**4.2.4 Metabolism of toxicants**

Three enzyme groups involved in metabolic resistance to pesticides: esterases, glutathione S-transferases (GST) and mixed function oxidaes (MFO). The following technique can be used for detection of these mechanisms.

#### **4.2.4.1 Esterase**

Esterases metabolize a variety of pesticides such as OP, carbamate, pyrethroids with ester linkages. "These enzymes confer resistance to pesticides in over 50 species of insects, ticks and mitesa" (Devorshak and Roe 1998; as cited in van Leeuwen et al., 2009). Detection and investigation of esterases-based mechanism can be achieved from synergistic bioassays and biochemical assays. For synergistic bioassays, some synergists such as DEF (S,S,Stributylphosphorotrithioate), TPP (O,O,O-triphenylphosphate), and IBP (O,O-bis[1-

Ecological Impacts of Pesticides in Agricultural Ecosystem 153

Genetic changes confer insecticide resistance in insects can affect their developmental time and reproductive potential. Resistance genes can alter some life table and physiology parameters of pests and thus causing a fitness cost (as cited in Gazavi et al., 2001). In some insect and mite species, genetic changes that enhance survival due to pesticides exposure reduce pest fitness in the absence of pesticides (Roff and Derose, 2001; Higginson et al., 2005). Because insecticides caused a huge selection on pest population in a short time, therefore susceptible and resistant populations are useful models for evaluating fitness trade-off (Crow, 1957; McKenzie, 1996; Higginson et al., 2005; Ghadamyari et al., 2008). Mutation associated with insecticide resistance can disturb pest physiology (MacCarroll et

Resistant and susceptible strains differ in some properties due to their adaptation to insecticides, such as developmental time, fecundity and fertility, overwintering success and sensitivity to alarm pheromone. Differences in the biological parameters affecting the net reproductive rate of insect populations are important for insecticide resistance management (IRM) (Haubruge and Arnaud 2001). Altered acetylcholinesterase (AChE) and esteraseassociated resistance in peach aphids have life history disadvantages compared with susceptible counterpart (Ghadamyari et al., 2008). Also, altered GABA receptor, esterase, MFO and GST associated abamectin resistance in *Tetranychus urticae* showed life history

Field and laboratory studies on different strains of *M. persicae* have been showed that adverse selection by pesticides caused "poorer winter survival, maladaptive behaviour and

Some resistance mechanisms can confer more fitness disadvantages to resistant strains than others. Some strains of *M. persicae* with over expressing high levels of carboxylesterase (due to structural gene amplification) show a reduced tendency to move away from senescing leaves compared with susceptible counterpart (Foster et al. 1997, 2003). This behaviour caused higher mortality during worst winter weather conditions and so can be regarded as a deleterious pleiotropic effect of pesticide resistance. Studies have been shown that peach– potato aphids caring both gene amplification and the knock down mutation has reduced

Many experiments measured reproductive fitness in the absence of pesticides in resistant and susceptible strains. The results of these experiments showed that individuals carrying resistant genes have lower reproductive rate than susceptible. When pressure of insecticide is diminished on resistant strain, the number of resistant individual in population quickly reduce due to fitness cost (Crow, 1957; Carrière and Roff, 1995; McKenzie, 1996; as cited in Arnaud et al., 2005). Although the majority of insecticide resistance strains show fitness cost (McKenzie, 1996; Foster et al., 2003; Berticat et al., 2004; as cited in Arnaud et al., 2005; Ghadamyari et al., 2008), a few researches present species with no fitness cost (McKenzie, 1996; Oppert et al., 2000; McCant et al., 2005). For example "some resistant strains of mosquitoes in absence of pesticides showed only one quarter of the reproductive potential of susceptible strains" (Georghiou and Taylor, 1977). *Varroa destructor* has little or no reproductive fitness cost associated with pyrethroid resistance in Texas (Martin et al., 2002).

**4.3 Fitness costs associated with pesticide resistance** 

reduced reproductive fitness" (Foster et al., 2000).

response to alarm pheromone (Foster et al., 2003).

**4.3.2 Reduced reproductive success** 

al., 2000).

disadvantages.

