal issues, worker safety, and environmental impacts. Non-chemical or residue-free alternatives also provide opportunities to yield products with attributes closer to their natural sensory and nutritional properties. Furthermore, because physical processes are

once released in specific areas can reduce the impact of local/regional infestations.

and is inadequate to fit with the logistics of marketing fresh agricultural products.

governments as well as to industry and regulators worldwide.

be resisted by consumers and by increased limiting regulations.

resource for insect control.

solely based on the use of energy, they are naturally free of residues and therefore can serve the needs of both conventional and organic agriculture.

Since 2002, research at the University of California, Davis established the use of RF power for disinfestation as well as for many novel sanitation and preservation purposes for a variety of food, non-food and agricultural commodities. Since then, RF processing has been established as a novel methodology able to provide new alternatives for chemical-free disinfestation, disinfection and enzyme deactivation effects on various commodities (Lagunas-Solar, 2003; Lagunas-Solar, Zeng & Essert, 2003; Lagunas-Solar, Zeng, Essert et al. 2005a; Lagunas-Solar, Cullor, Zeng, et al. 2005b; Lagunas-Solar, Zeng, Essert et al. 2006a). RF disinfestation, in particular, was proven as an effective, rapid, and a reliable chemical-free alternative to pesticides and capable of large-scale processing.

Radiofrequency waves using designated, single frequencies are approved for industrial, scientific and medical uses by national (US Federal Communication Commission, FCC) and international organizations. Currently, limited but increasing commercial use in all these areas to heat-treat and dry a variety of commodities is underway. Radiofrequency power provides well-controlled, volumetric (internal) and rapid heating of a diverse variety of food and non-food commodities. Appropriate food and non-food products to be processed and heated with RF power are generally known as dielectrics (poor electric conductors) and include pests, microbes, foods and non-food agricultural commodities such as soil, packaging and wood (pallets) products.

Dielectric properties are directly related to the material's chemical (molecular) composition and due to the presence and relative abundance of dipoles like water and/or induced dipoles like proteins, lipids, and carbohydrates. Therefore, the material's ability to absorb RF power and convert it to thermal power resides at the molecular level. Because molecules are well distributed and organized within and on the surface of dielectric materials, the effect of absorbing RF power occurs throughout its volume and to a lesser extent on its surface (lower concentrations) where temperatures are slightly lower than its internal volume (< 1oC). For this reason, RF processing is said to be a volumetric process, comparable to microwave heating, but in contrast with any other conventional surface thermal process known today. By comparison, the volumetric nature of RF processing provides with unique opportunities to reduce the needed thermal load (i.e. temperature over time) required for an intended effect as heat losses by radiation are larger at the surface. This volumetric property applies equally to arthropod and microbial pests as well as to the host commodity and its package.

The RF disinfestation process is rapid (seconds to minutes) and proven effective when reaches lethal thermal levels (50-60oC). These levels are sufficient to provide thermal loads able to irreversibly disrupt essential and common metabolic pathways and to affect all biological stages of arthropod (and other) pests. Furthermore, as the interaction of RF photons with molecules is frequency dependent, at specific frequencies insect pests exhibit a higher heating rate than the host commodity allowing a somewhat selective heating process to be realized. This selective process minimizes processing time and lowers the overall thermal load applied to the commodity thus decreasing the potential for any adverse effects on its quality attributes.

The fundamental physical concepts and the rationale behind the RF disinfestation process, including the interactive energy-transfer and conversion mechanisms (RF to thermal power) with arthropod pests are explained below.

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 237

This expression indicates that all photons in the electromagnetic spectrum come as discrete quantities named "quanta" and moving at the speed of light. It also indicates that photon

Because frequency (f in Hz) and wavelength (λ in m) of an electromagnetic wave are related

*E hc* /

Biological materials - including foods, microbes, arthropods and many agricultural products, are non-magnetic in nature, therefore, only the electric field component of an electromagnetic wave is able to interact and strongly affect the polar and induced polar

In the presence of an oscillating electric field (changing polarity at a set frequency), the interactive mechanisms of the electric field with RF active molecules (i.e. dielectrics or poor electric conductors) include: (1) reorientation of permanent dipoles (i.e. water); (2) inducing dipoles by polarization of bound charges (proteins, carbohydrates, lipids); and (3) forcing the drift (displacement) of electronic and ionic conduction charges (mineral nutrients)

The above interactive mechanisms only act at the molecular level and thus the effects of RF processing is based solely on the material's chemical composition in which permanent dipoles (i.e. water) play a major role while other lower concentration non-polar or weakly polar molecules are activated in proportion to the magnitude of the electric field. Initially, and without an electric field, polar and non-polar molecules in any material are randomly oriented due to thermal excitation, which forces their multi-directional movement and

When an electric field is applied, dipole (polar) molecules tend to re-orient and become aligned according to the direction of the electric field in a phenomenon known as "orientation polarization". Still, orientation is opposed by thermal excitation and therefore, the net orientation effect is proportional to the intensity of the electric field once it

In non-polar molecules, the electric forces separate positive and negative charges a small distance thus inducing temporal dipoles. This type of induced dipole exists only when the electric field is present and occurs via electronic (displacement of electrons) or atomic (displacement of charged atoms) mechanisms known collectively as "distortion

In both cases with orientation or distortion polarization, the charges in dipoles or in induced dipoles do not cancel and, therefore, new internal electric fields are formed. Distortion polarization is temperature dependent while orientation polarization is inversely

overcomes the random distribution of the active molecules in the RF field.

(2)

(3)

h is the Planck's constant (6.626 x 10-34 Joules sec or 4.136 x 10-15 eV sec); and

energy is always a multiple of Planck's constant times its frequency (cycles/sec).

indicating that photon energy E is inversely proportional to its wavelength λ.

f is the photon frequency (Hz or cycles/sec).

*c f*

**2.2 Interactions of RF photons with matter** 

to the speed of light as

molecules in the product.

(Klauenberg & Miklavcic, 2000).

spatial distribution.

polarization".

formula 1 can also be expressed as

#### **2. Physics of RF power**

#### **2.1 RF photons and the electromagnetic spectrum**

Radiofrequency photons belong to the electromagnetic spectrum of radiant energy. The electromagnetic spectrum covers a very large range of wave photons with frequencies ranging from 106 to 1020 Hz (1 Hz = 1 cycle/sec) and wavelengths from 103 to 10-12 m. As shown below in Figure 1, this range covers radiowaves (~106 to 1010 Hz), microwaves (~1010 to 1012 Hz), infrared, visible and ultraviolet radiation (~1012 to 1016 Hz) and soft, hard X rays and gamma rays (1016 to 1020 Hz).

Fig. 1. Electromagnetic spectrum (simplified).

Radiofrequency power is, however, a small segment of the radiowaves region with an arbitrarily defined range of frequencies between ~ 1 MHz (300 m wavelengths) to 300 MHz (1 m wavelengths). In the defined frequency range, the RF photon energy is in the 6.6 x 10-28 to 6.6 x 10-26 J/photon (or 4.1 x 10-9 to 4.1 x 10-7 eV/photon). Therefore, RF processing involves photons of very low energy and long wavelength and therefore absorbing dipole or induced dipole molecules can only experience excitation effects (i.e. vibrational and rotational) but will not lose electrons to cause ionization or the formation of free radicals.1

Radiofrequency waves are produced by rapid electrical oscillations and generally are able to penetrate deep into various materials, but are reflected by electric conductors and by the ionized layers in the upper atmosphere. Like all other photons in the electromagnetic spectrum, RF photons consists of electric and magnetic waves oscillating at right angles to the direction of propagation (i.e. transverse waves) and moving through space at the speed of light (c = 2.998 x 108 m/sec). The combination of electric and magnetic fields originates an electromagnetic field.

The relationship between the RF photon energy and its frequency is given by Einstein's classical expression as:

$$E = hf \tag{1}$$

where: E is the photon energy (Joules);

<sup>1</sup> Chemical bond energies are in the range of 1 to 10 eV per bond. Therefore, RF photons (1 to 100MHz) carry one billionths to 100 millionths less energy than is required to break a single bond. Free radicals are extremely reactive (short lived) chemical species capable of inducing chemical reactions. Their formation is associated exclusively with sources of ionizing radiation (> 1 eV/photon).

h is the Planck's constant (6.626 x 10-34 Joules sec or 4.136 x 10-15 eV sec); and

f is the photon frequency (Hz or cycles/sec).

This expression indicates that all photons in the electromagnetic spectrum come as discrete quantities named "quanta" and moving at the speed of light. It also indicates that photon energy is always a multiple of Planck's constant times its frequency (cycles/sec).

Because frequency (f in Hz) and wavelength (λ in m) of an electromagnetic wave are related to the speed of light as

$$
\mathfrak{c} = f \mathfrak{A} \tag{2}
$$

formula 1 can also be expressed as

236 Insecticides – Basic and Other Applications

Radiofrequency photons belong to the electromagnetic spectrum of radiant energy. The electromagnetic spectrum covers a very large range of wave photons with frequencies ranging from 106 to 1020 Hz (1 Hz = 1 cycle/sec) and wavelengths from 103 to 10-12 m. As shown below in Figure 1, this range covers radiowaves (~106 to 1010 Hz), microwaves (~1010 to 1012 Hz), infrared, visible and ultraviolet radiation (~1012 to 1016 Hz) and soft, hard X rays

Radiofrequency power is, however, a small segment of the radiowaves region with an arbitrarily defined range of frequencies between ~ 1 MHz (300 m wavelengths) to 300 MHz (1 m wavelengths). In the defined frequency range, the RF photon energy is in the 6.6 x 10-28 to 6.6 x 10-26 J/photon (or 4.1 x 10-9 to 4.1 x 10-7 eV/photon). Therefore, RF processing involves photons of very low energy and long wavelength and therefore absorbing dipole or induced dipole molecules can only experience excitation effects (i.e. vibrational and rotational) but will not lose electrons to cause ionization or the formation of free radicals.1 Radiofrequency waves are produced by rapid electrical oscillations and generally are able to penetrate deep into various materials, but are reflected by electric conductors and by the ionized layers in the upper atmosphere. Like all other photons in the electromagnetic spectrum, RF photons consists of electric and magnetic waves oscillating at right angles to the direction of propagation (i.e. transverse waves) and moving through space at the speed of light (c = 2.998 x 108 m/sec). The combination of electric and magnetic fields originates an

The relationship between the RF photon energy and its frequency is given by Einstein's

1 Chemical bond energies are in the range of 1 to 10 eV per bond. Therefore, RF photons (1 to 100MHz) carry one billionths to 100 millionths less energy than is required to break a single bond. Free radicals are extremely reactive (short lived) chemical species capable of inducing chemical reactions. Their

formation is associated exclusively with sources of ionizing radiation (> 1 eV/photon).

*E hf* (1)

**2. Physics of RF power** 

and gamma rays (1016 to 1020 Hz).

Fig. 1. Electromagnetic spectrum (simplified).

electromagnetic field.

classical expression as:

where: E is the photon energy (Joules);

**2.1 RF photons and the electromagnetic spectrum** 

$$\mathbf{E} = \hbar \mathbf{c} \;/\; \mathcal{X} \tag{3}$$

indicating that photon energy E is inversely proportional to its wavelength λ.

#### **2.2 Interactions of RF photons with matter**

Biological materials - including foods, microbes, arthropods and many agricultural products, are non-magnetic in nature, therefore, only the electric field component of an electromagnetic wave is able to interact and strongly affect the polar and induced polar molecules in the product.

In the presence of an oscillating electric field (changing polarity at a set frequency), the interactive mechanisms of the electric field with RF active molecules (i.e. dielectrics or poor electric conductors) include: (1) reorientation of permanent dipoles (i.e. water); (2) inducing dipoles by polarization of bound charges (proteins, carbohydrates, lipids); and (3) forcing the drift (displacement) of electronic and ionic conduction charges (mineral nutrients) (Klauenberg & Miklavcic, 2000).

The above interactive mechanisms only act at the molecular level and thus the effects of RF processing is based solely on the material's chemical composition in which permanent dipoles (i.e. water) play a major role while other lower concentration non-polar or weakly polar molecules are activated in proportion to the magnitude of the electric field. Initially, and without an electric field, polar and non-polar molecules in any material are randomly oriented due to thermal excitation, which forces their multi-directional movement and spatial distribution.

When an electric field is applied, dipole (polar) molecules tend to re-orient and become aligned according to the direction of the electric field in a phenomenon known as "orientation polarization". Still, orientation is opposed by thermal excitation and therefore, the net orientation effect is proportional to the intensity of the electric field once it overcomes the random distribution of the active molecules in the RF field.

In non-polar molecules, the electric forces separate positive and negative charges a small distance thus inducing temporal dipoles. This type of induced dipole exists only when the electric field is present and occurs via electronic (displacement of electrons) or atomic (displacement of charged atoms) mechanisms known collectively as "distortion polarization".

In both cases with orientation or distortion polarization, the charges in dipoles or in induced dipoles do not cancel and, therefore, new internal electric fields are formed. Distortion polarization is temperature dependent while orientation polarization is inversely

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 239

restrict drifting and thus resist movements of electric currents. These types of losses are represented by a relative complex dielectric constant (Ɛ\*) which is given by the expression

In this expression, Ɛ" is a measure of the dissipation losses per cycle and is known as the "dielectric loss factor" and j is the imaginary unit. The dielectric loss factor measures the ability of a material to convert electric energy to thermal energy purely based on polarization effects (i.e. no resistance heating) and is always positive and much smaller than the relative complex dielectric constant (Ɛ\*) (Mudgett, 1986; Nyfors & Vainikainen, 1989). Both relative complex dielectric constant and dielectric loss factors are related to the absolute dielectric constant in vacuum (Ɛo = 8.85 x 10-12 F/m). For clarity, the use of the word "relative" is omitted from this point and therefore Ɛ\*, Ɛ' and Ɛ" will be known simply as complex dielectric constant, dielectric constant and dielectric loss factor, respectively. While most products have small dielectric loss factors, it increases rapidly with temperature but only slightly with pressure. However, these factors can vary drastically with operating

Finally, dielectric constants are the factor by which a dielectric material increases the capacitance of a parallel-plate RF system (i.e. RF cavity, see section 3.1 below) in relation to its capacitance in vacuum or air under the same electric field conditions. Examples of Ɛ\* values for selected materials are given in Table 1, below (Clarke, 2006). Worth noting is that Ɛ\* values for codling moth (71.5; 84.5) and Mexican fruit fly (90; 141) are exceptionally high and similar to water and much larger than values for some host materials (i.e. nuts). Thus, RF disinfestation applications with nuts or similar products, selective (higher) heating of insects - as compared with heating of the host commodity, can be realized and is advantageous for effective insect control while lowering overall thermal loads applied to the

frequencies but are independent of the applied electric-field magnitude.

host commodity. This phenomenon is further explained in section 3.5.2, below.

**Material (Moisture %) Temperature (oC) Frequency Ɛ\***

20 20 (60)

15

Table 1. Values of complex dielectric constants for selected materials (\*).

Water 100 > 1MHz 80 Ethanol 20 10 MHz 24 Sand (dry) 20 1 2.5

Almonds (5%) 20 (60) 27 MHz 5.9 (6.0) Codling moth 20 (60) 27 MHz 71.5 (84.5) Mexican fruit fly 20 (60) 27 MHz 90 (141)

Paper Fiber 20 1 MHz 4.5 Polyethylene (non polar) 20 50 Hz/1 GHz 2.3 Polycarbonate (polar) 20 1 MHz 3.0 (\*) From: National Physical Laboratory (www.kayelaby.npl.co.uk/general\_physics/2\_6/2\_6\_5.html)

10 MHz 10 (27) MHz

15 1 MHz/10 MHz 3.2

2.0 5.0 (4.9)

4.3

Ɛ\* = Ɛ' - j Ɛ" (5)

(Metaxas & Meredith, 1983):

Walnut (0%) Walnut (17%)

Douglass fir (11%) Compressed

and Wang et al., (2003)

proportional to temperature as RF active molecules must overcome the randomness from thermal excitation.

Furthermore, all polarization effects can only operate up to a limiting frequency after which if frequency increases, orientation polarization effects tend to disappear as the inertial effect of permanent polar molecules prevent reversal of their direction of motion and thus their inertial movement (i.e. momentum) cannot be overcome. The RF process is thus frequency dependent and can be optimized at certain selective frequencies matching the dielectric properties of a material (Lagunas-Solar, Zeng & Essert, 2003).

In arthropod pests, as in all biological systems, water (free and bound) and to a lesser extend proteins, lipids, carbohydrates are the major chemical constituents while mineral nutrients are at trace levels. Water is a natural permanent dipole but its degree of freedom depends on its chemical environ with free (unbound) water being the most active dipole to interact with oscillating electric fields. Bound water, on the other hand, because of its binding (coordination) with other molecules, may still be active but is somewhat restricted to respond to electric field oscillations. Proteins, including enzymes, lipids and carbohydrates are polarizable under a voltage difference and therefore become temporal induced dipoles able to experience electric field interactions and be actively involved in generating thermal energy within the material. Inorganic ions (i.e. mineral nutrients) are always charged and can be displaced by the electric fields and generate small electric currents which converts to heat through resistance (Ohm's law). Overall, although at different levels, all constituents may be actively re-oriented or displaced generating thermal energy by combination of the above different interactive mechanisms.

Although most permanent and induced dipoles are not free to drift, displacements of conduction charges or free ions under the influence of an electric field is a classical phenomenon known as ionic conductivity. Conduction effects (Jc in Amperes/m2) are related directly to both conductivity (σ in Siemens/m2) and the net electric field E (Amperes/Siemens) (Lea & Burke, 1998).

#### **2.3 Mechanisms of RF heating**

The ability to induce polarization effects in a material by an applied electric field and the creation of new, transient electric fields and currents within the material is characterized by a quantity noted as Ɛ and called "dielectric constant" or "permittivity" (Klauenberg & Miklavcic, 2000). Therefore, the dielectric constant measures how easily a material is polarized to store electric energy.

However, dielectric constants are measured in relation to vacuum or air (Ɛo = 1.00000 and 1.00054, respectively) as they represent the ability of a material to store electric energy (i.e. capacitance) at a given voltage as compared to vacuum or air. Therefore, relative dielectric constants for a material are given by

$$\mathsf{E}' = \left| \triangle\_{\mathsf{a}} \right| / \left| \triangleleft \right| \tag{4}$$

where Ɛ' is the relative dielectric constant and Êa and Ê are the applied and the net electric field strengths (vectors), respectively.