**4.3.1 Maladaptive behavior** 

methylethyl] S-phenylmethylphosphorothioate) were used to inhibit esterases (Raffa & Priester, 1985 as cited in Jensen, 2000). However, the synergistic bioassays are useful to achieve valuable data, but DEF in higher concentrations inhibits MFO and esterase activity (Scott, 1990).

In biochemical assay, increased esterase activity in resistant insect can be checked with some artificial substrates such as α- and ß- naphthyl acetates, α- and ß- naphtyl butyrates, α- and ß- naphtyl propionates and p- nitrophenyl acetates. These substrates hydrolyzed by general esterases and the involvement of esterase in resistance must be checked by more than one substrate. Also, the specific elevated esterase can be detected with immunological methods and an antiserum for example antiserum of E4 carboxylesterase *M. persicae (*Devonshire et al., 1986). An affinity purified immunoglobulin G (*IgG*) fraction from this antiserum has been used in a immunoplate assay to distinguish between the different resistant strains of *M. persicae* (Devonshire et al., 1986).

#### **4.2.4.2 GST**

The GSTs are involved in the detoxification of a wide range of xenobiotics including insecticides, fungicides, acaricides and herbicides (Salinas & Wong, 1999). GSTs catalyze the conjugation of hydrophobic electrophile compounds such as pesticides and their metabolites with the thiol group of reduced glutathione (GSH) (Habig *et al*., 1974).

 Elevated GST activity has been associated with resistance to the major classes of pesticides and the involvement of GSTs in resistance to insecticides is well reviewed in Enayati et al., (2005). Because GSTs can metabolize a wide variety of xenobiotics such as insecticides and plant allelochemicals, increased GST activity may be due to exposure of insect with foreign compound in environment not resistance mechanisms. Diethyl maleate (DEM) is used as synergist for inhibiting of GSTs involved in resistance in bioassays.

GST activity can be measured from direct measurement of the conjugation of reduced glutathione with non-fluorescent monochlorobimane (MCB), l-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) as substrates using a spectrophotometer at 340 nm.

#### **4.2.4.3 Cytochrome P450**

Cytochrome P450 plays important role in metabolism of pesticides and confers resistance to many classes of pesticides in mite, insect, weeds and fungi. These enzymes widely distributed in fungi, bacteria, yeast, insect, mite, weeds and invertebrate. Piperonyl butoxide (PBO) and sesamex was used in synergistic bioassays for involvement of MFO in resistance. However, PBO can inhibit both MFO and esterases-based resistance mechanisms in some insect and mite species (Gunning et al., 1999; Young et al., 2005).

There are many P450-monooxygenase isoenzymes with different substrate specificity within an insect. Therefore, in measuring activity of MFO- based resistance mechanisms must use different substrates and methods (Rose et al.*,* 1991). Different biochemical methods have been used to study P450-monooxygenase activity in insects and mites. One of these methods is measuring the total amount of heme containing protein using a heme-peroxidase assay (Brogdon et al., 1997). Another method uses the aldrin as substrate to measure P450 monooxygenase activity. *O*-demethylase can be detected using *p*-nitroanisole as substrate using spectrophotometer. O-deethylation activity of the artificial substrates, 7 ethoxycoumarin (7-EC) and ethoxy-4-trifluoromethylcoumarin, by MFO can be measured with fluorometric microplate assay (van Pottelberge et al., 2009).

methylethyl] S-phenylmethylphosphorothioate) were used to inhibit esterases (Raffa & Priester, 1985 as cited in Jensen, 2000). However, the synergistic bioassays are useful to achieve valuable data, but DEF in higher concentrations inhibits MFO and esterase activity

In biochemical assay, increased esterase activity in resistant insect can be checked with some artificial substrates such as α- and ß- naphthyl acetates, α- and ß- naphtyl butyrates, α- and ß- naphtyl propionates and p- nitrophenyl acetates. These substrates hydrolyzed by general esterases and the involvement of esterase in resistance must be checked by more than one substrate. Also, the specific elevated esterase can be detected with immunological methods and an antiserum for example antiserum of E4 carboxylesterase *M. persicae (*Devonshire et al., 1986). An affinity purified immunoglobulin G (*IgG*) fraction from this antiserum has been used in a immunoplate assay to distinguish between the different resistant strains of

The GSTs are involved in the detoxification of a wide range of xenobiotics including insecticides, fungicides, acaricides and herbicides (Salinas & Wong, 1999). GSTs catalyze the conjugation of hydrophobic electrophile compounds such as pesticides and their metabolites

 Elevated GST activity has been associated with resistance to the major classes of pesticides and the involvement of GSTs in resistance to insecticides is well reviewed in Enayati et al., (2005). Because GSTs can metabolize a wide variety of xenobiotics such as insecticides and plant allelochemicals, increased GST activity may be due to exposure of insect with foreign compound in environment not resistance mechanisms. Diethyl maleate (DEM) is used as

GST activity can be measured from direct measurement of the conjugation of reduced glutathione with non-fluorescent monochlorobimane (MCB), l-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) as substrates using a spectrophotometer at

Cytochrome P450 plays important role in metabolism of pesticides and confers resistance to many classes of pesticides in mite, insect, weeds and fungi. These enzymes widely distributed in fungi, bacteria, yeast, insect, mite, weeds and invertebrate. Piperonyl butoxide (PBO) and sesamex was used in synergistic bioassays for involvement of MFO in resistance. However, PBO can inhibit both MFO and esterases-based resistance mechanisms in some

There are many P450-monooxygenase isoenzymes with different substrate specificity within an insect. Therefore, in measuring activity of MFO- based resistance mechanisms must use different substrates and methods (Rose et al.*,* 1991). Different biochemical methods have been used to study P450-monooxygenase activity in insects and mites. One of these methods is measuring the total amount of heme containing protein using a heme-peroxidase assay (Brogdon et al., 1997). Another method uses the aldrin as substrate to measure P450 monooxygenase activity. *O*-demethylase can be detected using *p*-nitroanisole as substrate using spectrophotometer. O-deethylation activity of the artificial substrates, 7 ethoxycoumarin (7-EC) and ethoxy-4-trifluoromethylcoumarin, by MFO can be measured

with the thiol group of reduced glutathione (GSH) (Habig *et al*., 1974).

synergist for inhibiting of GSTs involved in resistance in bioassays.

insect and mite species (Gunning et al., 1999; Young et al., 2005).

with fluorometric microplate assay (van Pottelberge et al., 2009).

(Scott, 1990).

**4.2.4.2 GST** 

340 nm.

**4.2.4.3 Cytochrome P450** 

*M. persicae* (Devonshire et al., 1986).

#### **4.3 Fitness costs associated with pesticide resistance**

Genetic changes confer insecticide resistance in insects can affect their developmental time and reproductive potential. Resistance genes can alter some life table and physiology parameters of pests and thus causing a fitness cost (as cited in Gazavi et al., 2001). In some insect and mite species, genetic changes that enhance survival due to pesticides exposure reduce pest fitness in the absence of pesticides (Roff and Derose, 2001; Higginson et al., 2005). Because insecticides caused a huge selection on pest population in a short time, therefore susceptible and resistant populations are useful models for evaluating fitness trade-off (Crow, 1957; McKenzie, 1996; Higginson et al., 2005; Ghadamyari et al., 2008). Mutation associated with insecticide resistance can disturb pest physiology (MacCarroll et al., 2000).

Resistant and susceptible strains differ in some properties due to their adaptation to insecticides, such as developmental time, fecundity and fertility, overwintering success and sensitivity to alarm pheromone. Differences in the biological parameters affecting the net reproductive rate of insect populations are important for insecticide resistance management (IRM) (Haubruge and Arnaud 2001). Altered acetylcholinesterase (AChE) and esteraseassociated resistance in peach aphids have life history disadvantages compared with susceptible counterpart (Ghadamyari et al., 2008). Also, altered GABA receptor, esterase, MFO and GST associated abamectin resistance in *Tetranychus urticae* showed life history disadvantages.

Field and laboratory studies on different strains of *M. persicae* have been showed that adverse selection by pesticides caused "poorer winter survival, maladaptive behaviour and reduced reproductive fitness" (Foster et al., 2000).