In real practice, the ratio by which each mechanism intervenes in storing electric energy is accompanied by effective dissipation losses due to thermal excitation, inertial motions and due to the different binding forces in lattices or accompanying the RF active chemicals. These losses force molecules to lag behind the frequency of the oscillating electric field or

proportional to temperature as RF active molecules must overcome the randomness from

Furthermore, all polarization effects can only operate up to a limiting frequency after which if frequency increases, orientation polarization effects tend to disappear as the inertial effect of permanent polar molecules prevent reversal of their direction of motion and thus their inertial movement (i.e. momentum) cannot be overcome. The RF process is thus frequency dependent and can be optimized at certain selective frequencies matching the dielectric

In arthropod pests, as in all biological systems, water (free and bound) and to a lesser extend proteins, lipids, carbohydrates are the major chemical constituents while mineral nutrients are at trace levels. Water is a natural permanent dipole but its degree of freedom depends on its chemical environ with free (unbound) water being the most active dipole to interact with oscillating electric fields. Bound water, on the other hand, because of its binding (coordination) with other molecules, may still be active but is somewhat restricted to respond to electric field oscillations. Proteins, including enzymes, lipids and carbohydrates are polarizable under a voltage difference and therefore become temporal induced dipoles able to experience electric field interactions and be actively involved in generating thermal energy within the material. Inorganic ions (i.e. mineral nutrients) are always charged and can be displaced by the electric fields and generate small electric currents which converts to heat through resistance (Ohm's law). Overall, although at different levels, all constituents may be actively re-oriented or displaced generating thermal energy by combination of the

Although most permanent and induced dipoles are not free to drift, displacements of conduction charges or free ions under the influence of an electric field is a classical phenomenon known as ionic conductivity. Conduction effects (Jc in Amperes/m2) are related directly to both conductivity (σ in Siemens/m2) and the net electric field E

The ability to induce polarization effects in a material by an applied electric field and the creation of new, transient electric fields and currents within the material is characterized by a quantity noted as Ɛ and called "dielectric constant" or "permittivity" (Klauenberg & Miklavcic, 2000). Therefore, the dielectric constant measures how easily a material is

However, dielectric constants are measured in relation to vacuum or air (Ɛo = 1.00000 and 1.00054, respectively) as they represent the ability of a material to store electric energy (i.e. capacitance) at a given voltage as compared to vacuum or air. Therefore, relative dielectric

where Ɛ' is the relative dielectric constant and Êa and Ê are the applied and the net electric

In real practice, the ratio by which each mechanism intervenes in storing electric energy is accompanied by effective dissipation losses due to thermal excitation, inertial motions and due to the different binding forces in lattices or accompanying the RF active chemicals. These losses force molecules to lag behind the frequency of the oscillating electric field or

Ɛ' = │Êa│ / │Ê│ (4)

properties of a material (Lagunas-Solar, Zeng & Essert, 2003).

above different interactive mechanisms.

(Amperes/Siemens) (Lea & Burke, 1998).

**2.3 Mechanisms of RF heating** 

polarized to store electric energy.

constants for a material are given by

field strengths (vectors), respectively.

thermal excitation.

restrict drifting and thus resist movements of electric currents. These types of losses are represented by a relative complex dielectric constant (Ɛ\*) which is given by the expression (Metaxas & Meredith, 1983):

$$
\mathfrak{E}^\* = \mathfrak{E}' \text{ - j } \mathfrak{E}'' \tag{5}
$$

In this expression, Ɛ" is a measure of the dissipation losses per cycle and is known as the "dielectric loss factor" and j is the imaginary unit. The dielectric loss factor measures the ability of a material to convert electric energy to thermal energy purely based on polarization effects (i.e. no resistance heating) and is always positive and much smaller than the relative complex dielectric constant (Ɛ\*) (Mudgett, 1986; Nyfors & Vainikainen, 1989).

Both relative complex dielectric constant and dielectric loss factors are related to the absolute dielectric constant in vacuum (Ɛo = 8.85 x 10-12 F/m). For clarity, the use of the word "relative" is omitted from this point and therefore Ɛ\*, Ɛ' and Ɛ" will be known simply as complex dielectric constant, dielectric constant and dielectric loss factor, respectively.

While most products have small dielectric loss factors, it increases rapidly with temperature but only slightly with pressure. However, these factors can vary drastically with operating frequencies but are independent of the applied electric-field magnitude.

Finally, dielectric constants are the factor by which a dielectric material increases the capacitance of a parallel-plate RF system (i.e. RF cavity, see section 3.1 below) in relation to its capacitance in vacuum or air under the same electric field conditions. Examples of Ɛ\* values for selected materials are given in Table 1, below (Clarke, 2006). Worth noting is that Ɛ\* values for codling moth (71.5; 84.5) and Mexican fruit fly (90; 141) are exceptionally high and similar to water and much larger than values for some host materials (i.e. nuts). Thus, RF disinfestation applications with nuts or similar products, selective (higher) heating of insects - as compared with heating of the host commodity, can be realized and is advantageous for effective insect control while lowering overall thermal loads applied to the host commodity. This phenomenon is further explained in section 3.5.2, below.


(\*) From: National Physical Laboratory (www.kayelaby.npl.co.uk/general\_physics/2\_6/2\_6\_5.html) and Wang et al., (2003)

Table 1. Values of complex dielectric constants for selected materials (\*).

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 241

where the ratio ΔT/t is the rate of heating expressed as a function of processing parameters (f, E) and the other factors are associated with the product properties (Ɛ" and d), where d

Reaching all pests in a volume of material to be disinfested is an important feature of RF disinfestation as the process must be effective over large volumes of material to assure reliable control with adequate throughputs. Temperature distribution and depth of penetration are thus important aspects that need to be considered for RF disinfestation of

In standard volumes of boxed or palletized materials processed with a parallel-plate capacitor (see section 3, below), the intensity of the electric field is largely unaffected by the load and it contributes to similar energy absorption throughout the material. In addition,

An electromagnetic wave incident on the surface of a dielectric material can either be reflected (i.e. reflected wave) or be transmitted into the material (i.e. transmitted wave). In good dielectrics (including its package), a great fraction of the wave energy is transmitted but is gradually attenuated as it is converted to thermal energy. The extent (length) of the wave transmitted into the material is known as "penetration depth" (Dp) and is arbitrarily defined as the distance from the surface to the point (plane) at which its energy is reduced to

Because the effective loss tangent (tan δeff = Ɛ"eff/ Ɛ'); the penetration depth can be

2 ( ) tan 2 *<sup>p</sup>*

*c c <sup>D</sup>*

 

load) as some limitations are expected by penetration depth factors.

' 1/2 "

*f f*

Penetration depth (Dp in meters) is therefore proportional to the dielectric constant (Ɛ') and inversely proportional to the dielectric loss factor (Ɛ"eff) and to the frequency of oscillation of the electric field. In general, at frequencies below 100 MHz, penetration depth is of the order of meters unless the dielectric loss factors are exceedingly high (Metaxas & Meredith, 1983). Despite its penetration, however, the energy distribution and thus the thermal profiles of the RF heated material must be taken into account when the process's efficacy requires a pre-

For disinfestation applications, however, the threshold temperature to assure lethal effects in all insects and mites at any biological stage is rather small (50-60oC) and requires a short time (< 1 min). This allows the use of RF disinfestation in large-volume containers (i.e. pallets at 2 x 2 x 2.2 m high) as material handling techniques can also be applied to improve temperature homogeneity to narrower ranges (but assuring reaching a threshold thermal

However, as explained above, changes in the dielectric behavior of the load due to increased temperature (i.e. increased dielectric loss factors) induce rapid and significant changes in the fraction of the electric energy being absorbed and converted to thermal power. Unattended, these factors could lead to severe localized, uneven heating of the packaged commodity with potential loss of quality. Therefore, process controls need to be focused into

  ' 1/2

(10)

( )

*eff eff*

 

rms)/d (9)

ΔT/t = (2πƐofƐ"effE2

**2.5 Temperature distribution and depth of penetration in RF processing** 

(kg/m3) is its density.

e-1 (1/2.71 or 37%).

approximated by

large volumes of commodities.

depth of penetration is an important added factor.

defined or a narrow temperature range.

#### **2.4 RF power dissipation as thermal power**

The ability of molecules within a material to store electric energy from an operating RF system is the first step towards an effective heating process able to induce a desirable biological effect (i.e. disinfestation). As indicated above, this is expressed by the complex dielectric constant which combines dielectric properties defined by molecular composition. Therefore, the conversion of RF power into thermal power is directly related to the polarization and ionic conduction mechanisms described above. However, the fractional contribution of each interactive mechanism is determined by the frequency (Hz) of the oscillating electric field.

At low frequencies, all dipole molecules (permanent and induced) have sufficient time to follow and adjust to the reversal cycles of the oscillating electric fields. In this case, no or negligible energy dissipation losses occur due to orientation polarization effects. Under this condition, the dielectric constant is at its maximum value and the dielectric material is capable of storing a maximum energy from the applied electric field. The RF heating is then mostly due to combined polarization and ionic conduction effects.

As frequency increases, dipoles gradually lose their ability to fully adjust to the oscillations in polarity of the electric field and polarization effects lag behind and contribute less to the total polarization. To minimize this lagging, the electric field transfers its energy to the dipoles forcing them to respond faster. However, this electric-field forced adjustment reaches a limit at which no further corrections occur. At this point, lags in dipolar polarization become larger forcing the dielectric constant to fall in value while dielectric loss factors increases. Under this scenario, RF heating depends less on polarization effects but more on ionic conduction effects (displacements or drifting of charged molecules and ions) leading to resistance heating. This variation in mechanism is therefore highly influenced by commodity temperature.

The total RF power dissipation into a sample is derived from the fundamental laws of electromagnetism. For a steady-state sinusoidal electric field, the time-average of RF thermal power dissipation per unit volume of the sample is given by:

$$\mathbf{P\_v = 2\pi E\_o \mathcal{E} \mathcal{E}''\_{\rm eff} E\_{\rm rms}} \tag{6}$$

where Pv is in watt per cubic meter (W/m3; or [Joules/sec]/m3); f is the frequency of the oscillating electric field (Hz); Ɛ" is the dielectric loss factor and Erms is the root-mean-square of the applied electric field in Volts per meter (V/m) (Metaxas & Meredith, 1983).

The total amount of heat Q (Joules) needed for a mass m (kg) of a dielectric material to increase its temperature from an initial value (Ti) to a final temperature (Tf) (i.e. ΔT = Tf – Ti) is given by the classical expression

$$\mathbf{Q} = \mathbf{m} \mathbf{C}\_{\mathbf{P}} (\mathbf{T}\_{\mathbf{f}} - \mathbf{T}\_{\mathbf{i}}) \tag{7}$$

where Cp is the specific heat of the material (Joules/kgoC).

Power per unit volume in formula 6 can be combined with the energy required as given in formula 7 to provide a combined formula (formula 8) leading to RF throughput as determined with RF processing parameters:

$$\text{mC}\_{\text{p}} \text{(T}\_{\text{l}} - \text{T}\_{\text{i}} \text{)} / \text{Vt} = 2 \text{mC}\_{\text{o}} \text{f} \text{C}'''\_{\text{efl}} \text{E}\_{\text{rms}} \tag{8}$$

which can be expressed as

$$
\Delta \text{T/t} = \left( \text{Tr} \mathbb{E}\_{\text{o}} \text{f} \mathbb{E}'' \, \_{\text{eff}} \mathbb{E}\_{\text{rms}} \right) / \text{d} \tag{9}
$$

where the ratio ΔT/t is the rate of heating expressed as a function of processing parameters (f, E) and the other factors are associated with the product properties (Ɛ" and d), where d (kg/m3) is its density.

#### **2.5 Temperature distribution and depth of penetration in RF processing**

240 Insecticides – Basic and Other Applications

The ability of molecules within a material to store electric energy from an operating RF system is the first step towards an effective heating process able to induce a desirable biological effect (i.e. disinfestation). As indicated above, this is expressed by the complex dielectric constant which combines dielectric properties defined by molecular composition. Therefore, the conversion of RF power into thermal power is directly related to the polarization and ionic conduction mechanisms described above. However, the fractional contribution of each interactive mechanism is determined by the frequency (Hz) of the

At low frequencies, all dipole molecules (permanent and induced) have sufficient time to follow and adjust to the reversal cycles of the oscillating electric fields. In this case, no or negligible energy dissipation losses occur due to orientation polarization effects. Under this condition, the dielectric constant is at its maximum value and the dielectric material is capable of storing a maximum energy from the applied electric field. The RF heating is then

As frequency increases, dipoles gradually lose their ability to fully adjust to the oscillations in polarity of the electric field and polarization effects lag behind and contribute less to the total polarization. To minimize this lagging, the electric field transfers its energy to the dipoles forcing them to respond faster. However, this electric-field forced adjustment reaches a limit at which no further corrections occur. At this point, lags in dipolar polarization become larger forcing the dielectric constant to fall in value while dielectric loss factors increases. Under this scenario, RF heating depends less on polarization effects but more on ionic conduction effects (displacements or drifting of charged molecules and ions) leading to resistance heating. This variation in mechanism is therefore highly influenced by

The total RF power dissipation into a sample is derived from the fundamental laws of electromagnetism. For a steady-state sinusoidal electric field, the time-average of RF thermal

 Pv = 2πƐofƐ"effE2rms (6) where Pv is in watt per cubic meter (W/m3; or [Joules/sec]/m3); f is the frequency of the oscillating electric field (Hz); Ɛ" is the dielectric loss factor and Erms is the root-mean-square

The total amount of heat Q (Joules) needed for a mass m (kg) of a dielectric material to increase its temperature from an initial value (Ti) to a final temperature (Tf) (i.e. ΔT = Tf – Ti)

Power per unit volume in formula 6 can be combined with the energy required as given in formula 7 to provide a combined formula (formula 8) leading to RF throughput as

Q = mCp(Tf – Ti) (7)

rms (8)

of the applied electric field in Volts per meter (V/m) (Metaxas & Meredith, 1983).

mostly due to combined polarization and ionic conduction effects.

power dissipation per unit volume of the sample is given by:

where Cp is the specific heat of the material (Joules/kgoC).

mCp(Tf – Ti)/Vt = 2πƐofƐ"effE2

**2.4 RF power dissipation as thermal power** 

oscillating electric field.

commodity temperature.

is given by the classical expression

which can be expressed as

determined with RF processing parameters:

Reaching all pests in a volume of material to be disinfested is an important feature of RF disinfestation as the process must be effective over large volumes of material to assure reliable control with adequate throughputs. Temperature distribution and depth of penetration are thus important aspects that need to be considered for RF disinfestation of large volumes of commodities.

In standard volumes of boxed or palletized materials processed with a parallel-plate capacitor (see section 3, below), the intensity of the electric field is largely unaffected by the load and it contributes to similar energy absorption throughout the material. In addition, depth of penetration is an important added factor.

An electromagnetic wave incident on the surface of a dielectric material can either be reflected (i.e. reflected wave) or be transmitted into the material (i.e. transmitted wave).

In good dielectrics (including its package), a great fraction of the wave energy is transmitted but is gradually attenuated as it is converted to thermal energy. The extent (length) of the wave transmitted into the material is known as "penetration depth" (Dp) and is arbitrarily defined as the distance from the surface to the point (plane) at which its energy is reduced to e-1 (1/2.71 or 37%).

Because the effective loss tangent (tan δeff = Ɛ"eff/ Ɛ'); the penetration depth can be approximated by

$$D\_p = \frac{c}{2\pi f \left(\boldsymbol{\varepsilon}^\circ\right)^{1/2} \tan \delta\_{\rm eff}} = \frac{c \left(\boldsymbol{\varepsilon}^\circ\right)^{1/2}}{2\pi f \boldsymbol{\varepsilon}\_{\rm eff}^\circ} \tag{10}$$

Penetration depth (Dp in meters) is therefore proportional to the dielectric constant (Ɛ') and inversely proportional to the dielectric loss factor (Ɛ"eff) and to the frequency of oscillation of the electric field. In general, at frequencies below 100 MHz, penetration depth is of the order of meters unless the dielectric loss factors are exceedingly high (Metaxas & Meredith, 1983). Despite its penetration, however, the energy distribution and thus the thermal profiles of the RF heated material must be taken into account when the process's efficacy requires a predefined or a narrow temperature range.

For disinfestation applications, however, the threshold temperature to assure lethal effects in all insects and mites at any biological stage is rather small (50-60oC) and requires a short time (< 1 min). This allows the use of RF disinfestation in large-volume containers (i.e. pallets at 2 x 2 x 2.2 m high) as material handling techniques can also be applied to improve temperature homogeneity to narrower ranges (but assuring reaching a threshold thermal load) as some limitations are expected by penetration depth factors.

However, as explained above, changes in the dielectric behavior of the load due to increased temperature (i.e. increased dielectric loss factors) induce rapid and significant changes in the fraction of the electric energy being absorbed and converted to thermal power. Unattended, these factors could lead to severe localized, uneven heating of the packaged commodity with potential loss of quality. Therefore, process controls need to be focused into

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 243

The RF cavity operates with equally charged plates (top positive and bottom negative) formed when a voltage difference is applied. Electric field lines (red) are directed towards the negative (ground) plate and are equally spaced and parallel to each other. Transverse waves (not shown) are perpendicular to the electric field. When activated, however, by placing a material (load) in between, the electric field geometry is changed as field lines are distorted (i.e. fringe effects especially at low frequencies) due to the load and its package, and its intensity is decreased because of new charges created in the load. The presence of air gaps in between and on top of the packaged dielectric load also contributes to field distortion and localization effects. Therefore, an active RF cavity needs to be properly designed and managed in order to minimize the above effects and maintain field

A schematic of the major features of a RF power system is shown in Figure 3, below, while a

Fig. 2. A static parallel-plate capacitor (RF cavity).

homogeneity and thus treat with adequate uniformity.

Fig. 3. Schematics of a conveyorized RF processing system.

version of an operating commercial-scale prototype is shown in Figure 4.

maintaining adequate RF power densities to be applied and by controlling product temperatures during the process. Besides, product geometry, package material and its geometry, and air gaps within the material clearly contribute further to different power densities being generated in their volumes and thus temperature variations are to be expected. As lethal thermal loads in insects and mites are low (50-60oC; ~ 1 min), process effectiveness is assured by reaching the relatively low lethal thermal loads needed. This occurs at levels below than those affecting quality in the host commodity and is due to the higher metabolic complexity of arthropod pests as compared with the much simpler metabolism of the host food (i.e. insects in grains) or agricultural commodity (i.e. insects in pallets).

Heat transfer and temperature distribution across a material in RF disinfestation is critical to assure effectiveness and both phenomena have been studied intensively (Giles, Moore & Bounds, 1970). In the absence of any significant mass transfer (e.g. evaporation), the temperature distribution in the medium obeys the heat diffusion law and is given by:

$$\frac{\partial T}{\partial t} = \alpha\_T \nabla^2 T + \frac{\delta P}{d\varepsilon\_p} \tag{11}$$

where α*T* is the thermal diffusivity of the medium (m²/s), T is temperature, *δP* is the localized power density (W/m³), is the Laplacian operator, d is density and cp the specific heat (Metaxas & Meredith, 1983). The thermal diffusivity measures the ability of a material to conduct thermal energy relative to its ability to store thermal energy. Materials of large *α<sup>T</sup>* will respond quickly to changes in their thermal environment, while materials of small *α<sup>T</sup>* will respond more sluggishly and take more time to reach a new temperature equilibrium condition.