#### **4.3.1 Maladaptive behavior**

Some resistance mechanisms can confer more fitness disadvantages to resistant strains than others. Some strains of *M. persicae* with over expressing high levels of carboxylesterase (due to structural gene amplification) show a reduced tendency to move away from senescing leaves compared with susceptible counterpart (Foster et al. 1997, 2003). This behaviour caused higher mortality during worst winter weather conditions and so can be regarded as a deleterious pleiotropic effect of pesticide resistance. Studies have been shown that peach– potato aphids caring both gene amplification and the knock down mutation has reduced response to alarm pheromone (Foster et al., 2003).

#### **4.3.2 Reduced reproductive success**

Many experiments measured reproductive fitness in the absence of pesticides in resistant and susceptible strains. The results of these experiments showed that individuals carrying resistant genes have lower reproductive rate than susceptible. When pressure of insecticide is diminished on resistant strain, the number of resistant individual in population quickly reduce due to fitness cost (Crow, 1957; Carrière and Roff, 1995; McKenzie, 1996; as cited in Arnaud et al., 2005). Although the majority of insecticide resistance strains show fitness cost (McKenzie, 1996; Foster et al., 2003; Berticat et al., 2004; as cited in Arnaud et al., 2005; Ghadamyari et al., 2008), a few researches present species with no fitness cost (McKenzie, 1996; Oppert et al., 2000; McCant et al., 2005). For example "some resistant strains of mosquitoes in absence of pesticides showed only one quarter of the reproductive potential of susceptible strains" (Georghiou and Taylor, 1977). *Varroa destructor* has little or no reproductive fitness cost associated with pyrethroid resistance in Texas (Martin et al., 2002).

Ecological Impacts of Pesticides in Agricultural Ecosystem 155

Populations of *S. zeamais* with different levels of susceptibility to insecticides were used a model for evaluating the mechanisms of fitness cost associated resistance. Demographic and competition studies carried out on different strains of *S. zeamais* susceptible and resistant to pyrethroids showed fitness costs associated with insecticide resistance in some strains and

The susceptible, resistant no-cost and resistant cost strains showed some differences in some

**I.** Some differences in the accumulation and consumption of fuel nutrients macromolecules were observed between *S. zeamais* pyrethriod-resistant and susceptible strains. These differences caused *S. zeamais* could be able detoxify insecticides without reduction in its reproductive potential. Pyrethroid- resistance cost strain has greater stored total proteins and carbohydrates compared with susceptible and resistant cost strains (Arau´jo et al., 2008a, 2008b). Finally Arau´jo et al. (2008a, 2008b) concluded that

increased energy reserves may be due to increased digestive enzyme activities. **II.** The pyrethroid -resistant strains showed increased serine- and cysteine-proteolytic and cellulolytic activity. Also, kinetic parameters of these enzymes were different in susceptible, resistant no-cost and resistant cost strains. These differences suggested that cysteine-proteinase and cellulase activities were more important for justifying the cost of insecticide resistance in *S. zeamais* strains (Arau´jo et al., 2008b). The activity of carbohydrases specially amylase was higher in the resistant no-cost strains suggesting that a more efficient energy storage may justify the fitness costs due to the over

**III.** The pyrethroid-resistant no-cost strain of *S. zeamais* show higher grain loss, higher respiration rate, higher body mass, and larger energy reserve cells than the pyrethroidresistant cost strain and susceptible strains (Guedes et al., 2006). These advantages cause resistant no-cost strain has additional reserved energy for detoxifying insecticides

Chemical pesticides are used to control target pests. Extensive use of pesticides after World War II has substantially increased the agricultural production. However non target organisms including human and wildlife are affected. Pesticides are bioactive molecules that interfere with vital biochemical and physiological processes in organisms. Some are lethal to exposed organisms and many can cause disorder at sub lethal level. Extensive research is necessary to clarify the side effects of pesticides on organisms. About 3 billion kg of pesticides is applied each year with a purchase price rose to \$47 billion in 2008, worldwide

without any adverse effect on their life table parameters (Guedes et al., 2006). Some resistant strains of green peach potato aphid in the UK showed various fitness costs, such as reduced overwintering ability, lower rate of movement away from senescing plant leaves at low temperatures, reduced responses to alarm pheromones and reduced reproduction (Foster et al., 2000, 2002, 2005; Ghadamyari et al., 2008). Foster et al., 2000 concluded that differences in behavior reducing *M. persicae* survival due to pleiotropic effects of the *kdr* mechanism and over expression of E4 and FE4 gene (Foster et al., 2000) Also behavioural characteristics is associated with knock down resistance in the *M. domestica*

no fitness costs in other strain (Fragoso et al., 2005; Oliveira et al., 2007).

expression of detoxify enzymes (Lopes et al., 2010).