In RF processing, materials are usually heterogeneous, and therefore *αT* plays an important role because different parts absorb RF power at different rates. For homogenous materials, *αT* is less important in temperature distribution, and *δP* can be approximated as *Pv* (formula 6) for the temperature analysis. However, thermal diffusivity (or thermal mass effect) of insects and mites is not known despite the many reported studies on thermoregulation of common habitats with its surroundings. However, the rapid heating of insects with RF power (Nelson & Charity, 1972) suggests an appreciable value of thermal diffusivity for insects.

Finally, due to its direct heating effects at molecular levels, RF heating is independent of temperature differences and heat transfer coefficients, although both these factors will influence the subsequent dynamic distribution of thermal energy within the volume of the material (Hill et al., 1969).

#### **3. Principles of RF processing**

#### **3.1 The RF cavity – A parallel-plate capacitor**

In order to best realize and apply the above mechanisms in a controlled and safe RF disinfestation process, a parallel-plate capacitor is used with materials to be treated placed in between and named the "load". The process can be performed either statically (batch mode) or continuously (conveyorized mode). This type of capacitor is known as a "RF cavity" and is shown schematically in Figure 2, below.

Fig. 2. A static parallel-plate capacitor (RF cavity).

maintaining adequate RF power densities to be applied and by controlling product temperatures during the process. Besides, product geometry, package material and its geometry, and air gaps within the material clearly contribute further to different power densities being generated in their volumes and thus temperature variations are to be expected. As lethal thermal loads in insects and mites are low (50-60oC; ~ 1 min), process effectiveness is assured by reaching the relatively low lethal thermal loads needed. This occurs at levels below than those affecting quality in the host commodity and is due to the higher metabolic complexity of arthropod pests as compared with the much simpler metabolism of the host food (i.e. insects in grains) or agricultural commodity (i.e. insects in

Heat transfer and temperature distribution across a material in RF disinfestation is critical to assure effectiveness and both phenomena have been studied intensively (Giles, Moore & Bounds, 1970). In the absence of any significant mass transfer (e.g. evaporation), the temperature distribution in the medium obeys the heat diffusion law and is given by:

> 2 *T*

*T P <sup>T</sup> t dc*

where α*T* is the thermal diffusivity of the medium (m²/s), T is temperature, *δP* is the localized power density (W/m³), is the Laplacian operator, d is density and cp the specific heat (Metaxas & Meredith, 1983). The thermal diffusivity measures the ability of a material to conduct thermal energy relative to its ability to store thermal energy. Materials of large *α<sup>T</sup>* will respond quickly to changes in their thermal environment, while materials of small *α<sup>T</sup>* will respond more sluggishly and take more time to reach a new temperature equilibrium

In RF processing, materials are usually heterogeneous, and therefore *αT* plays an important role because different parts absorb RF power at different rates. For homogenous materials, *αT* is less important in temperature distribution, and *δP* can be approximated as *Pv* (formula 6) for the temperature analysis. However, thermal diffusivity (or thermal mass effect) of insects and mites is not known despite the many reported studies on thermoregulation of common habitats with its surroundings. However, the rapid heating of insects with RF power (Nelson & Charity, 1972) suggests an appreciable value of thermal diffusivity for

Finally, due to its direct heating effects at molecular levels, RF heating is independent of temperature differences and heat transfer coefficients, although both these factors will influence the subsequent dynamic distribution of thermal energy within the volume of the

In order to best realize and apply the above mechanisms in a controlled and safe RF disinfestation process, a parallel-plate capacitor is used with materials to be treated placed in between and named the "load". The process can be performed either statically (batch mode) or continuously (conveyorized mode). This type of capacitor is known as a "RF

*p*

(11)

pallets).

condition.

insects.

material (Hill et al., 1969).

**3. Principles of RF processing** 

**3.1 The RF cavity – A parallel-plate capacitor** 

cavity" and is shown schematically in Figure 2, below.

The RF cavity operates with equally charged plates (top positive and bottom negative) formed when a voltage difference is applied. Electric field lines (red) are directed towards the negative (ground) plate and are equally spaced and parallel to each other. Transverse waves (not shown) are perpendicular to the electric field. When activated, however, by placing a material (load) in between, the electric field geometry is changed as field lines are distorted (i.e. fringe effects especially at low frequencies) due to the load and its package, and its intensity is decreased because of new charges created in the load. The presence of air gaps in between and on top of the packaged dielectric load also contributes to field distortion and localization effects. Therefore, an active RF cavity needs to be properly designed and managed in order to minimize the above effects and maintain field homogeneity and thus treat with adequate uniformity.

A schematic of the major features of a RF power system is shown in Figure 3, below, while a version of an operating commercial-scale prototype is shown in Figure 4.

Fig. 3. Schematics of a conveyorized RF processing system.

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 245

created to be converted to heat. However, due to lattice limitations, when the frequency is at the maximum equilibrium between rotation and inertial restrictions, it is said to be at a "Debye resonance" at which there is maximum conversion to heat. If operating frequency is beyond the ability of the molecules to react due to inertial motion, the process loose overall energy-conversion efficiency. This suggests that specific materials, due to their own unique chemical composition will present an optimal frequency at which to operate with maximum energy-use efficiency. In materials with complex or different composition (i.e. pest and host) is therefore possible to establish selective RF heating effects and establish a process with

In addition to polarization mechanisms, a dielectric material can also be heated by the resistance to direct ionic conduction or drift mechanism as given by Ohm's law and that states that the current (I in Amperes) through a conductor between two points is directly proportional to the voltage difference (V in volts) across the two points and inversely proportional to the resistance (R in Ohms). The heating level through these mechanisms depends on the electric conductivity of the material which is generally low as dielectric (i.e.

Finally, because these mechanisms occur with equal intensity between the RF cavities (i.e. same electric field intensity) and are only dependent on the material's chemical composition, RF heating is in principle homogeneous and a volumetric (internal) method in contrast with all other surface heating methods known today. However, at a microscopic scale within biological materials, some differences do occur due to variations in chemical composition and moisture levels. These differences allow for the enhancement of the rate of heating with distinct materials and are the basis for selective RF heating effects (Zimmerman, Pilwat &

For disinfestation purposes, RF power provides a unique mean to heat an arthropod pest (small mass or volume) inside a host commodity (large mass or volume) volumetrically (internally) and with penetrating RF waves. This behavior is opposite to the use of conventional surface-heat methods such as infrared, dry and wet steam, or hot water where the host's surface becomes a physical barrier to the applied thermal energy. In all latter cases, the distribution of the applied heat to reach the entire volume depends on heattransport mechanisms and time. In addition, heat is only applied at its surface. Furthermore, under these conditions, many commodities experience undesirable changes that lower product value. In contrast, because of its penetration, RF waves are effective in reaching deeply internalized pests such as eggs and larva deposited in internal cavities by borer insects, a situation in which the effectiveness of fumigants is restricted by the presence of

Radiofrequency processing is volumetric heating and its energy transfers directly to the product without the need of intermediate transfer mechanism such as conduction, radiation, or convection. This allows RF energy to be transfer to the load much faster and more effectively. The amount of input energy can be controlled by reducing the input power or switching the system on and off in order to achieve precisely the final temperature. These characteristics allow the RF process to be operated within low and high thermal boundaries, called "thermal windows". Thermal windows for RF disinfestation as compared with other biological effects (i.e. pasteurization and enzyme deactivation) are given in Figure 5, below.

minimal energy input to the lesser dielectric component (Lagunas-Solar et al. 2006).

poor conductor) properties prevail.

**3.2 Advantages of RF disinfestation** 

air-locks impeding penetration of fumigants.

Riemann, 1974).

Fig. 4. RF system for batch processing (13.15 MHz, 10 kW). Designed by UC Davis & RF Biocidics Inc.

In this latter system, the RF cavity is shielded in all directions with a metallic enclosure (shown in light blue) so as to prevent propagation or reflections of RF waves outside its boundaries and thus eliminate the potential to expose workers, the surrounding environs or interfere with other radiowaves. This basic configuration, singly or in modules, is able to operate and meet the conditions to generate and delivery RF energy safely and efficiently to food and agricultural commodities at commercial-scale levels.

The parallel-plate configuration shown in Figure 2 (above) is said to be in a static condition in which no material (other than air or vacuum) is placed in between and therefore the electric field lines are equally spaced and parallel to each other while the overall electric field is uniform except at the edges. However, when a product (load) (i.e. a dielectric) is introduced and the electric field is rapidly oscillated (changing electric polarities at every cycle) with a certain frequency, the dielectric product (load) is now capable of absorbing RF energy by a combination of the above mentioned molecular mechanisms and convert it to thermal power.

The main characteristic of RF processing (RF heating) is therefore, based on the high frequency alternating oscillating electric fields interacting with the dielectric medium (dipoles and induced dipoles) in between the plates and generating thermal energy (heat). RF heating is therefore, also known as "high frequency capacitive heating" (Piyasena et al., 2003), although as the medium in between the plates is also a dielectric material, the process is often referred as "high frequency dielectric heating" (Zhao et al., 2000).

The generation of thermal energy is due to the ability of the applied oscillating electric field to polarize and re-orient internal electric fields of charges formed in the load (material). The rotating electric field exerts torques on permanent and induced dipoles to force them into flip-flop motions. During the rapid cycling, friction and heat is generated between polarized molecules (permanent or induced dipoles) and their neighbors including lattice losses as they move. The higher the frequency of oscillations the greater is the energy available or created to be converted to heat. However, due to lattice limitations, when the frequency is at the maximum equilibrium between rotation and inertial restrictions, it is said to be at a "Debye resonance" at which there is maximum conversion to heat. If operating frequency is beyond the ability of the molecules to react due to inertial motion, the process loose overall energy-conversion efficiency. This suggests that specific materials, due to their own unique chemical composition will present an optimal frequency at which to operate with maximum energy-use efficiency. In materials with complex or different composition (i.e. pest and host) is therefore possible to establish selective RF heating effects and establish a process with minimal energy input to the lesser dielectric component (Lagunas-Solar et al. 2006).

In addition to polarization mechanisms, a dielectric material can also be heated by the resistance to direct ionic conduction or drift mechanism as given by Ohm's law and that states that the current (I in Amperes) through a conductor between two points is directly proportional to the voltage difference (V in volts) across the two points and inversely proportional to the resistance (R in Ohms). The heating level through these mechanisms depends on the electric conductivity of the material which is generally low as dielectric (i.e. poor conductor) properties prevail.

Finally, because these mechanisms occur with equal intensity between the RF cavities (i.e. same electric field intensity) and are only dependent on the material's chemical composition, RF heating is in principle homogeneous and a volumetric (internal) method in contrast with all other surface heating methods known today. However, at a microscopic scale within biological materials, some differences do occur due to variations in chemical composition and moisture levels. These differences allow for the enhancement of the rate of heating with distinct materials and are the basis for selective RF heating effects (Zimmerman, Pilwat & Riemann, 1974).

#### **3.2 Advantages of RF disinfestation**

244 Insecticides – Basic and Other Applications

Fig. 4. RF system for batch processing (13.15 MHz, 10 kW). Designed by UC Davis & RF

food and agricultural commodities at commercial-scale levels.

In this latter system, the RF cavity is shielded in all directions with a metallic enclosure (shown in light blue) so as to prevent propagation or reflections of RF waves outside its boundaries and thus eliminate the potential to expose workers, the surrounding environs or interfere with other radiowaves. This basic configuration, singly or in modules, is able to operate and meet the conditions to generate and delivery RF energy safely and efficiently to

The parallel-plate configuration shown in Figure 2 (above) is said to be in a static condition in which no material (other than air or vacuum) is placed in between and therefore the electric field lines are equally spaced and parallel to each other while the overall electric field is uniform except at the edges. However, when a product (load) (i.e. a dielectric) is introduced and the electric field is rapidly oscillated (changing electric polarities at every cycle) with a certain frequency, the dielectric product (load) is now capable of absorbing RF energy by a combination of the above mentioned molecular mechanisms and convert it to

The main characteristic of RF processing (RF heating) is therefore, based on the high frequency alternating oscillating electric fields interacting with the dielectric medium (dipoles and induced dipoles) in between the plates and generating thermal energy (heat). RF heating is therefore, also known as "high frequency capacitive heating" (Piyasena et al., 2003), although as the medium in between the plates is also a dielectric material, the process

The generation of thermal energy is due to the ability of the applied oscillating electric field to polarize and re-orient internal electric fields of charges formed in the load (material). The rotating electric field exerts torques on permanent and induced dipoles to force them into flip-flop motions. During the rapid cycling, friction and heat is generated between polarized molecules (permanent or induced dipoles) and their neighbors including lattice losses as they move. The higher the frequency of oscillations the greater is the energy available or

is often referred as "high frequency dielectric heating" (Zhao et al., 2000).

Biocidics Inc.

thermal power.

For disinfestation purposes, RF power provides a unique mean to heat an arthropod pest (small mass or volume) inside a host commodity (large mass or volume) volumetrically (internally) and with penetrating RF waves. This behavior is opposite to the use of conventional surface-heat methods such as infrared, dry and wet steam, or hot water where the host's surface becomes a physical barrier to the applied thermal energy. In all latter cases, the distribution of the applied heat to reach the entire volume depends on heattransport mechanisms and time. In addition, heat is only applied at its surface. Furthermore, under these conditions, many commodities experience undesirable changes that lower product value. In contrast, because of its penetration, RF waves are effective in reaching deeply internalized pests such as eggs and larva deposited in internal cavities by borer insects, a situation in which the effectiveness of fumigants is restricted by the presence of air-locks impeding penetration of fumigants.

Radiofrequency processing is volumetric heating and its energy transfers directly to the product without the need of intermediate transfer mechanism such as conduction, radiation, or convection. This allows RF energy to be transfer to the load much faster and more effectively. The amount of input energy can be controlled by reducing the input power or switching the system on and off in order to achieve precisely the final temperature. These characteristics allow the RF process to be operated within low and high thermal boundaries, called "thermal windows". Thermal windows for RF disinfestation as compared with other biological effects (i.e. pasteurization and enzyme deactivation) are given in Figure 5, below.

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 247

produce homogeneous heating because of the limited penetration of the shorter wavelength and the complex non-uniform standing wave patterns. The penetration depth of microwave is in the order of 5 cm to 10 cm for bodies with high water content, and may be higher (in several tens of centimeters) for other drier materials (Orfeuil, 1987). In addition, the electric field inside the microwave oven is not uniform due to the nature of standing waves. In fact, the enclosed electric field and power density vary with the location and the sample's shape and size. Non-uniform electric field patterns and variable power densities often lead to local (or uneven) heating in the material. Besides, the power density in microwave heating are much higher than in RF heating (due to much higher operational frequencies) and is associated with non-uniform electric fields. Therefore microwave heating normally causes

In contrast, the RF process is operated at frequencies much lower than conventional microwaves hence the penetration of RF energy is greater, usually higher than 1 m and even several tens of meters at low frequencies (Orfeuil, 1987). Furthermore, the electric fields generated between two parallel plates are very uniform; therefore, RF transversal waves interact and heat the material more homogeneously (Wig *et al.*, 1999; Mitcham *et al.*,

Today, conventional or emerging alternatives face several restrictions or their use is associated with many safety concerns many of which prompted the development of RF disinfestation as well. The contributing factors from the industry perspective are

Methyl bromide fumigation was for many decades the preferred treatment applied to many stored food commodities. It was used worldwide to meet quarantine and phytosanitary restrictions and quality requirements as mandated by global agriculture markets. Current alternative methods used to control insects in grains include the use of insecticides (e.g. Malathion), fumigants (e.g. phosphine, carbon dioxide) and temperature treatment (Bond,

Malathion (American Cyanamid Co., USA) is one of the safest organophosphate insecticides. Nevertheless, existing regulations demands that the treated grains should not be sold for at least 7 days nor should be eaten within 60 days after treatment to avoid

Phosphine gas is very toxic to human therefore its application requires strict controls, even

Other pesticides in use include Chloropicrin, 1,3-dichloropropene, Telone/Vapam, sulfaryl

However, all pesticides available and those mentioned in particular are of global concern due to the potential for causing detrimental effects on animals, air, water and soil as well as

Conventional carbon dioxide fumigation of grains usually referred as modified atmospheres requires a lengthy treatment (i.e. days to weeks) therefore its cost is high as well as its

**3.4 Comparison with conventional disinfestation technologies** 

**3.4.1 Chemical pesticides issues and concerns** 

potential toxic effects from residues left.

fluoride and hydrogen cyanide.

though there is no residue left to the treated grains.

potentially impacting public health and workers safety.

impact on the logistics of product distribution to markets.

local hot spots to the commodity.

2004).

2007).

summarized below.

Fig. 5. Thermal Windows (colored arrows) for RF processing effects.

A thermal window represents the differential thermal sensitivity between living organisms (highly-heat sensitive) and the more heat-tolerant properties of agricultural products. Therefore, operating within a product's thermal window minimizes the impact on the host commodity. This is a critical advantage of RF processing over any conventional (surface) heating method as disinfestation effects can be well controlled because of the high sensitivity of arthropod pests to thermal energy and the higher heat tolerance of most affected foods and agricultural commodities.

By comparison with conventional heating processes, overheating the surface is very common because energy is first applied to the surface and then is conducted to its interior. Because energy loss from the surface (by radiation and/or convection) is unavoidable, significant and fast, these processes often require additional heat input on the surface in order to produce internal temperatures high enough to achieve a uniform biological effect. The host, however, received higher and usually damaging thermal loads on its surface.

As a result, the upper boundary of the thermal window (especially for the surface) is frequently exceeded causing unacceptable changes in the physiological, sensory, and quality of foods. In RF disinfestation, the surface temperature is usually lower than the internal temperature due to the heat loss from surface radiation and due to evaporation. This can be effectively prevented during RF processing by reducing evaporation (e.g. high humidity inside the chamber), by adding moisture before processing and by providing good radiation reflectors in the RF cavity design.

#### **3.3 RF and microwave processing**

Frequently, microwave heating is confused with RF heating. While fundamentally similar, microwave heating (also an energy source in the electromagnetic spectrum) is operated at 915 MHz (*λ* = 0.3 m) and 2,450 MHz (*λ* = 0.1 m), that is with higher frequency and shorter waves than RF. For most commercial scales (i.e. large amounts) of foods and agricultural products, microwave heating is not adequate also has many disadvantages in aspects of heating homogeneity, energy penetration, and energy-use efficiency. First, it does not produce homogeneous heating because of the limited penetration of the shorter wavelength and the complex non-uniform standing wave patterns. The penetration depth of microwave is in the order of 5 cm to 10 cm for bodies with high water content, and may be higher (in several tens of centimeters) for other drier materials (Orfeuil, 1987). In addition, the electric field inside the microwave oven is not uniform due to the nature of standing waves. In fact, the enclosed electric field and power density vary with the location and the sample's shape and size. Non-uniform electric field patterns and variable power densities often lead to local (or uneven) heating in the material. Besides, the power density in microwave heating are much higher than in RF heating (due to much higher operational frequencies) and is associated with non-uniform electric fields. Therefore microwave heating normally causes local hot spots to the commodity.

In contrast, the RF process is operated at frequencies much lower than conventional microwaves hence the penetration of RF energy is greater, usually higher than 1 m and even several tens of meters at low frequencies (Orfeuil, 1987). Furthermore, the electric fields generated between two parallel plates are very uniform; therefore, RF transversal waves interact and heat the material more homogeneously (Wig *et al.*, 1999; Mitcham *et al.*, 2004).

#### **3.4 Comparison with conventional disinfestation technologies**

Today, conventional or emerging alternatives face several restrictions or their use is associated with many safety concerns many of which prompted the development of RF disinfestation as well. The contributing factors from the industry perspective are summarized below.

#### **3.4.1 Chemical pesticides issues and concerns**

246 Insecticides – Basic and Other Applications

A thermal window represents the differential thermal sensitivity between living organisms (highly-heat sensitive) and the more heat-tolerant properties of agricultural products. Therefore, operating within a product's thermal window minimizes the impact on the host commodity. This is a critical advantage of RF processing over any conventional (surface) heating method as disinfestation effects can be well controlled because of the high sensitivity of arthropod pests to thermal energy and the higher heat tolerance of most

By comparison with conventional heating processes, overheating the surface is very common because energy is first applied to the surface and then is conducted to its interior. Because energy loss from the surface (by radiation and/or convection) is unavoidable, significant and fast, these processes often require additional heat input on the surface in order to produce internal temperatures high enough to achieve a uniform biological effect. The host, however, received higher and usually damaging thermal loads on its surface. As a result, the upper boundary of the thermal window (especially for the surface) is frequently exceeded causing unacceptable changes in the physiological, sensory, and quality of foods. In RF disinfestation, the surface temperature is usually lower than the internal temperature due to the heat loss from surface radiation and due to evaporation. This can be effectively prevented during RF processing by reducing evaporation (e.g. high humidity inside the chamber), by adding moisture before processing and by providing good radiation

Frequently, microwave heating is confused with RF heating. While fundamentally similar, microwave heating (also an energy source in the electromagnetic spectrum) is operated at 915 MHz (*λ* = 0.3 m) and 2,450 MHz (*λ* = 0.1 m), that is with higher frequency and shorter waves than RF. For most commercial scales (i.e. large amounts) of foods and agricultural products, microwave heating is not adequate also has many disadvantages in aspects of heating homogeneity, energy penetration, and energy-use efficiency. First, it does not

Fig. 5. Thermal Windows (colored arrows) for RF processing effects.

affected foods and agricultural commodities.

reflectors in the RF cavity design.

**3.3 RF and microwave processing** 

Methyl bromide fumigation was for many decades the preferred treatment applied to many stored food commodities. It was used worldwide to meet quarantine and phytosanitary restrictions and quality requirements as mandated by global agriculture markets. Current alternative methods used to control insects in grains include the use of insecticides (e.g. Malathion), fumigants (e.g. phosphine, carbon dioxide) and temperature treatment (Bond, 2007).

Malathion (American Cyanamid Co., USA) is one of the safest organophosphate insecticides. Nevertheless, existing regulations demands that the treated grains should not be sold for at least 7 days nor should be eaten within 60 days after treatment to avoid potential toxic effects from residues left.

Phosphine gas is very toxic to human therefore its application requires strict controls, even though there is no residue left to the treated grains.

Other pesticides in use include Chloropicrin, 1,3-dichloropropene, Telone/Vapam, sulfaryl fluoride and hydrogen cyanide.

However, all pesticides available and those mentioned in particular are of global concern due to the potential for causing detrimental effects on animals, air, water and soil as well as potentially impacting public health and workers safety.

Conventional carbon dioxide fumigation of grains usually referred as modified atmospheres requires a lengthy treatment (i.e. days to weeks) therefore its cost is high as well as its impact on the logistics of product distribution to markets.

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 249

macromolecules and denature their normal biological order and function. The most essential thermal damage that leads to cell death is the denaturation of enzymes, especially some critical enzymes responsible for DNA and RNA replications in the cell (Roti Roti, 1982). Thermal energy or heat can cause non-repairable denaturation of DNA, RNA, and sometimes create structural DNA lesions (sections of DNA contain elementary damage

Heat also transfers its energy to make molecules more energetic which leads to weaker hydrogen bonds and hydrophobic interactions sustaining the cell membrane, and eventually causes its disruption or collapse. The disruption of cell membrane leads to uncontrollable material exchange between the cell and its environment, which causes the cell to lose its optimum microenvironment required for its metabolisms and the cell dies eventually (Bowler & Fuller, 1987). Heat can also destroy storage materials in cells such as lipids, fats,

Thermal energy from RF power can increase insect body temperature high enough to be lethal and destroy them (disinfestation) by causing cellular damages (i.e. cell death or dysfunctional) or body dehydration. The thermal death due to cellular damages of this multi-cellular organism is not usually the consequence of massive cell death per unit time, but it may due to the loss or disruption of cells in a certain critical tissues (Denlinger &

Differences in species and developmental stages are also likely to influence the site of lethal thermal wounding. The more complex the biological system, the more susceptible it is to high thermal stress. Therefore, it is expected that macromolecules (e.g. proteins, DNA, RNA, lipid, fat, etc.) are more resistant to thermal stress than cellular organelles (e.g. mitochondria, nucleus, Golgi complex, etc.), cellular organelles are more resistant than cells, cells are more resistant than tissues, and tissues are more resistant than the whole organism (Ushakov, 1964; Prosser, 1986). Hence for a multi-cellular organism, lethal wounding may be inflicted from cellular damages of an organization with a high level of complexity. The above concepts explains the prevalence of the concept "living dead" in the insect control, which means organisms are still alive but will not survive and reproduce due to cellular thermal injuries (Bowler, 1963; Chen, Lee & Denlinger, 1990). Therefore, as insect's biology is more complex than unicellular organisms (e.g. bacteria, fungi), they are

High temperature can also be lethal to insects by causing dehydration and promoting desiccation. Above a certain temperature, the critical transition temperature, the rates that insects lose water from their bodies increase dramatically (Yoder & Denlinger, 1991). Critical transition temperature values commonly range from 30 to 60°C for different species and developmental stages (Hadley, 1994). Most insects contain about 60 to 70% water in their body weights, and many can tolerate a loss of 20 to 30% of water for brief periods. The loss of water will increase the osmotic stress and concurrently increase the solute concentration within the body, presumably leading to irreversible cell damages. This also increases RFinduced ionic conduction effects in insects thus enhancing thermal energy production and

The main mechanism of disinfestation in RF selective heating is also thermal stress (i.e. heat). In the selective RF heating, a proper operating frequency is selected so that the effective dielectric loss factor (Ɛ") of the target material is close to its maximum value and

sites) that cause the loss of cellular genetic information (Ward, 1985).

and carbohydrates by oxidation.

expectedly more susceptible to thermal stress.

thermal stress favoring lethality.

**3.5.2 RF selective heating of insects** 

Yocum, 1998).

#### **3.4.2 Conventional heat processing**

Conventional high-temperature treatments of grains, such as hot air or hot water immersion and dry or wet steam are usually less effective to internally hidden eggs or pupae inside grain kernels. As adequate lethal temperature for insect pests need to be applied throughout the volume of the commodity, surface overheating and diminishing quality attributes usually occurs due to slow dynamics of heat transport from the outside to the core of grain kernels. Overheating also leads to the deterioration of grain quality and viability.

Because of the above, there is a clear need to develop and establish better, less or noninvasive alternatives to disinfect grains and other commodities to overcome safety concerns associated to invasive methods (leaving residues). Highly desirable is the long-stated need to reduce risks to consumers, workers and the environment as indicated by international organizations (UNEP 1998; WMO 2003).

#### **3.5 Mechanisms of RF Disinfestation**

As RF disinfestation is to initiate energy-transfer mechanisms at the molecular level, there are two possible mechanisms for the inactivation/control of insect and mites using RF power: thermal and non-thermal effects. The thermal effect of RF power is essential to the destruction of microorganisms and many studies have proven its validity (Goldblith & Wang, 1967; Fujikawa et al., 1992; Kozempel, Annous, Cook et al., 1998). The energy absorption from RF power can raise the temperature of contaminant organisms high and fast enough to induce irreversible (i.e. non repairable) biochemical damage to cells such as the denaturation of enzymes, proteins, DNA, RNA, or of other vital cellular components, as well as disruption of cell membranes (Heddleson & Doores, 1994). Reports of potential nonthermal effects (effects unrelated to heat stress) with higher-frequency dielectric heating (basically at microwave frequencies) are still controversial. While some researchers have announced these effects (Burton, 1949; Olsen, 1965; Fung & Cunningham, 1980; Cross & Fung, 1982), other researches have concluded there is little or no non-thermal effect on cells (Goldblith & Wang, 1967; Carroll & Lopez, 1969; Rosenberg & Bögl, 1987; Knutson *et al.*, 1987). However, using high-peak power RF technologies capable of delivering ultra-short pulses with very high instant power (>MW/pulse) remain as a potentially successful approach for disinfestation and in particular for fresh produce and other high-thermally sensitive commodities (Lagunas-Solar, Zeng & Essert, 2003).

#### **3.5.1 RF disinfestation thermal effects**

The cell is the fundamental unit of all living matter. Living cells (prokaryotes and eukaryotes) are basically composed of high-molecular-weight polymeric compounds (macromolecules) such as proteins, DNA, RNA, polysaccharides, lipids, and storage materials such as fats, glycogen, polyhydroxybutyrate, etc. (Madigan, Martinko & Parker, 2000). These macromolecules are only functional in the proper three-dimensional structures. The structural property is affected by thermal energy and is especially important for enzymes as they are very effective biological catalysts and involved in most of cellular reactions (Shuler and Kargi, 1992).

Because RF power generates heat at the molecular level, RF energy can effectively increase the kinetic energy of molecules and make these molecules vibrate more rapidly and violently. These molecular vibrations, up to a point, are strong enough to disrupt weak intermolecular forces, such as hydrogen bonds, salt bridges, disulfide bonds, and non-polar hydrophobic interactions in secondary, tertiary and quaternary structures of macromolecules and denature their normal biological order and function. The most essential thermal damage that leads to cell death is the denaturation of enzymes, especially some critical enzymes responsible for DNA and RNA replications in the cell (Roti Roti, 1982). Thermal energy or heat can cause non-repairable denaturation of DNA, RNA, and sometimes create structural DNA lesions (sections of DNA contain elementary damage sites) that cause the loss of cellular genetic information (Ward, 1985).

Heat also transfers its energy to make molecules more energetic which leads to weaker hydrogen bonds and hydrophobic interactions sustaining the cell membrane, and eventually causes its disruption or collapse. The disruption of cell membrane leads to uncontrollable material exchange between the cell and its environment, which causes the cell to lose its optimum microenvironment required for its metabolisms and the cell dies eventually (Bowler & Fuller, 1987). Heat can also destroy storage materials in cells such as lipids, fats, and carbohydrates by oxidation.

Thermal energy from RF power can increase insect body temperature high enough to be lethal and destroy them (disinfestation) by causing cellular damages (i.e. cell death or dysfunctional) or body dehydration. The thermal death due to cellular damages of this multi-cellular organism is not usually the consequence of massive cell death per unit time, but it may due to the loss or disruption of cells in a certain critical tissues (Denlinger & Yocum, 1998).

Differences in species and developmental stages are also likely to influence the site of lethal thermal wounding. The more complex the biological system, the more susceptible it is to high thermal stress. Therefore, it is expected that macromolecules (e.g. proteins, DNA, RNA, lipid, fat, etc.) are more resistant to thermal stress than cellular organelles (e.g. mitochondria, nucleus, Golgi complex, etc.), cellular organelles are more resistant than cells, cells are more resistant than tissues, and tissues are more resistant than the whole organism (Ushakov, 1964; Prosser, 1986). Hence for a multi-cellular organism, lethal wounding may be inflicted from cellular damages of an organization with a high level of complexity.

The above concepts explains the prevalence of the concept "living dead" in the insect control, which means organisms are still alive but will not survive and reproduce due to cellular thermal injuries (Bowler, 1963; Chen, Lee & Denlinger, 1990). Therefore, as insect's biology is more complex than unicellular organisms (e.g. bacteria, fungi), they are expectedly more susceptible to thermal stress.

High temperature can also be lethal to insects by causing dehydration and promoting desiccation. Above a certain temperature, the critical transition temperature, the rates that insects lose water from their bodies increase dramatically (Yoder & Denlinger, 1991). Critical transition temperature values commonly range from 30 to 60°C for different species and developmental stages (Hadley, 1994). Most insects contain about 60 to 70% water in their body weights, and many can tolerate a loss of 20 to 30% of water for brief periods. The loss of water will increase the osmotic stress and concurrently increase the solute concentration within the body, presumably leading to irreversible cell damages. This also increases RFinduced ionic conduction effects in insects thus enhancing thermal energy production and thermal stress favoring lethality.

#### **3.5.2 RF selective heating of insects**

248 Insecticides – Basic and Other Applications

Conventional high-temperature treatments of grains, such as hot air or hot water immersion and dry or wet steam are usually less effective to internally hidden eggs or pupae inside grain kernels. As adequate lethal temperature for insect pests need to be applied throughout the volume of the commodity, surface overheating and diminishing quality attributes usually occurs due to slow dynamics of heat transport from the outside to the core of grain

Because of the above, there is a clear need to develop and establish better, less or noninvasive alternatives to disinfect grains and other commodities to overcome safety concerns associated to invasive methods (leaving residues). Highly desirable is the long-stated need to reduce risks to consumers, workers and the environment as indicated by international

As RF disinfestation is to initiate energy-transfer mechanisms at the molecular level, there are two possible mechanisms for the inactivation/control of insect and mites using RF power: thermal and non-thermal effects. The thermal effect of RF power is essential to the destruction of microorganisms and many studies have proven its validity (Goldblith & Wang, 1967; Fujikawa et al., 1992; Kozempel, Annous, Cook et al., 1998). The energy absorption from RF power can raise the temperature of contaminant organisms high and fast enough to induce irreversible (i.e. non repairable) biochemical damage to cells such as the denaturation of enzymes, proteins, DNA, RNA, or of other vital cellular components, as well as disruption of cell membranes (Heddleson & Doores, 1994). Reports of potential nonthermal effects (effects unrelated to heat stress) with higher-frequency dielectric heating (basically at microwave frequencies) are still controversial. While some researchers have announced these effects (Burton, 1949; Olsen, 1965; Fung & Cunningham, 1980; Cross & Fung, 1982), other researches have concluded there is little or no non-thermal effect on cells (Goldblith & Wang, 1967; Carroll & Lopez, 1969; Rosenberg & Bögl, 1987; Knutson *et al.*, 1987). However, using high-peak power RF technologies capable of delivering ultra-short pulses with very high instant power (>MW/pulse) remain as a potentially successful approach for disinfestation and in particular for fresh produce and other high-thermally

The cell is the fundamental unit of all living matter. Living cells (prokaryotes and eukaryotes) are basically composed of high-molecular-weight polymeric compounds (macromolecules) such as proteins, DNA, RNA, polysaccharides, lipids, and storage materials such as fats, glycogen, polyhydroxybutyrate, etc. (Madigan, Martinko & Parker, 2000). These macromolecules are only functional in the proper three-dimensional structures. The structural property is affected by thermal energy and is especially important for enzymes as they are very effective biological catalysts and involved in most of cellular

Because RF power generates heat at the molecular level, RF energy can effectively increase the kinetic energy of molecules and make these molecules vibrate more rapidly and violently. These molecular vibrations, up to a point, are strong enough to disrupt weak intermolecular forces, such as hydrogen bonds, salt bridges, disulfide bonds, and non-polar hydrophobic interactions in secondary, tertiary and quaternary structures of

kernels. Overheating also leads to the deterioration of grain quality and viability.

**3.4.2 Conventional heat processing** 

organizations (UNEP 1998; WMO 2003).

**3.5 Mechanisms of RF Disinfestation** 

sensitive commodities (Lagunas-Solar, Zeng & Essert, 2003).

**3.5.1 RF disinfestation thermal effects** 

reactions (Shuler and Kargi, 1992).

The main mechanism of disinfestation in RF selective heating is also thermal stress (i.e. heat). In the selective RF heating, a proper operating frequency is selected so that the effective dielectric loss factor (Ɛ") of the target material is close to its maximum value and

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 251

these pests can absorb a larger proportion of the available RF energy delivered. By comparison, the host commodity is expected to have complex dielectric constants in the range of 3 to 6 for low-moisture foods (nuts, seeds, grains) or up to 60 to 70 for highmoisture foods (i.e. fruits) although considerable higher dielectric loss factors (>200) for insects have been reported under the same processing conditions (frequency) (Ikediala et al., 2000). The difference in dielectric properties between insects and host generates lower

As arthropods (arachnids as well) have similar chemical composition, selective heating effects have been demonstrated with ants, aphids, beetles, borers, bugs, fruit flies, moths, thrips, mites and arachnids confirming the validity of the selective heating process in

Therefore, disinfestation appears as an effective RF application that can heat arthropod pests rapidly (45 to 65oC; 3-4 min) inducing lethal conditions that are well tolerated by a large variety of foods. As proven in various laboratory-scale experimentations, this approach is being developed for commercial-scale applications with RF systems designed and

A full control of all life cycles of Angoumois grain moths (*Sitotroga cerealella* [Oliver]) and lesser grain borers *Rhyzopertha dominica* [F.]), in laboratory-scale experimentation with rough

Samples of rough rice (13.5% and 11.0% moisture) were obtained from Pacific International Rice Mills Inc., (Woodland, CA) and were infested in separate batches (~ 10 kg each) with adult grain moth *Sitotroga cerealella* (13.5% batch) and with both *Sitotroga cerealella* and adult

After approximately one month at 28-30oC (35-40% relative humidity) both colonies were well established showing abundant populations of all biological stages. RF disinfestation was conducted at the University of California, Davis using several processing conditions with 500 W of RF power at 20.3 MHz (Lagunas-Solar et al., 2008). Samples were treated at the same temperature (60oC) but with different times (5 and 30 min; 1 and 2 h) so as to vary thermal loads (temperature x time) delivered. Effectiveness of the RF disinfestation process was determined by assaying the emergence of adult insects found over ~40 days of periodic observations. However, as no adult insects survived any of the initial treatments, adult emergence was assumed to be due to the presence of surviving eggs, larva and/or pupas. Results from replicates in triplicate (control and treated) are shown in Figures 6 and 7,

The response of grain moth *Sitotroga cerealella* and lesser-grain borer *Rhyzopertha dominica* to the same RF processing conditions were different indicating that other parameters need to

*As expected, Sitotroga cerealella* was found to be more sensitive to the RF disinfestation process as these insects are normally on the outside surface of the grain. While disinfestation effects were observed at all conditions (Figure 6), some adult emergence (~ 16%) was observed in the 60oC/5 min samples after ~40-day incubation and observation period. This was attributed to the partial survival of eggs at different eclosion stages prompting a delayed emergence of adult insects. In all other treatments (60oC/30min; 60oC/1h; 60oC/2h) the thermal loads were sufficiently high to cause a full control of all stages of *Sitotroga* 

different food hosts as well as soil and wood products (unpublished results).