**5. Pesticides residue in the environment** 

biochemical parameters as follow:

(Foster et al., 2003).

(Pimentel, 2009; Frabotta, 2009).

*Tetranychus urticae* resistance to abamectin showed reduced reproductive success compared with susceptible populations and the rm of susceptible population was higher than rm of resistant population (unpublished data).

In contrast, in *Tribolium castaneum "*resistant to malathion, susceptible male individuals show reduced reproductive success compared with resistant lines" (Arnaud et al., 2005). Foster et al. (2000) showed reduced reproductive success in *M. persicae* expressing the highest levels of carboxylesterase.

#### **4.3.3 Reduced overwintering ability**

Winter field trials by Foster et al., 1996 showed that UK *M. persicae* clones expressing high levels of esterase-based resistance (i.e. R2 and R3) present higher mortality than their susceptible (S) and -R1 counterparts during worst weather conditions (Foster et al., 1996). In *Heliothis virescens*, resistance to Cry1Ac is recessive and associated to cadherin gene (Morin et al., 2003), research showed fitness costs associated resistance affecting overwintering success and survival on non-Bt cotton (Carriere and Tabashnik, 2001). The frequency of pink bollworm resistance to Bt cotton has not increased in the field compared with laboratory that show fitness cost (Tabashnik et al., 2003). Monitoring *Culex pipiens* mosquitoes overwintering in a cave in southern France (in an area where OP insecticides are widely used) showed a "decrease in the frequency of insecticide-resistant mosquitoes compared with susceptible counterpart, indicating a huge fitness cost" (Gazave et al., 2001). In the pink bollworm, *Pectinophora gosypiella*, has been shown fitness costs at low temperatures associated with resistance to Bt and this can delay resistance of this pest to Bt cotton (Carrie`re et al., 2001; Tabashnik et al., 2005).

#### **4.3.4 Why insecticide resistance caused a fitness cost?**

The occurrence of fitness costs in insecticide resistant strain is reported for many pests such as *M. persicae* (Ghadamyari et al., 2008), *T. urticae* (unpublished data) and *Sitophilus zeamais*  (Coleoptera: Curculionidae) (Arau´jo et al., 2008a, 2008b; Guedes et al., 2006). Populations of *T. urticae*, *S. zeamais* and *M. persicae* with different levels of resistance to different pesticides have shown to be good subjects and models for evaluating the physiological base of fitness cost associated pesticide resistance.

Recently, some attempt was done to show the relationship between the energy consumption and the energy reserves available for metabolism of pesticides. The energy consumption could be measured using the electron transport activity (at the mitochondrial level), while reserve energy for metabolism could be achieved by measuring total lipids, protein and sugar contents by spectrophotometric method. The differences between energy consumption and the energy reserves represent the energy available for growth and biomarker of fitness cost in resistant populations. Our research on fitness cost of *T. urticae* resistant to abamectin showed that no significant differences were presented between the amounts of fuel nutrients macromolecules (carbohydrate, protein and lipid) in the resistant and susceptible populations of *T. urticae*, the amount of energy consumed was higher for resistant population when compared to its susceptible counterpart. Also the susceptible population exhibits a significantly higher *rm* than the resistant population. These suggested that the resistant population may be less fit than the susceptible compartment.

The following theory was presented by Guedes et al., 2006, Arau´jo et al., 2008a, 2008b and Lopes et al., 2010 and we attempt to discuss their theory with their justifying fitness cost in *S. zeamais*.