**4.1 Experimental results of RF disinfestation of rough (paddy) rice** 

(paddy) rice as host was reported (Lagunas-Solar et al., 2008).

lesser-grain borer *Rhyzopertha dominica* (11.0% batch).

be considered in establishing an optimized process.

*cerealella*, as no adult emerged in the treated samples.

thermal effects on the commodity (Kunze, 1979).

engineered for full optimization.

below.

the load (material) can be heated fast. Because different materials have different dielectric properties (i.e. dielectric constant [Ɛ'] and effective dielectric loss factor [Ɛ"]) - both of which depend on the composition and frequency, they interact and convert RF energy into heat at different rates at the same frequency.

This leads to the potential that different materials in the same load can have different heating rates, depending on the values of their effective loss factor (Ɛ") at that frequency. If an appropriate frequency is chosen so that contaminant organisms (e.g. arthropods, arachnids) can absorb RF energy faster than host material, those organisms can be heated much faster than other components in the same load (Lagunas-Solar *et al.*, 2006; Lagunas-Solar et al., 2008). As a result, insects/mites are destroyed by heat while the host commodity is unaffected. This treatment is proposed for somewhat thermally resistance fresh products (i.e. tomatoes, avocados, apples, grapes, and broccoli) which can tolerate some low thermal loads but sufficiently high to be effective for disinfestation applications using a controlled- thermal RF treatment.

While theoretically applicable to selective RF heating of microorganisms, their small size prevents adequate absorption of the penetrating RF energy waves and thus there is no evidence today for the availability of this selective mechanism for microorganisms.

Finally, the above and other technological and consumer factors prompted the investigation on the use of RF power for disinfestation of various commodities by several authors (Ikediala et al., 2000; Wang et al., 2003; Mitcham et al., 2004; Wang et al., 2007a; Wang et al., 2007b). Results and conclusions of all these studies corroborated the advantages of RF disinfestation over available techniques and also helped identify remaining challenges (Prakash & Rao, 2002).

#### **4. Case study: RF disinfestation of rough (paddy) rice**

During long-term storage, insects can cause considerable damage to grains (and to other products, i.e. nuts), with weight and nutritional losses reducing yields and quality which reduces market values. Furthermore, deterioration of grains intended for seedling purposes may cause further losses in quality and viability (germination) thus affecting future yields in crop production.

Under current storage (bulk) conditions over long periods of time, the presence of even a few viable colonies of insect pests may result in the emergence of much larger populations as the storage conditions are favorable to insect reproduction and propagation due to the abundant presence of nutrients and lack of antagonistic organisms. In rough (paddy) rice, two major insects Angoumois grain moths (*Sitotroga cerealella* [Oliver]) and lesser grain borers (*Rhyzopertha dominica* [F.]) represent major threats as primary grain insects whose larvae feed entirely inside the kernel of the grain and eat from inside becoming more tolerant to fumigation as diffusion of gas into kernels is severely restricted or blocked by the presence of air locks (pockets). Therefore, infestation with primary insects are critically more damaging to stored grains than secondary insects that eat grains from outside and are more easily controlled with conventional fumigation or heat treatments.

As explained above (see section 3.5.2), selective heating of arthropod pests is feasible via a differential heating mechanism based upon the higher ionic conductivity in pests (see Table 1). Therefore, all biological stages of arthropod pests do heat faster than the host commodity leading to their effective biological inactivation (Wang et al., 2003). As shown in table 1 (above), insects such as codling moths and Mexican fruit flies have large dielectric constants (Ɛ\* 71.5-84.5 and 90 to 141; respectively at 27 MHz). Therefore, when treated with RF power these pests can absorb a larger proportion of the available RF energy delivered. By comparison, the host commodity is expected to have complex dielectric constants in the range of 3 to 6 for low-moisture foods (nuts, seeds, grains) or up to 60 to 70 for highmoisture foods (i.e. fruits) although considerable higher dielectric loss factors (>200) for insects have been reported under the same processing conditions (frequency) (Ikediala et al., 2000). The difference in dielectric properties between insects and host generates lower thermal effects on the commodity (Kunze, 1979).

As arthropods (arachnids as well) have similar chemical composition, selective heating effects have been demonstrated with ants, aphids, beetles, borers, bugs, fruit flies, moths, thrips, mites and arachnids confirming the validity of the selective heating process in different food hosts as well as soil and wood products (unpublished results).

Therefore, disinfestation appears as an effective RF application that can heat arthropod pests rapidly (45 to 65oC; 3-4 min) inducing lethal conditions that are well tolerated by a large variety of foods. As proven in various laboratory-scale experimentations, this approach is being developed for commercial-scale applications with RF systems designed and engineered for full optimization.

#### **4.1 Experimental results of RF disinfestation of rough (paddy) rice**

250 Insecticides – Basic and Other Applications

the load (material) can be heated fast. Because different materials have different dielectric properties (i.e. dielectric constant [Ɛ'] and effective dielectric loss factor [Ɛ"]) - both of which depend on the composition and frequency, they interact and convert RF energy into heat at

This leads to the potential that different materials in the same load can have different heating rates, depending on the values of their effective loss factor (Ɛ") at that frequency. If an appropriate frequency is chosen so that contaminant organisms (e.g. arthropods, arachnids) can absorb RF energy faster than host material, those organisms can be heated much faster than other components in the same load (Lagunas-Solar *et al.*, 2006; Lagunas-Solar et al., 2008). As a result, insects/mites are destroyed by heat while the host commodity is unaffected. This treatment is proposed for somewhat thermally resistance fresh products (i.e. tomatoes, avocados, apples, grapes, and broccoli) which can tolerate some low thermal loads but sufficiently high to be effective for disinfestation applications

While theoretically applicable to selective RF heating of microorganisms, their small size prevents adequate absorption of the penetrating RF energy waves and thus there is no

Finally, the above and other technological and consumer factors prompted the investigation on the use of RF power for disinfestation of various commodities by several authors (Ikediala et al., 2000; Wang et al., 2003; Mitcham et al., 2004; Wang et al., 2007a; Wang et al., 2007b). Results and conclusions of all these studies corroborated the advantages of RF disinfestation over available techniques and also helped identify remaining challenges

During long-term storage, insects can cause considerable damage to grains (and to other products, i.e. nuts), with weight and nutritional losses reducing yields and quality which reduces market values. Furthermore, deterioration of grains intended for seedling purposes may cause further losses in quality and viability (germination) thus affecting future yields in

Under current storage (bulk) conditions over long periods of time, the presence of even a few viable colonies of insect pests may result in the emergence of much larger populations as the storage conditions are favorable to insect reproduction and propagation due to the abundant presence of nutrients and lack of antagonistic organisms. In rough (paddy) rice, two major insects Angoumois grain moths (*Sitotroga cerealella* [Oliver]) and lesser grain borers (*Rhyzopertha dominica* [F.]) represent major threats as primary grain insects whose larvae feed entirely inside the kernel of the grain and eat from inside becoming more tolerant to fumigation as diffusion of gas into kernels is severely restricted or blocked by the presence of air locks (pockets). Therefore, infestation with primary insects are critically more damaging to stored grains than secondary insects that eat grains from outside and are more

As explained above (see section 3.5.2), selective heating of arthropod pests is feasible via a differential heating mechanism based upon the higher ionic conductivity in pests (see Table 1). Therefore, all biological stages of arthropod pests do heat faster than the host commodity leading to their effective biological inactivation (Wang et al., 2003). As shown in table 1 (above), insects such as codling moths and Mexican fruit flies have large dielectric constants (Ɛ\* 71.5-84.5 and 90 to 141; respectively at 27 MHz). Therefore, when treated with RF power

evidence today for the availability of this selective mechanism for microorganisms.

**4. Case study: RF disinfestation of rough (paddy) rice** 

easily controlled with conventional fumigation or heat treatments.

different rates at the same frequency.

using a controlled- thermal RF treatment.

(Prakash & Rao, 2002).

crop production.

A full control of all life cycles of Angoumois grain moths (*Sitotroga cerealella* [Oliver]) and lesser grain borers *Rhyzopertha dominica* [F.]), in laboratory-scale experimentation with rough (paddy) rice as host was reported (Lagunas-Solar et al., 2008).

Samples of rough rice (13.5% and 11.0% moisture) were obtained from Pacific International Rice Mills Inc., (Woodland, CA) and were infested in separate batches (~ 10 kg each) with adult grain moth *Sitotroga cerealella* (13.5% batch) and with both *Sitotroga cerealella* and adult lesser-grain borer *Rhyzopertha dominica* (11.0% batch).

After approximately one month at 28-30oC (35-40% relative humidity) both colonies were well established showing abundant populations of all biological stages. RF disinfestation was conducted at the University of California, Davis using several processing conditions with 500 W of RF power at 20.3 MHz (Lagunas-Solar et al., 2008). Samples were treated at the same temperature (60oC) but with different times (5 and 30 min; 1 and 2 h) so as to vary thermal loads (temperature x time) delivered. Effectiveness of the RF disinfestation process was determined by assaying the emergence of adult insects found over ~40 days of periodic observations. However, as no adult insects survived any of the initial treatments, adult emergence was assumed to be due to the presence of surviving eggs, larva and/or pupas. Results from replicates in triplicate (control and treated) are shown in Figures 6 and 7, below.

The response of grain moth *Sitotroga cerealella* and lesser-grain borer *Rhyzopertha dominica* to the same RF processing conditions were different indicating that other parameters need to be considered in establishing an optimized process.

*As expected, Sitotroga cerealella* was found to be more sensitive to the RF disinfestation process as these insects are normally on the outside surface of the grain. While disinfestation effects were observed at all conditions (Figure 6), some adult emergence (~ 16%) was observed in the 60oC/5 min samples after ~40-day incubation and observation period. This was attributed to the partial survival of eggs at different eclosion stages prompting a delayed emergence of adult insects. In all other treatments (60oC/30min; 60oC/1h; 60oC/2h) the thermal loads were sufficiently high to cause a full control of all stages of *Sitotroga cerealella*, as no adult emerged in the treated samples.

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 253

As compared with controls (1330 adults/40 days); in the 60oC/5min batch 490 adults/40 days were observed for ~37% emergence (~63% control). As thermal load was increased, the 60oC/30 min batch showed only 190 adults/40 days (~14% emergence; 86% control). With either 60oC/1h and 60oC/2h processes, a 100% control were observed as no adults emerged during the 40-day observation period. It was concluded that the ability of *Rhyzopertha dominica* to bore into grains and deposit eggs from which larva emerged, provided

As stated earlier, RF disinfestation is applicable under similar conditions to all arthropod pests as the interactive mechanisms utilized operate with similar molecules present in all arthropods and thus is independent from the biological speciation, developmental stages, or behavioral patterns. Optimization of the RF disinfestation process is also straightforward as thermal loads required for full control (i.e. 60oC/1h for both insects) can be achieved rapidly by increasing RF power. As only 500 W were used in previous experimentation, an operation at 10 kW would only require a 5-min processing time. Other commercial-scale conditions with increased RF power (i.e. 25-50 kW) are also possible and available (www.rfbiocidics.com) for processing larger throughputs (> 2-4 tons/h) while taking full

The application of RF power in disinfestation applications should also consider the potential effects of the applied thermal load to the host commodity. Therefore, the potential for changes of quality attributes in RF treated rough (paddy) rice was also studied and the results are summarized in Table 2, below. These measurements were conducted using

**Rough (paddy) rice samples** 

Commercial application of RF disinfestation is already taking place on various commodities and is combined with simultaneous disinfection (pasteurization) and enzyme deactivation effects.2 This combination of desirable and simultaneous sanitation effects is unique to RF processing and is only dependent on the applied thermal loads (see Figure 5, above). In

**(%) Controls Batch 1 (50oC) Batch 2 (60oC) Batch 3 (70oC)**  Moisture 13.5 0.1 13.5 0.1 13.5 0.1 13.5 0.1 Whole kernel 79.3 1.1 81.1 7.9 78.3 0.1 77.9 0.8 Total rice 68.1 0.3 68.3 0.1 68.2 0.1 68.0 0.1 Dockage 16.9 4.8 11.7 1.0 12.4 1.6 13.2 1.7 Brown rice 81.1 0.4 81.1 0.4 81.3 0.2 81.3 0.1 Whiteness 44.2 0.2 44.1 0.2 44.2 0.2 44.3 0.3 \* Mean values and standard deviations for triplicate measurements with 1-kg samples. Data courtesy of California Rice Association.

additional barriers and protection due to the internalized condition of the pest.

advantage of this emerging chemical-free alternative.

standard commercial laboratory tests and indicated no adverse effects.

Table 2. Quality attributes of RF disinfested rough (paddy) rice\*

2 RF Biocidics Inc., Vacaville, CA 95688 USA (www.rfbiocidics.com)

**4.2 Quality attributes of host commodity** 

**Quality attributes** 

**5. RF process economics** 

*Rhyzopertha dominica* showed higher tolerance under the same processing conditions.

Fig. 6. RF disinfestation of *Sitotroga cereallela* grain moths in rough (paddy) rice.

Fig. 7. RF disinfestation of *Rhyzopertha dominica* (lesser grain borers) in rough (paddy) rice.

As compared with controls (1330 adults/40 days); in the 60oC/5min batch 490 adults/40 days were observed for ~37% emergence (~63% control). As thermal load was increased, the 60oC/30 min batch showed only 190 adults/40 days (~14% emergence; 86% control). With either 60oC/1h and 60oC/2h processes, a 100% control were observed as no adults emerged during the 40-day observation period. It was concluded that the ability of *Rhyzopertha dominica* to bore into grains and deposit eggs from which larva emerged, provided additional barriers and protection due to the internalized condition of the pest.

As stated earlier, RF disinfestation is applicable under similar conditions to all arthropod pests as the interactive mechanisms utilized operate with similar molecules present in all arthropods and thus is independent from the biological speciation, developmental stages, or behavioral patterns. Optimization of the RF disinfestation process is also straightforward as thermal loads required for full control (i.e. 60oC/1h for both insects) can be achieved rapidly by increasing RF power. As only 500 W were used in previous experimentation, an operation at 10 kW would only require a 5-min processing time. Other commercial-scale conditions with increased RF power (i.e. 25-50 kW) are also possible and available (www.rfbiocidics.com) for processing larger throughputs (> 2-4 tons/h) while taking full advantage of this emerging chemical-free alternative.

#### **4.2 Quality attributes of host commodity**

252 Insecticides – Basic and Other Applications

*Rhyzopertha dominica* showed higher tolerance under the same processing conditions.

Fig. 6. RF disinfestation of *Sitotroga cereallela* grain moths in rough (paddy) rice.

Fig. 7. RF disinfestation of *Rhyzopertha dominica* (lesser grain borers) in rough (paddy) rice.

The application of RF power in disinfestation applications should also consider the potential effects of the applied thermal load to the host commodity. Therefore, the potential for changes of quality attributes in RF treated rough (paddy) rice was also studied and the results are summarized in Table 2, below. These measurements were conducted using standard commercial laboratory tests and indicated no adverse effects.


Table 2. Quality attributes of RF disinfested rough (paddy) rice\*

#### **5. RF process economics**

Commercial application of RF disinfestation is already taking place on various commodities and is combined with simultaneous disinfection (pasteurization) and enzyme deactivation effects.2 This combination of desirable and simultaneous sanitation effects is unique to RF processing and is only dependent on the applied thermal loads (see Figure 5, above). In

<sup>2</sup> RF Biocidics Inc., Vacaville, CA 95688 USA (www.rfbiocidics.com)

Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 255

Finally, because of its nature, RF disinfestation can be applied to conventionally- and

The author wishes to acknowledge the scientific and technical assistance from Mr. Timothy Essert, Mrs. Cecilia Piña U., Drs. Nolan Zeng and Tin Truong from the University of California, Davis. The author also wishes to acknowledge the research support, encouragement and vision of Dr. Robert G. Flocchini, former director of Crocker Nuclear Laboratory, University of California, Davis. Sponsorship from Allied Minds Inc. Boston, MA, Ishida Ltd. Kyoto, Japan, Hortifrut S.A., Santiago, Chile, during the various stages of

Bond, E.J. 2007. Manual for fumigation for insect control. FAO Plant Production and

Bowler, K. 1963. A Study of the Factors Involved in Acclimatization to Temperature and

Calderon, M. 1990. Food Preservation by Modified Atmospheres. CRC Press

Carroll, D.E. and Lopez, A. 1969. Lethality of Radio Frequency Energy Upon

Chen, C.P., Lee, R.E., and Denlinger, D.L. 1990. A Comparison of the Response of Tropical

Clarke, R.N. 2006. Dielectric Properties of Materials. Kaye & Laby: Tables of Physical &

Cross, G.A. and Fung, D.Y.C. 1982. The Effect of Microwaves on Nutrient Value of Foods.

Denlinger, D.L. and Yocum, G.D. 1998. Physiology of Heat Sensitivity (Editors: G.J.

Giles, P.G., Moore, E.E., and Bounds, L. 1970. Investigation of Heat Penetration of Food Sample at Various Frequencies. Journal of Microwave Power, 5(1): 40-48.

http://www.kayelaby.npl.co.uk/general\_physics/2\_6/2\_6\_5.html

Critical Reviews in Food Science and Nutrition, 16: 355-381.

Foods. Journal of Food Protection, 43(8): 641-650.

Hallman and D.L. Denlinger). Westview Press, Colorado, USA. Fröhlich, H. 1958. Theory of Dielectrics, 2nd Edition. Oxford University Press. London. Fujikawa, H., Ushioda, H., and Kudo, Y. 1992. Kinetics of *Escherichia coli* Destruction by Microwave Irradiation. Applied and Environmental Microbiology, 58(3): 920-924. Fung, D.Y.C and Cunningham, E.E. 1980. Effect of Microwaves on Microorganisms in

Bowler, K. and Fuller, B.J. 1987. Temperature and Animal Cells. Cambridge, England. Burton, H. 1949. A Survey of Literature on Bacterial Effects of Short Electromagnetic

Death at High Temperatures in *Astacus pallipes*; II. Experiments at the Tissue Level.

Waves. Shinfield, England. National Institute for Research in Dairying Shinfield.

Microorganisms in Liquid, Buffered, and Alcoholic Food System. Journal of Food

and Temperate Flies (Diptera: Sarcohagidae) to Cold and Heat Stress. Journal of Comparative Physiology B: Biochemical, Systematic, and Environmental

Chemical Constants. National Physical Laboratory, © Crown Copy Right 2006.

Protection, Paper 54. FAO. www.fao.org/docrep/x5042E00.htm

Journal of Cellular and Comparative Physiology, 62: 133-146.

(www.crcpress.com/product/isbn/97808493365690)

organically-produced food commodities.

this research is also greatly appreciated.

N.I.R.D. Paper No, 1041.

Science, 34(4): 320-324.

Physiology, 160 (5): 543-547.

**7. Acknowledgements** 

**8. References** 

addition, and because of its penetration, RF power is capable of processing commodities in its package (boxes, bags) thus avoiding recontamination and facilitating logistics of operation. Therefore, due to its chemical-free nature and to the combined effects, RF processing offers many advantages over single-effect technologies including those based upon applications of conventional surface-heat sources (i.e. dry or wet heat, vapor, steam, etc.). Despite its unique advantages and multiple controlling effects, the current commercial application of RF disinfestation is priced competitively in comparison with the cost of using chemical pesticides or any other physical process.

As a new and emerging option for sanitation of foods and agricultural commodities, RF operating facilities are being established to operate at or near high agriculture production areas or near key distribution centers and facilities in which RF processing can be part of the overall chain of production and distribution for local, regional and overseas markets.

### **6. Conclusions**

The application of RF power to disinfestation provides a rapid and effective chemical-free alternative capable of replacing the use of chemical and biological pesticides and as alternative to other conventional heating processes during post-harvest management of various foods and agricultural commodities. As a physical, electricity-based process, its operation is based on well-known, designed and engineered systems capable of safe and large-scale applications. Disinfestation efficacy requires reaching a relatively low thermalload level as RF is a volumetric heating process with interactions and heating effects starting at the molecular level and somewhat selectively. It can be readily applied to arthropods and arachnids with equal effectiveness using thermal loads well below the threshold for impacting host's quality.

The RF process - with similar and even higher thermal loads, has been demonstrated at a commercial scale for various different commodities including nut products (no effects on free fatty acids, peroxide values), other grains (Quinoa, edible seeds (Chia, pumpkins, sunflower), spices (paprika, cumin, cardamom, nutmeg, coriander, etc.) and flours (brown rice, oat, wheat, flaxseed). Therefore, RF disinfestation is an emerging process with broad applications to many potentially infested commodities and can even be extended to disinfest some heat-tolerant fruits and vegetables as the required thermal load is low and the RF disinfestation process is rapid.

Furthermore, additional energy-use savings can be realized as less RF energy would be needed to control insects (a very small load) as compared with the larger mass (load) represented by the commodity. It is postulated that this approach would result in significant operational cost reductions for RF-based disinfestation applications of a variety of foods such as grains, nut products, flours, beans, spices, and agricultural commodities such as wood products (pallets), soil and soil amendments, and tobacco.

As the needs for non-chemical (residue-free), non-thermal technologies for disinfestation (and disinfection as well) continues to be a goal in production agriculture, a new nonthermal, residue-free process named metabolic stress has recently emerged and is soon to initiate commercialization (Lagunas-Solar, Essert, Piña et al., 2006b; Lagunas-Solar & Essert, 2011). Metabolic stress, singly or in combination with RF processing, is expected to overcome some of the limitations of RF disinfestation and be able to treat commercial levels of thermally-sensitive commodities in particular fresh fruits and vegetables.3

<sup>3</sup> RF Biocidics Inc., (www.rfbiocidics.com)

Finally, because of its nature, RF disinfestation can be applied to conventionally- and organically-produced food commodities.

#### **7. Acknowledgements**

254 Insecticides – Basic and Other Applications

addition, and because of its penetration, RF power is capable of processing commodities in its package (boxes, bags) thus avoiding recontamination and facilitating logistics of operation. Therefore, due to its chemical-free nature and to the combined effects, RF processing offers many advantages over single-effect technologies including those based upon applications of conventional surface-heat sources (i.e. dry or wet heat, vapor, steam, etc.). Despite its unique advantages and multiple controlling effects, the current commercial application of RF disinfestation is priced competitively in comparison with the cost of using

As a new and emerging option for sanitation of foods and agricultural commodities, RF operating facilities are being established to operate at or near high agriculture production areas or near key distribution centers and facilities in which RF processing can be part of the

The application of RF power to disinfestation provides a rapid and effective chemical-free alternative capable of replacing the use of chemical and biological pesticides and as alternative to other conventional heating processes during post-harvest management of various foods and agricultural commodities. As a physical, electricity-based process, its operation is based on well-known, designed and engineered systems capable of safe and large-scale applications. Disinfestation efficacy requires reaching a relatively low thermalload level as RF is a volumetric heating process with interactions and heating effects starting at the molecular level and somewhat selectively. It can be readily applied to arthropods and arachnids with equal effectiveness using thermal loads well below the threshold for

The RF process - with similar and even higher thermal loads, has been demonstrated at a commercial scale for various different commodities including nut products (no effects on free fatty acids, peroxide values), other grains (Quinoa, edible seeds (Chia, pumpkins, sunflower), spices (paprika, cumin, cardamom, nutmeg, coriander, etc.) and flours (brown rice, oat, wheat, flaxseed). Therefore, RF disinfestation is an emerging process with broad applications to many potentially infested commodities and can even be extended to disinfest some heat-tolerant fruits and vegetables as the required thermal load is low and the RF

Furthermore, additional energy-use savings can be realized as less RF energy would be needed to control insects (a very small load) as compared with the larger mass (load) represented by the commodity. It is postulated that this approach would result in significant operational cost reductions for RF-based disinfestation applications of a variety of foods such as grains, nut products, flours, beans, spices, and agricultural commodities such as

As the needs for non-chemical (residue-free), non-thermal technologies for disinfestation (and disinfection as well) continues to be a goal in production agriculture, a new nonthermal, residue-free process named metabolic stress has recently emerged and is soon to initiate commercialization (Lagunas-Solar, Essert, Piña et al., 2006b; Lagunas-Solar & Essert, 2011). Metabolic stress, singly or in combination with RF processing, is expected to overcome some of the limitations of RF disinfestation and be able to treat commercial levels

wood products (pallets), soil and soil amendments, and tobacco.

of thermally-sensitive commodities in particular fresh fruits and vegetables.3

overall chain of production and distribution for local, regional and overseas markets.

chemical pesticides or any other physical process.

**6. Conclusions** 

impacting host's quality.

disinfestation process is rapid.

3 RF Biocidics Inc., (www.rfbiocidics.com)

The author wishes to acknowledge the scientific and technical assistance from Mr. Timothy Essert, Mrs. Cecilia Piña U., Drs. Nolan Zeng and Tin Truong from the University of California, Davis. The author also wishes to acknowledge the research support, encouragement and vision of Dr. Robert G. Flocchini, former director of Crocker Nuclear Laboratory, University of California, Davis. Sponsorship from Allied Minds Inc. Boston, MA, Ishida Ltd. Kyoto, Japan, Hortifrut S.A., Santiago, Chile, during the various stages of this research is also greatly appreciated.

#### **8. References**


Non-Chemical Disinfestation of Food and Agricultural Commodities with Radiofrequency Power 257

Lagunas-Solar, M.C. and Essert, T.K. (Inventors). 2011. Disinfection and Disinfestation of

Lea, S.M. and Burke, J.R. 1998. Physics: The Nature of Things. Brooks/Cole Publishing

Metaxas, R. and Meredith, R.J. 1983. Industrial Microwave Heating. Peter Peregrinus Ltd.,

Mitcham, E.J, Veltman, R.H., Feng, X., de Castro, E., Johnson, J.A., Simpson, T.L., Biasi,

Mudgett, R.E. 1986. Electrical Properties of Foods. M.A. Rao, and S.S.H. Rizvi (Eds.). Engineering Properties of Foods. Marcel Dekker, New York, 329-390. Nelson, S.O. and Charity, L.F. 1972. Frequency Dependence of Energy Absorption by

Nyfors, E. and Vainikainen, P. 1989. Industrial Microwave Sensors. Artech House,

Orefeuil, M. 1987. Electric Process Heating: Technologies/Equipment/Applications.

Pimentel, D., Lach, L., Zuniga, R., and Morrison, D. 1999. Environmental and Economic

Piyasena P., Dussault, C., Koutchma, T., Ramaswamy, H.S., and Awuah, G.B. 2003. Radio

Roti Roti, J.L. 1982. Heat-induced Cell Death and Radiosensitization: Molecular

Shuler, M.L. and Kargi, F. 1992. Bioprocess Engineering Basic Concepts. Prentice Hall, Inc.,

United Nations Environmental Program (UNEP). 1998. Montreal Protocol on Substances

USDA Federal Grain Inspection Service. 1994. Rice Inspection Handbook. Washington

Ushakov, B. 1964. Thermostability of Cells and Proteins of Poikilotherms and Its

Significance in Speciation. Physiological Reviews, 44: 518-559.

Review. Critical Reviews in Food Science and Nutrition, 43(6): 587-606. Prakash, A. and Rao, J. 2002. Integrated Management of Rice Storage Insects. The Central Rice Research Institute (CRRI), Cuttack, India. http://www.crriicar.org/ Prosser, C.L. 1986. Adaptational Biology: Molecules to Organisms. Wiley, New York, USA. Rosenberg, U. and Bögl, W. 1987. Microwave Pasteurization, Sterilization, Blanching, and

Pest Control in the Food Industry. Food Technology, 41(6): 92-99.

by Hyperthermia, Drugs and Radiation: 3-10.

Urbain W.M. 1986. Food Irradiation, Academic Press, Orlando FL.

D.C.: USDA Agricultural Marketing Service.

Englewood Cliffs, New Jersey, USA.

that Deplete the Ozone Layer.

Olsen, C.M. 1965. Microwaves Inhibit Bread Mold. Food Engineering, 37(7): 51-53.

College of Agriculture and Life Sciences, Cornell University.

Agriculture 23(5): 647-654.

Engineers, 15(6): 1099-1102.

Battelle Press, Columbus, Ohio, USA.

Company, Pacific Grove, California, USA.

Patent No. 7,975,427.

London, UK.

100.

Norwood.

Rough Rice with Full Retention of Quality Attributes. Applied Engineering in

Foods, Perishables and Other Commodities. University of California, assignee. US

W.V., Wang, S., and Tang, J. 2004. Application of Radio Frequency Treatments to Control Insects in In-Shell Walnuts. Postharvest Biology and Technology, 33: 93-

Insects and Grains. Transactions of the American Society of Agricultural

Costs Associated with Non-indigenous Species in the United States. Report.

Frequency Heating of Foods: Principles, Applications and Related Properties-A

Mechanisms. Proceedings of the Third International Symposium: Cancer Therapy


Goldblith, S.A. and Wang, D.I.C. 1967. Effect of Microwaves on *Escherichia coli* and *Bacillus* 

Hadley, N.R. 1994. Water Relations of Terrestial Arthropods. Academic Press, San Diego,

Heddleson, R.A. and Doores, S. 1994. Factors Affecting Microwave Heating of Foods and

Hill, N., Vaughan, W.E., Price, A.H., and Davies, M. 1969. Dielectric Properties and

Jeschke, P. 2004. The unique role of fluorine in the design of active ingredients for modern

Kasevich, R. S. 1998. Understand the Potential of Radiofrequency Energy. Chemical

Klauenberg, B.J. and Miklavcic, D. 2000. Radio Frequency Radiation Dosimetry and Its

Knutson, K.M., Marth, E.H., and Wagner, M.K. 1987. Microwave Heating of Food.

Kozempel, M.F., Annous, B.A., Cook, R.D., Scullen, O.J., and Whiting, R.C. 1998.

Kunze, O.R. 1979. Fissuring of The Rice After Heated Air Drying. Transactions of the

Lagunas-Solar, M.C. 2003. Development of New Physical Methods as Alternative Clean

Lagunas-Solar, M. C., Zeng, N.X., and Essert, T.K. (Inventors). 2003. Method for Inhibiting

Lagunas-Solar, M.C., Zeng, N.X., Essert, T.K., Truong, T.D., Piña, C., Cullor, J.S., Smith,

Lagunas-Solar, M.C., Cullor, J.S., Zeng, N.X., Truong, T.D., Essert, T. K., Smith, W.L., and

Lagunas-Solar, M.C., Essert, T.K., Pina U, C., Zeng, N.X., and Truong, T.D. 2006b.

Lagunas-Solar, M.C., Pan, Z., Zeng, N.X., Truong, T.D., Khir, R., and Amaratunga, K.S.P.

Radiofrequency Power. Journal of Dairy Science, 88 (11): 4120-4131. Lagunas-Solar, M.C., Zeng, N.X., Essert, T.K., Truong, T.D., and Piña, C. 2006a.

American Society of Agricultural Engineers, 22(5): 1197-1207.

Relationship to the Biological Effects of Electromagnetic Fields. Kluwer Academic

Inactivation of Microorganisms with Microwaves at Reduced Temperatures.

Technologies for Production Agriculture in the XXI Century. Agro-Ciencia 19(1):

Pathogenic and Spoilage Organisms in Products. University of California,

W.L., and Larraín, R. 2005a. Disinfection of Fishmeal with Radiofrequency Heating for Improved Quality and Energy Efficiency. Journal of the Science of

Pina, C. 2005b. Disinfection of Dairy and Animal Farm Wastewater with

Radiofrequency Power Disinfects and Disinfests Food, Soils and Wastewater.

Metabolic Stress Disinfection and Disinfestation (MSDD): A New, Non-thermal, Residue-free Process for Fresh Agricultural Products. Journal of the Science of

2008. Application of Radiofrequency Power for Non-chemical Disinfestation of

Microwave Induced Destruction of Foodborne Pathogens – A Review. Journal of

*subtilis*. Applied Microbiology, 15(6): 1371-1375.

Molecular Behavior. Von Nostrand, New York, USA.

crop production. ChemBioChem 5: 570-589.

Publishers, Dordrecht, The Netherlands.

Lebensm Wiss Technology, 20: 101-110.

Journal of Food Protection, 61(5): 582-585.

assignee. US Patent No. 6,638,475.

Food and Agriculture, 85 (13): 2273-2280.

California Agriculture, 60(4): 192-199.

Food and Agriculture, 86: 1814-1825.

Food Protection, 57 (11): 1025-1037.

Engineering Progress, 94(1): 75-81.

California, USA.

57-73.

Rough Rice with Full Retention of Quality Attributes. Applied Engineering in Agriculture 23(5): 647-654.


**13** 

*Turkey* 

**for Insecticides** 

**Zero-Inflated Regression Methods** 

*3Siirt University, Faculty of architecture and engineering,* 

*Computer Engineering Department, Siirt,* 

Abdullah Yeşilova1, M. Salih Özgökçe2 and Ylmaz Kaya3

*1Yuzuncu Yil University, Faculty of Agriculture, Biometry & Genetic Unit, Van, 2Yuzuncu Yil University, Faculty of Agriculture, Plant Protection Department, Van* 

The numerical abundance of many species sharing the same ecosystem very different levels of the organism and are in constant change, depending on many factors. Due to the heterogeneous strucspeciese of the life cycles of organisms and abiotic resources in the environment based on census population densities derived from overdispersion (variance is higher than means in Poisson distribution) (Cox, 1983; Cameron and Trivedi, 1998) and a large number of zero values (zero-inflated data) is observed (Yeşilova et al, 2011). In such a case, zero-inated Poisson (ZIP) regression model is a appropriate approach for analyzing a dependent variable having excess zero observations (Lambert, 1992; Böhning, 1998; Böhning et al, 1999; Yau and Lee, 2001; Lee et al, 2001; Khoshgoftaar et al, 2005; Yeşilova et al, 2010). Zero-inflation is also likely in data sets, excess zero observations. In such cases, a zeroinated negative binominal (ZINB) regression model is an alternative method (Ridout et al, 2001; Yau, 2001; Cheung, 2002; Jansakul, 2005; Long and Frese, 2006; Hilbe, 2007; Yeşilova et al, 2009; Yeşilova et al, 2010). Morever, The Poisson hurdle model and negative binomial hurdle model (Rose and Martin, 2006; Long and Frese, 2006; Hilbe, 2007; Yeşilova et al, 2009; Yeşilova et al, 2010), and zero-inflated generalized Poisson (ZIGP) model (Consul, 1989, Consul and Famoye, 1992; Czado et al., 2007) are widely used in the analysis of zero-inflated

In this part, the analysis of data with many zeros for *Notonecta viridis* Delcourt (Heteroptera: Notonectidae) and Chironomidae species (Diptera) were carried out by means of using the models of Poisson Regression (PR), negative binomial (NB) regression, zero-inflated Poisson (ZIP) regression, zero-inflated negative binomial (ZINB) regression and negative binomial

The study was based on periodical samplings of the coastal band of Van Lake, conducted between July-September 2005 and May-September 2006. Samples were taken at totally twenty sampling points as streams entrance (6 points), settlement coastlines (7 points) and naspeciesal coastlines (7 points). Samples were taken according to Hansen et al. (2000). The

**1. Introduction** 

data.

hurdle (NBH) model.

**Samplings** 


### **Zero-Inflated Regression Methods for Insecticides**

Abdullah Yeşilova1, M. Salih Özgökçe2 and Ylmaz Kaya3 *1Yuzuncu Yil University, Faculty of Agriculture, Biometry & Genetic Unit, Van, 2Yuzuncu Yil University, Faculty of Agriculture, Plant Protection Department, Van 3Siirt University, Faculty of architecture and engineering, Computer Engineering Department, Siirt, Turkey* 

#### **1. Introduction**

258 Insecticides – Basic and Other Applications

Wang S., Tang J., Johnson J.A., Mitcham E., Hansen J.D., Hallman G., Drake S.R., and Wang

Wang S., Monzon M., Johnson J.A., Mitcham E.J., and Tang J. 2007b. Industrial-scale radio

Ward, J.F. 1985. Biochemistry of DNA Lesions. Radiation Research-Supplement, 104: S103-

Wig, T., Tang, J., Younce, F., Hallberg, L., Sunne, C.P., and Koral, T. 1999. Radio Frequency

World Meteorological Organization (WMO). 2003. Scientific Assessment of Ozone

Yoder, J.A., and Denlinger, D.L. 1991. Water Balance in Flesh Fly Pupae and Water Vapor

Zhao, Y., Flugstad, B., Kolbe, E., Park, J.W., and Wells, J.H. 2000. Using Capacitive (Radio

Zimmermann, U., Pilwat, G., and Riemann, F. 1974. Dielectric Breakdown of Cell

and Microwave Treatments. Byosystems Engineering 85 (2): 201-212. Wang S., Monzon M., Johnson J.A., Mitcham E.J., and Tang J. 2007a. Industrial-scale radio

energy efficiency. Postharvest Biology and Technology 45: 240-246.