*Tetranychus urticae* resistance to abamectin showed reduced reproductive success compared with susceptible populations and the rm of susceptible population was higher than rm of

In contrast, in *Tribolium castaneum "*resistant to malathion, susceptible male individuals show reduced reproductive success compared with resistant lines" (Arnaud et al., 2005). Foster et al. (2000) showed reduced reproductive success in *M. persicae* expressing the

Winter field trials by Foster et al., 1996 showed that UK *M. persicae* clones expressing high levels of esterase-based resistance (i.e. R2 and R3) present higher mortality than their susceptible (S) and -R1 counterparts during worst weather conditions (Foster et al., 1996). In *Heliothis virescens*, resistance to Cry1Ac is recessive and associated to cadherin gene (Morin et al., 2003), research showed fitness costs associated resistance affecting overwintering success and survival on non-Bt cotton (Carriere and Tabashnik, 2001). The frequency of pink bollworm resistance to Bt cotton has not increased in the field compared with laboratory that show fitness cost (Tabashnik et al., 2003). Monitoring *Culex pipiens* mosquitoes overwintering in a cave in southern France (in an area where OP insecticides are widely used) showed a "decrease in the frequency of insecticide-resistant mosquitoes compared with susceptible counterpart, indicating a huge fitness cost" (Gazave et al., 2001). In the pink bollworm, *Pectinophora gosypiella*, has been shown fitness costs at low temperatures associated with resistance to Bt and this can delay resistance of this pest to Bt cotton

The occurrence of fitness costs in insecticide resistant strain is reported for many pests such as *M. persicae* (Ghadamyari et al., 2008), *T. urticae* (unpublished data) and *Sitophilus zeamais*  (Coleoptera: Curculionidae) (Arau´jo et al., 2008a, 2008b; Guedes et al., 2006). Populations of *T. urticae*, *S. zeamais* and *M. persicae* with different levels of resistance to different pesticides have shown to be good subjects and models for evaluating the physiological base of fitness

Recently, some attempt was done to show the relationship between the energy consumption and the energy reserves available for metabolism of pesticides. The energy consumption could be measured using the electron transport activity (at the mitochondrial level), while reserve energy for metabolism could be achieved by measuring total lipids, protein and sugar contents by spectrophotometric method. The differences between energy consumption and the energy reserves represent the energy available for growth and biomarker of fitness cost in resistant populations. Our research on fitness cost of *T. urticae* resistant to abamectin showed that no significant differences were presented between the amounts of fuel nutrients macromolecules (carbohydrate, protein and lipid) in the resistant and susceptible populations of *T. urticae*, the amount of energy consumed was higher for resistant population when compared to its susceptible counterpart. Also the susceptible population exhibits a significantly higher *rm* than the resistant population. These suggested

The following theory was presented by Guedes et al., 2006, Arau´jo et al., 2008a, 2008b and Lopes et al., 2010 and we attempt to discuss their theory with their justifying fitness cost in

that the resistant population may be less fit than the susceptible compartment.

resistant population (unpublished data).

highest levels of carboxylesterase.

**4.3.3 Reduced overwintering ability** 

(Carrie`re et al., 2001; Tabashnik et al., 2005).

cost associated pesticide resistance.

*S. zeamais*.

**4.3.4 Why insecticide resistance caused a fitness cost?** 

Populations of *S. zeamais* with different levels of susceptibility to insecticides were used a model for evaluating the mechanisms of fitness cost associated resistance. Demographic and competition studies carried out on different strains of *S. zeamais* susceptible and resistant to pyrethroids showed fitness costs associated with insecticide resistance in some strains and no fitness costs in other strain (Fragoso et al., 2005; Oliveira et al., 2007).

The susceptible, resistant no-cost and resistant cost strains showed some differences in some biochemical parameters as follow:


Some resistant strains of green peach potato aphid in the UK showed various fitness costs, such as reduced overwintering ability, lower rate of movement away from senescing plant leaves at low temperatures, reduced responses to alarm pheromones and reduced reproduction (Foster et al., 2000, 2002, 2005; Ghadamyari et al., 2008). Foster et al., 2000 concluded that differences in behavior reducing *M. persicae* survival due to pleiotropic effects of the *kdr* mechanism and over expression of E4 and FE4 gene (Foster et al., 2000) Also behavioural characteristics is associated with knock down resistance in the *M. domestica* (Foster et al., 2003).