Sterilization of Military Group Rations. AIChE Annual Meeting.

quality. Postharvest Biology and Technology 45: 247-253.

Journal of Food Process Engineering, 23: 25-55.

Membranes. Biophysical Journal, 14(11): 881-889.

S111.

273-286.

Switzerland, 498pp.

Y. 2003. Dielectric Properties of Fruits and Insect Pests as related to Radiofrequency

frequency treatments for insect control in walnuts. I: Heating uniformity and

frequency treatments for insect control in walnuts. II: Insect mortality and product

Depletion: 2002 Global Ozone Research Monitoring Project Report # 47, Geneva,

Absorption Associated with Diapause. The Journal of Experimental Biology, 157:

Frequency) Dielectric Heating in Food Processing and Preservation – A Review.

The numerical abundance of many species sharing the same ecosystem very different levels of the organism and are in constant change, depending on many factors. Due to the heterogeneous strucspeciese of the life cycles of organisms and abiotic resources in the environment based on census population densities derived from overdispersion (variance is higher than means in Poisson distribution) (Cox, 1983; Cameron and Trivedi, 1998) and a large number of zero values (zero-inflated data) is observed (Yeşilova et al, 2011). In such a case, zero-inated Poisson (ZIP) regression model is a appropriate approach for analyzing a dependent variable having excess zero observations (Lambert, 1992; Böhning, 1998; Böhning et al, 1999; Yau and Lee, 2001; Lee et al, 2001; Khoshgoftaar et al, 2005; Yeşilova et al, 2010). Zero-inflation is also likely in data sets, excess zero observations. In such cases, a zeroinated negative binominal (ZINB) regression model is an alternative method (Ridout et al, 2001; Yau, 2001; Cheung, 2002; Jansakul, 2005; Long and Frese, 2006; Hilbe, 2007; Yeşilova et al, 2009; Yeşilova et al, 2010). Morever, The Poisson hurdle model and negative binomial hurdle model (Rose and Martin, 2006; Long and Frese, 2006; Hilbe, 2007; Yeşilova et al, 2009; Yeşilova et al, 2010), and zero-inflated generalized Poisson (ZIGP) model (Consul, 1989, Consul and Famoye, 1992; Czado et al., 2007) are widely used in the analysis of zero-inflated data.

In this part, the analysis of data with many zeros for *Notonecta viridis* Delcourt (Heteroptera: Notonectidae) and Chironomidae species (Diptera) were carried out by means of using the models of Poisson Regression (PR), negative binomial (NB) regression, zero-inflated Poisson (ZIP) regression, zero-inflated negative binomial (ZINB) regression and negative binomial hurdle (NBH) model.

#### **Samplings**

The study was based on periodical samplings of the coastal band of Van Lake, conducted between July-September 2005 and May-September 2006. Samples were taken at totally twenty sampling points as streams entrance (6 points), settlement coastlines (7 points) and naspeciesal coastlines (7 points). Samples were taken according to Hansen et al. (2000). The

Zero-Inflated Regression Methods for Insecticides 261

*y x i i yi <sup>y</sup> <sup>y</sup> ii i <sup>i</sup> <sup>i</sup>*

log 1 0

*Iy <sup>e</sup> <sup>i</sup> i i <sup>i</sup>*

<sup>1</sup> log 1 <sup>0</sup> !

*<sup>e</sup> <sup>i</sup> <sup>i</sup> <sup>i</sup> Iy <sup>i</sup> <sup>i</sup> yi*

log 1 0

*LL <sup>i</sup> <sup>İ</sup> Iyi y y i i i i*

*I* . , given in equation (5) is the indicator function for the specified event. Then

log *B*

log <sup>1</sup>

*y x <sup>y</sup> <sup>y</sup> i i <sup>i</sup> <sup>i</sup> <sup>i</sup> <sup>y</sup> i i*

 

*<sup>i</sup>* parameters can be obtained following link functions,

In equations 6 and 7, B(nxp) and G(nxq) are covariate matrixes.

1 Pr( )

 

**2.4 Zero inflated negative binomial regression** 

ZINB regression model is [18],

unknown parameter vectors with px1 and qx1 dimension (Yau, 2006).

*Iy <sup>e</sup> <sup>i</sup> i i <sup>i</sup> <sup>n</sup>*

*<sup>n</sup> <sup>y</sup> LL <sup>i</sup>*

 

(1 )exp( ), 0

 

(4)

(5)

(6)

*<sup>i</sup>* and

are respectively

(8)

(1 )exp( ) !, 0

*<sup>i</sup>* represents the possibility of extra zeros' existence. Log likelihood function

 

 (7)

 and 

*G*

 

11 , 0

1

 

(1 ) , 0 <sup>1</sup> <sup>1</sup> ! <sup>1</sup>

 

*<sup>y</sup> <sup>y</sup> <sup>i</sup> <sup>i</sup> <sup>i</sup>*

*<sup>y</sup> iii i*

 

 

log 1 <sup>1</sup> <sup>0</sup> log log !

 

 

*<sup>y</sup> i i ii*

**2.3 Zero inflated poisson regression** 

for ZIP model is (Yau, 2006),

Pr( )

ZIP regression is [13],

In equation (4),

and

invertebrates were collected with a standard sweep net (30 cm width, 1 mm mesh) (Southwood, 1978; Rosenberg, 1997; Hansen et. al, 2000; Yeşilova et al., 2011).

Notonectid identification was made by Dmitry A. Gapon (Zoological Institute RAS, Universitetskaya nab., 1, St. Petersburg, Russia).

#### **2. Methods**

#### **2.1 Poisson regression**

The logarithm of mean of Poisson distribution ( ) is supposed to be a linear function of independent variables ( *<sup>i</sup> x* ) is,

$$\log\left(\mu\_i\right) = \left(\stackrel{\cdot}{\mathcal{X}\_i}\mathcal{B}\right)$$

Poisson Regression Model can be written as

$$\Pr(y\_i \mid \mu\_i, \mathbf{x}\_i) = \exp(-\mu\_i)\mu\_i^{y\_i} \Big/ y\_i! \Big/ \mathbf{y}\_i! \tag{1},\\
\mathbf{j} = \mathbf{0}, \mathbf{1}, \dots \tag{1}$$

In equation 1, *<sup>i</sup> y* denotes dependent variable having Poisson distribution. Likelihood function for PR model is, (Böhning, 1998)

$$LL\left(\mathcal{B}/\mathcal{Y}\_{\bar{1}}, \mathbf{x}\_{\bar{1}}\right) = \sum\_{i=1}^{n} \left[\mathcal{Y}\_{\bar{1}}\mathbf{x}\_{i\bar{1}}^{\dagger}\mathcal{B} - \exp\left(\mathbf{x}\_{i\bar{1}}^{\dagger}\mathcal{B}\right) - \ln\mathcal{Y}\_{\bar{1}}\mathbf{1}\right] \tag{2}$$

In equation 2, are unknown parameters. can be estimated by maximizing log likelihood function according to ML (Khoshgoftaar et al, 2005;Yau, 2006).

#### **2.2 Negative binomial regression**

NB regression model is,

$$\Pr\left(Y = y\_i \mid \mathbf{x}\_i\right) = \frac{\Gamma\left(y\_i + \frac{1}{a}\right)}{y\_i!\Gamma\left(\frac{1}{a}\right)} \frac{\left(a\,\mu\_i\right)^{y\_i}}{\left(1 + a\,\mu\_i\right)^{y\_i + \frac{1}{a}}} \; \alpha > 0 \tag{3}$$

In equation 3, is a arbitrary parameter and indicates overdispersion level. Log likelihood function for NB regression model is (Hilbe, 2007; Yau, 2006),

$$LL\left(\boldsymbol{\beta}, \alpha, y\right) = \sum\_{i=1}^{n} \begin{vmatrix} \frac{1}{\alpha} \log\left(1 + \alpha \,\mu\_{i}\right) \cdot y\_{i} \log\left(1 + \frac{1}{\alpha \,\mu\_{i}}\right) \\ + \log \Gamma\left(y\_{i} + \frac{1}{\alpha}\right) \cdot \log \Gamma\left(\frac{1}{\alpha}\right) \cdot \log y\_{i}! \end{vmatrix}$$

#### **2.3 Zero inflated poisson regression**

ZIP regression is [13],

260 Insecticides – Basic and Other Applications

invertebrates were collected with a standard sweep net (30 cm width, 1 mm mesh)

Notonectid identification was made by Dmitry A. Gapon (Zoological Institute RAS,

 ' log *<sup>i</sup> xi* 

Pr( / , ) exp( ) ! *<sup>i</sup> <sup>y</sup> ii ii <sup>i</sup> <sup>i</sup> y*

1

*n*

*i*

likelihood function according to ML (Khoshgoftaar et al, 2005;Yau, 2006).

Pr( / ) <sup>1</sup> ! <sup>1</sup>

*i i*

*Yy x*

function for NB regression model is (Hilbe, 2007; Yau, 2006),

*i*

, ,

   *x y* 

In equation 1, *<sup>i</sup> y* denotes dependent variable having Poisson distribution. Likelihood

' ' , exp ln !

1

1 1 log 1 - log 1

*<sup>y</sup> <sup>n</sup> <sup>i</sup> <sup>i</sup> <sup>i</sup> LL y*

*y*

*y*

 

*i*

1

 

*i*

*i*

*y*

 

is a arbitrary parameter and indicates overdispersion level. Log likelihood

<sup>1</sup> 1 1 log - log - log !

 

*y y i i*

 

*<sup>y</sup> <sup>i</sup>*

*i i*

*LL <sup>y</sup> x xx y y ii i i ii*

are unknown parameters.

) is supposed to be a linear function of

,yi=0,1,… (1)

can be estimated by maximizing log

0 (3)

(2)

(Southwood, 1978; Rosenberg, 1997; Hansen et. al, 2000; Yeşilova et al., 2011).

Universitetskaya nab., 1, St. Petersburg, Russia).

The logarithm of mean of Poisson distribution (

Poisson Regression Model can be written as

function for PR model is, (Böhning, 1998)

**2.2 Negative binomial regression** 

NB regression model is,

**2. Methods** 

In equation 2,

In equation 3,

**2.1 Poisson regression** 

independent variables ( *<sup>i</sup> x* ) is,

$$\Pr(y\_i/\alpha\_i) = \begin{cases} \pi\_i + (1 - \pi\_i)\exp(-\mu\_i), & y\_j = 0 \\ (1 - \pi\_i)\exp(-\mu\_i)\mu\_i^{y\_j} \Big/ y\_i!, & y\_j > 0 \end{cases} \tag{4}$$

In equation (4), *<sup>i</sup>* represents the possibility of extra zeros' existence. Log likelihood function for ZIP model is (Yau, 2006),

$$LL = \sum\_{i=1}^{n} \begin{pmatrix} I\_{\mathcal{Y}\_{\hat{i}}} = 0 \log \left( \pi i + (1 - \pi i)e^{-\mu\_{\hat{i}}} \right) \\ + I\_{\mathcal{Y}\_{\hat{i}}} > 0 \log \left( (1 - \pi i) \frac{\mu\_{\hat{i}}^{\mathcal{Y}\_{\hat{i}}} e^{-\mu\_{\hat{i}}}}{y\_{\hat{i}}!} \right) \end{pmatrix}$$

$$LL = \sum\_{\hat{i}=1}^{n} \begin{pmatrix} I\_{\mathcal{Y}\_{\hat{i}}} = 0 \log \left( \pi i + (1 - \pi i)e^{-\mu\_{\hat{i}}} \right) \\ + I\_{\mathcal{Y}\_{\hat{i}}} > 0 \begin{pmatrix} \log(1 - \pi i) \\ + y\_{\hat{i}} \log \mu\_{\hat{i}} - \mu\_{\hat{i}} - \log y\_{\hat{i}}! \end{pmatrix} \end{pmatrix} \tag{5}$$

*I* . , given in equation (5) is the indicator function for the specified event. Then *<sup>i</sup>* and *<sup>i</sup>* parameters can be obtained following link functions,

$$\log\left(\mu\right) = B\beta \tag{6}$$

and

$$\log\left(\frac{\pi}{1-\pi}\right) = G\gamma \tag{7}$$

In equations 6 and 7, B(nxp) and G(nxq) are covariate matrixes. and are respectively unknown parameter vectors with px1 and qx1 dimension (Yau, 2006).

#### **2.4 Zero inflated negative binomial regression**

ZINB regression model is [18],

$$\Pr(y\_{i}/\pi\_{i}) = \begin{cases} \pi\_{i} + \left(1 - \pi\_{i}\right)\left(1 + \alpha \mu\_{i}\right)^{-\alpha^{-1}}, & y\_{j} = 0 \\ (1 - \pi\_{i})\frac{\Gamma\left(y\_{i} + \frac{1}{\alpha}\right)}{y\_{i}!\Gamma\left(\frac{1}{\alpha}\right)} \frac{\left(\alpha \mu\_{i}\right)^{y\_{j}}}{\left(1 + \alpha \mu\_{i}\right)^{y\_{j} + \frac{1}{\alpha}}}, & y\_{j} > 0 \end{cases} \tag{8}$$

Zero-Inflated Regression Methods for Insecticides 263

exp 1 1 exp 1 *xb xb*

ln exp 1 1 exp 1 *xb xb*

*y* \* ln exp 1 exp *xb xb*

 ln 1 exp ln 1 *xb y*

ln 1 ln 1 *y*

ln 1 1 exp 1 *xb*

Akaiki Information Criteria (AIC) is goodness of criteria used for model selection. AIC,

 22 *AIC LL r* (11) In equations, LL indicates log likelihood, *r* indicates parameter number and n indicates

In this study, R statistical software program was used. Insect densities were included to the model as dependent variable. Besides years, months, species and station are included as independent variables to the model. The 66 (20.63%) of the 320 dependent variable were zero valued. The distribution of the insect densities was skewed to right because of excess

The log likelihood function for both parts of negative binomial Hurdle Model is,

L=cond*y* 0,ln 1 1 exp 1 , *xb*

and 1- *f* 0 is,

**2.6 Model selection** 

sample size.

**3. Results** 

zeros.

Model AIC PR 57846.00 ZIP 47791.71 NB 3176.40 ZINB 2819.800 PH 47791.71 **NBH 2803.206** 

Table 1. Model selection criteria for PR, NB, ZIP, ZINB, PH and NBH.

In equation (8), ( 0) indicates an overdispersion parameter. Log likelihood function for ZINB model is (Yau, 2006),

$$\begin{split} \text{LL}(\mu,\alpha,\pi;y) &= \sum\_{i} \left( \begin{aligned} {}^{I}y\_{i} &= 0 \log(\pi i) \\ {}^{I}(-1-\pi i) \left( {}^{1}\alpha \,\mu\_{i} \right)^{-\alpha} \end{aligned} \right) \\ &+ {}^{I}y\_{i} > 0 \log \left( (1-\pi\_{i}) \frac{\Gamma\left( y\_{i} + \frac{1}{\alpha} \right)}{y\_{i}!\Gamma\left( \frac{1}{\alpha} \right)} \left( {}^{0}\mu\_{i} \overline{\mu}\_{i} \right)^{y\_{i}} + \frac{1}{\alpha} \right) \right) \\ &= \sum\_{i} \left( {}^{I}y\_{i} = 0 \log(\pi\_{i} + (1-\pi\_{i})(1+\alpha\,\mu\_{i}))^{-\alpha^{-1}} \\ &+ {}^{I}y\_{i} > 0 \Big( \log(1-\pi\_{i}) - \frac{1}{\alpha} \log\left(1+\alpha\,\mu\_{i} \right) \\ &- y\_{i} \log\left(1+\frac{1}{\alpha\,\mu\_{i}} \right) + \log\Gamma\left(y\_{i} + \frac{1}{k} \right) \Big) \\ &- \log\Gamma\left(\frac{1}{\alpha} \right) - \log y\_{i}! \end{split} \tag{9}$$

*I* . , given in equation 9 is the indicator function for the specified event. The model descripted by Lambert (1992) can be given as,

$$
\log\left(\mu\right) = X\beta \quad \text{and} \quad \log\left(\frac{\pi}{1-\pi}\right) = G\gamma
$$

Here, X(nxp) and G(nxq) covariate matrixes, and are respectively unknown parameter vectors with px1 and qx1 dimension. Maximum likelihood estimations for , and can be obtained by using EM algorithm.

#### **2.5 Negative binomial hurdle model**

Log-likelihood for negative binomial hurdle model (Hilbe, 2007),

$$L = \ln\left(f\left(0\right)\right) + \left\{\ln\left[1 - f\left(0\right)\right] + \ln P\left(j\right)\right\} \tag{10}$$

In equation (10), *f* 0 indicates the probability of the binary part and *p j* indicates the probability of positive count. The probability of zero for logit model is,

$$f\left(0\right) = P\left(y = 0; \mathbf{x}\right) = 1/\left(1 + \exp\left(\mathbf{x} b \mathbf{1}\right)\right)$$

and 1- *f* 0 is,

262 Insecticides – Basic and Other Applications

 

*<sup>y</sup> yi <sup>i</sup> <sup>i</sup> Iy <sup>i</sup> <sup>i</sup>*

log( 1 1 0

*Iy i i <sup>i</sup> <sup>i</sup> <sup>i</sup>*

<sup>1</sup> log 1 log 1 <sup>0</sup>

*i i i y y*

<sup>1</sup> log log !

vectors with px1 and qx1 dimension. Maximum likelihood estimations for

log *X*

Log-likelihood for negative binomial hurdle model (Hilbe, 2007),

probability of positive count. The probability of zero for logit model is,

*Iy <sup>i</sup> <sup>i</sup> <sup>i</sup>*

1 1 log 1 log

*yi*

*I* . , given in equation 9 is the indicator function for the specified event. The model

and log <sup>1</sup>

 and 

In equation (10), *f* 0 indicates the probability of the binary part and *p j* indicates the

*f Py x* 0 0; 1 1 exp 1 *xb*

indicates an overdispersion parameter. Log likelihood function for

 

*G*

*L* ln 0 ln 1 0 ln *f f P j* (10)

 

1

(9)

are respectively unknown parameter

 , and can

*<sup>y</sup> <sup>y</sup> <sup>i</sup> <sup>i</sup> <sup>i</sup>*

*k*

log( 0

*Iy <sup>i</sup> <sup>i</sup>*

*<sup>i</sup> <sup>i</sup> <sup>i</sup>*

1

> 

log 1 <sup>0</sup> <sup>1</sup> <sup>1</sup> ! <sup>1</sup>

,,; 1 1 1

In equation (8), ( 0)

ZINB model is (Yau, 2006),

=

 

 

descripted by Lambert (1992) can be given as,

Here, X(nxp) and G(nxq) covariate matrixes,

be obtained by using EM algorithm.

**2.5 Negative binomial hurdle model** 

*LL y*

$$\exp(xb1) / \left(1 + \exp(xb1)\right)$$

The log likelihood function for both parts of negative binomial Hurdle Model is,

$$\begin{aligned} \text{L=cond}\left\{y &= 0, \ln\left(1/1 - \exp\left(xb1\right)\right), \\\\ \ln\left(\exp\left(xb1\right)\Big/\left(1 + \exp\left(xb1\right)\right)\right) \\\\ + y\*\ln\left(\exp\left(xb\right)\Big/\left(1 + \exp\left(xb\right)\right)\right) \\\\ -\ln\left(1 + \exp\left(xb\right)\right)\Big/a + \ln\Gamma\left(y + 1/a\right) \\\\ -\ln\Gamma\left(y + 1\right) - \ln\Gamma\left(1/a\right) \\\\ -\ln\left(1 - \left(1 + \exp\left(xb\right)\right)\left(-1/a\right)\right) \end{aligned}$$

#### **2.6 Model selection**

Akaiki Information Criteria (AIC) is goodness of criteria used for model selection. AIC,

$$\text{AIC} = -2\text{LL} + 2\text{r} \tag{11}$$

In equations, LL indicates log likelihood, *r* indicates parameter number and n indicates sample size.

#### **3. Results**

In this study, R statistical software program was used. Insect densities were included to the model as dependent variable. Besides years, months, species and station are included as independent variables to the model. The 66 (20.63%) of the 320 dependent variable were zero valued. The distribution of the insect densities was skewed to right because of excess zeros.


Table 1. Model selection criteria for PR, NB, ZIP, ZINB, PH and NBH.

Zero-Inflated Regression Methods for Insecticides 265

(Intercept) -3.92991 1.33906 -2.935 0.00334 \*\* 0.01964544 year 0.50266 0.33705 1.491 0.13587 1.653113 month 0.04405 0.11930 0.369 0.71197 1.045035 station 0.17250 0.02994 5.761 8.36e-09 \*\*\* 1.188272 species -0.44380 0.30013 -1.479 0.13923 0.6415937

ML parameter estimations and standard errors for zero-inflated negative binomial regression both count model and logit model were given in Table 6 and Table 7,

(Intercept) 9.47226 1.04897 9.030 < 2e-16 \*\*\* 12994.22 year -0.13254 0.20609 -0.643 0.520132 0.8758679 month -0.16895 0.09356 -1.806 0.070957 0.8445511 station -0.06233 0.01806 -3.452 0.000557 \*\*\* 0.9395728 species -2.21006 0.20855 -10.597 < 2e-16 \*\*\* 0.1096941

(Intercept) -1.21687 4.61145 -0.264 0.791872 0.2961557 year 1.64137 0.88380 1.857 0.063288 5.162237 month -0.23791 0.22636 -1.051 0.293246 0.7882736 station 0.18398 0.05485 3.354 0.000795 \*\*\* 1.201992 species -3.69139 3.88424 -0.950 0.341934 0.02493732

ML parameter estimations and standard errors for Poisson hurdle both count model and

(Intercept) 6.017745 0.056073 107.32 <2e-16 \*\*\* 410.6515 year 0.271101 0.013047 20.78 <2e-16 \*\*\* 1.311408 month 0.162333 0.005271 30.80 <2e-16 \*\*\* 1.176252 station -0.046859 0.001122 -41.76 <2e-16 \*\*\* 0.954222 species -2.002676 0.018382 -108.94 <2e-16 \*\*\* 0.1349736

Table 5. Parameter estimations and standard errors for ZIP logit model.

Table 6. Parameter estimations and standard errors for ZINB count model.

Table 7. Parameter estimations and standard errors for ZINB logit model.

Table 8. Parameter estimations and standard errors for PH count model.

logit model were given in Table 8 and Table 9, respectively.

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

respectively.

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

In PR analyses, deviance and Pearson Chi-square goodness of statistics higher than one (831.417 and 650.213, respectively). Thus, goodness of statistics represents that there is an overdispersion in insect densities. AIC model selection criteria for the models of PR, NB, ZIP, ZINB, PH, and NBH were given in Table 1. The model with the smallest AIC was NBH regression.

Maximum likelihood (ML) parameter estimations and standard errors for PR were given in Table 2.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 2. Parameter estimations and standard errors for Poisson regression.

ML parameter estimations and standard errors for negative binomial regression were given in Table 2.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 3. Parameter estimations and standard errors for negative binomial regression.

ML parameter estimations and standard errors for zero-inflated Poisson regression both count model and logit model were given in Table 4 and Table 5, respectively.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 4. Parameter estimations and standard errors for ZIP count model.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

264 Insecticides – Basic and Other Applications

In PR analyses, deviance and Pearson Chi-square goodness of statistics higher than one (831.417 and 650.213, respectively). Thus, goodness of statistics represents that there is an overdispersion in insect densities. AIC model selection criteria for the models of PR, NB, ZIP, ZINB, PH, and NBH were given in Table 1. The model with the smallest AIC was NBH

Maximum likelihood (ML) parameter estimations and standard errors for PR were given in

(Intercept) 6.179499 0.054470 113.449 <2e-16 \*\*\* 482.992 year 0.118847 0.013069 9.094 <2e-16 \*\*\* 1.125244 month 0.175298 0.005066 34.604 <2e-16 \*\*\* 1.191246 Station -0.081353 0.001124 -72.357 <2e-16 \*\*\* 0.921917 species -1.943212 0.018356 - 105.863 <2e-16 \*\*\* 0.1432735

ML parameter estimations and standard errors for negative binomial regression were given

(Intercept) 8.52318 0.99249 8.588 4.16e-16 \*\*\* 5029.119 year -0.15794 0.24824 -0.636 0.525 0.853901 month -0.08205 0.09168 -0.895 0.372 0.9212259 Station -0.08031 0.01949 -4.121 4.82e-05 \*\*\* 0.9228302 species -1.92518 0.22452 -8.575 4.56e-16 \*\*\* 0.1458495

Table 3. Parameter estimations and standard errors for negative binomial regression.

count model and logit model were given in Table 4 and Table 5, respectively.

Table 4. Parameter estimations and standard errors for ZIP count model.

ML parameter estimations and standard errors for zero-inflated Poisson regression both

(Intercept) 6.017745 0.056073 107.32 <2e-16 \*\*\* 410.6515 year 0.271101 0.013047 20.78 <2e-16 \*\*\* 1.311408 month 0.162333 0.005271 30.80 <2e-16 \*\*\* 1.176252 station -0.046859 0.001122 -41.76 <2e-16 \*\*\* 0.954222 species -2.002676 0.018382 -108.94 <2e-16 \*\*\* 0.1349736

Table 2. Parameter estimations and standard errors for Poisson regression.

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

regression.

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 2.

in Table 2.

Table 5. Parameter estimations and standard errors for ZIP logit model.

ML parameter estimations and standard errors for zero-inflated negative binomial regression both count model and logit model were given in Table 6 and Table 7, respectively.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 6. Parameter estimations and standard errors for ZINB count model.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 7. Parameter estimations and standard errors for ZINB logit model.

ML parameter estimations and standard errors for Poisson hurdle both count model and logit model were given in Table 8 and Table 9, respectively.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 8. Parameter estimations and standard errors for PH count model.

Zero-Inflated Regression Methods for Insecticides 267

Average insect density observed in the year 2005 has shown 17% decrease in reference to the year 2006. Insect densities observed at monthly sampling ranges depending on water temperaspeciese were increased with the rise of temperaspeciese, but specifically after the month of July such intensity was decreased at the rate of 16% ( -0.19128 *e* ~ 0.8434961 ) towards the month of September within the both years. It has been determined that insect intensities observed at different stations have shown differentiation at the rate of 5%. Chironomid larvae which are included in prey of notonectidae fed by different sources of food at aquatic environment have been found at rather lower density in reference to notonectid density. However, it is hard to guess that such decrement has been formed under the impact of notonectidae. Nevertheless notonectidae do not depend on a single host, their sources of food are rather wide range of variety. Small arthropods on the water surface, small crustaceans living in water, larvae of aquatic insects, snails, small fish or larvae of frog

[1] Böhning, D. Zero- Inflated Poisson Models and C. A. MAN. (1998). A Tutorial Collection

[2] Böhning, D., Dietz, E. and Schlattmann, P. (1999). The Zero-Inflated Poisson Model and

[3] Bruce, A.M., Pike, E.B. and Fisher, W.J. (1990). A review of treatment processes to meet

[4] Cameron, A.C. and Trivedi, P.K. (1998). Regression Analysis of Count Data. New York,

[5] Cheung, Y.B. (2002). Zero-Inflated Models for Regression Analysis of Count Data. *A Study of Growth and Development. Statistics in Medicine*, 21, 1461-1469. [6] Consul, P. C. Generalized Poisson distributions, Volume 99 of Statistics: Textbooks and

[7] Consul, P. C. and F. Famoye. (1992). Generalized Poisson regression model. Comm.

[9] Czado, C., Erhardt, V., Min, A., Wagner, S. (2007). Dispersion and zero-inflation level

[10] Hansen, J., Mki. Sato, R. Ruedy, A. Lacis & V. Oinas. (2000). Global warming in the

[12] Jansakul, N. (2005). Fitting a Zero-inflated Negative Binomial Model via R. In

[13] Khoshgoftaar, T.M., Gao, K. and Szabo, R.M. (2005). Comparing Software Fault

regression effects on the mean, Statistical Modelling. 7(2) : 125-153

[8] Cox, R. (1983). Some Remarks on Overdispersion. *Biometrika*, 70, 269-274.

twenty-first censpeciesy: An alternative scenario, [11] Hilbe, J.M. (2007). Negative Binomial Regression. Cambridge, UK.

*Journal of Systems Science*, 36(11), 705-715.

the EC Sludge Directive. *J. Inst. Wat. Environ. Management*, 4, 1-13.

the Decayed, Missing and Filled Teeth Index in Dental Epidemiology. *Journal of* 

Monographs. New York: Marcel Dekker Inc. Properties and applications 1989.p.1-

applied to patent outsourcing rates Zero-inflated generalized Poisson models with

Proceedings 20th International Workshop on Statistical Modelling. Sidney,

Predictions of Pure and Zero- inflated Poisson Regression Models. *International* 

are among their preys (Bruce et al., 1990).

of Evidence. *Biometrical Journal*, 40(7), 833-843.

*Royal Statistical Society*, *A*, 162, 195–209.

Statist. Theory Methods. 21(1), 89–109.

Cambridge University Pres.

Australia, 277-284.

**4. References** 

20.

ML parameter estimations and standard errors obtained for the NBH count model was given in Table 8. While stations and species were significant on the insect densities, the effect of years and the effect of months were not significant on the insect densities.

ML parameter estimations and standard errors obtained for the NBH logit model was given in Table 9. The effects months, years and species were not significant on the insect densities. However, the effect of station was significant on the insect densities.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 9. Parameter estimations and standard errors for PH logit model.

ML parameter estimations and standard errors obtained for negative binomial hurdle both count model and logit model were given in Table 10 and Table 11, respectively.

ML parameter estimations and standard errors obtained for the NBH count model was given in Table 10. While stations and species were significant on the insect densities, the effect of years and the effect of months were not significant on the insect densities.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 10. Parameter estimations and standard errors for NBH count model.

ML parameter estimations and standard errors obtained for the NBH logit model was given in Table 11. The effects months, years and species were not significant on the insect densities. However, the effect of station was significant on the insect densities.


\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

Table 11. Parameter estimations and standard errors for NBH logit model.

Average insect density observed in the year 2005 has shown 17% decrease in reference to the year 2006. Insect densities observed at monthly sampling ranges depending on water temperaspeciese were increased with the rise of temperaspeciese, but specifically after the month of July such intensity was decreased at the rate of 16% ( -0.19128 *e* ~ 0.8434961 ) towards the month of September within the both years. It has been determined that insect intensities observed at different stations have shown differentiation at the rate of 5%. Chironomid larvae which are included in prey of notonectidae fed by different sources of food at aquatic environment have been found at rather lower density in reference to notonectid density. However, it is hard to guess that such decrement has been formed under the impact of notonectidae. Nevertheless notonectidae do not depend on a single host, their sources of food are rather wide range of variety. Small arthropods on the water surface, small crustaceans living in water, larvae of aquatic insects, snails, small fish or larvae of frog are among their preys (Bruce et al., 1990).

#### **4. References**

266 Insecticides – Basic and Other Applications

ML parameter estimations and standard errors obtained for the NBH count model was given in Table 8. While stations and species were significant on the insect densities, the

ML parameter estimations and standard errors obtained for the NBH logit model was given in Table 9. The effects months, years and species were not significant on the insect densities.

(Intercept) 3.92991 1.33906 2.935 0.00334 \*\* 50.9024 year -0.50266 0.33705 -1.491 0.13587 0.6049194 month -0.04405 0.11930 -0.369 0.71197 0.9569061 station 0.17250 0.02994 -5.761 8.36e-09 \*\*\* 1.188272 species 0.44380 0.30013 1.479 0.13923 1.558619

ML parameter estimations and standard errors obtained for negative binomial hurdle both

ML parameter estimations and standard errors obtained for the NBH count model was given in Table 10. While stations and species were significant on the insect densities, the

(Intercept) 9.43372 1.26292 7.470 8.03e-14 \*\*\* 12502.95 year -0.19128 0.24381 -0.785 0.4327 0.8259013 month -0.17020 0.11124 -1.530 0.1260 0.8434961 station -0.04587 0.02096 -2.188 0.0287 \* 0.9551661 species -2.33333 0.25071 -9.307 < 2e-16 \*\*\* 0.0969723

ML parameter estimations and standard errors obtained for the NBH logit model was given in Table 11. The effects months, years and species were not significant on the insect

(Intercept) 3.92991 1.33906 2.935 0.00334 \*\* 50.9024 year -0.50266 0.33705 -1.491 0.13587 0.6049194 month -0.04405 0.11930 -0.369 0.71197 0.9569061 station -0.17250 0.02994 -5.761 8.36e-09 \*\*\* 0.8415583 species 0.44380 0.30013 1.479 0.13923 1.558619

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

Estimate Std. Error z value Pr(>|z|) *e*

effect of years and the effect of months were not significant on the insect densities.

However, the effect of station was significant on the insect densities.

Table 9. Parameter estimations and standard errors for PH logit model.

count model and logit model were given in Table 10 and Table 11, respectively.

effect of years and the effect of months were not significant on the insect densities.

Table 10. Parameter estimations and standard errors for NBH count model.

densities. However, the effect of station was significant on the insect densities.

Table 11. Parameter estimations and standard errors for NBH logit model.

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001

\*p<0.05, \*\*p<0.01, \*\*\*p<0.001


[14] Lambert, D. (1992). Zero-Inflated Poisson Regression, with an Application to Defects in

[15] Lee, A.H., Wang, K. and Yau, K. K. W. (2001). Analysis of Zero-Inflated Poisson Data Incorporating Extent of Exposure. *Biometrical Journal*, 43(8), 963-975. [16] Long, J.S. and Freese, J. (2006). Regression Models for Categorical Depentent Variable

[17] McCullagh, P. and Nelder, J. A. (1989). Generalized Linear Models*.* Second Edition,

[18] Ridout, M., Hinde, J. and Demetrio, C.G.B. (2001). A Score Test for a Zero-Inflated

[19] Rose, C.E, Martin, S.W., Wannemuehler, K.A. and Plikaytis, B.D. (2006). On the of

[20] Rosenberg, D. M., I. J. Davies, D.G. Cobb & A.P. Wiens. (1997). Protocols For

[21] Southwood, T.R.E. (1978). Ecological Methods, with Particular Reverence to the Study

[22] Yau, K.K.W. and Lee, A.H. (2001). Zero-Inflated Poisson Regression with Random

[23] Yau, Z. (2006). Score Tests for Generalization and Zore-Inflation in Count Data

[24] Yeşilova, A., Kaydan, B. and Kaya, Y. (2010). Modelling Insect-Egg Data with Excess

[25] Yeşilova, A. Y., Kaya, B., Kaki, İ.Kasap (2010)Analysis of Plant Protection Studies with

[26] Yeşilova, A., Özgökçe, M. S., Atlhan, R., Karaca, İ., Özgökçe, F., Yldz, Ş. and Kaydan,

Poisson regression. *Turkish Journal of Entomology.* 35(2).

Measuring Biodiversity: Benthic Macroinvertebrates in Fresh Waters,

Poisson Regression Model Against Zero-Inflated Negative Binomial Alteratves.

Zero-inflated and Hurdle Models for Medelling Vaccine Adverse event Count

http://www.emanrese.ca/eman/ecotools/protocols/freshwater/benthics/intro.ht

of Insect Populations, 2nd ed., Chapman and Hall, London and New York, 524 pp.

Effects to Evaluate an Occupational Injury Prevention Programme. *Statistics in* 

Modeling. Unpublished Ph. D. Dissertation, University of South Caroline,

Zeros using Zero-inflated Regression Models. *Hacettepe Journal of Mathematics and* 

Excess Zeros Using Zero-Inflated and Negative Bi Binomial Hurdle Models *G.U.* 

B. and Kaya, Y. (2011). Investigation of the effects of physico-chemical environmental conditions on population fluctuations of *Notonecta viridis* Delcourt, 1909 (Hemiptera: Notonectidae) in Van Lake by using zero-inflated generalized

Manaufacspeciesin. *Technometrics*, 34(1), 1-13.

Using Stata. A Stata Pres Publication, USA.

Data. *Journal of Biopharmaceutical Statistics*, 16, 463-481.

Chapmann and Hall, London.

*Biometrics*, 57, 219-233.

ml, (Data accessed: 10.10.

*Medicine*, 20, 2907-2920.

*Statistics*. 39(2),273-282.

*Journal of Science .*

Columbia.

### *Edited by Sonia Soloneski and Marcelo Larramendy*

It is our hope that this book will be of interest and use not only to scientists, but also to the food-producing industry, governments, politicians and consumers as well. If we are able to stimulate this interest, albeit in a small way, we have achieved our goal.

Insecticides - Basic and Other Applications

Insecticides

Basic and Other Applications

*Edited by Sonia Soloneski and Marcelo Larramendy*

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