ISJ 5: 97-xxx, 2008 ISJ 5: 97-102, 2008 ISSN 1824-307X RESEARCH REPORT Resistance mechanisms to oxydemeton-methyl in Tetranychus urticae Koch (Acari: Tetranychidae) M Ghadamyari, JJ Sendi Department of Plant Protection, College of Agriculture, University of Guilan, Rasht, Iran Accepted July 21, 2008 Abstract The resistance mechanisms to oxydemeton-methyl were surveyed in two Iranian strains of the two spotted spider mite, Tetranychus urticae Koch. Bioassay was carried out on two strains, collected from Tehran and Rasht using dipping method. The results of bioassay indicated that resistance ratio was 20.47 for resistant strain. The activity of esterase and glutathione S-transferase in resistant and susceptible strains showed that one of resistance mechanisms to oxydemeton-methyl was esterase- based resistance and glutathione S-transferase. The esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-naphtyl acetate (α-NA) and β-naphtyl acetate (β-NA) respectively. The kinetic characteristics acetylcholinesterase (AChE) showed that the AChE of resistant strain had lower affinity to artificial substrates; acetylthiocholine and butyrylthiocholine than that of susceptible strain. I50 of oxydemeton-methyl for resistant and susceptible strains were 2.68×10-6 M and 7.79×10-7 M respectively. The results suggested that AChE of resistant strain is insensitive to oxydemeton-methyl and ratio of AChE insensitivity of resistant to susceptible strain were 3.49 and 7.8-fold to oxydemeton-methyl and paraoxon, respectively. Key words: Tetranychus urticae; oxydemeton-methyl; esterase; insensitive acetylcholinesterase; glutathione S- transferase Introduction The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is an important agricultural pest with a global distribution. Its phytophagous nature, high reproductive potential and short life cycle facilitate rapid resistance development to many acaricides often after a few applications (Cranham and Helle 1985; Keena and Granett, 1990; Devine et al., 2001; Stumpf and Nauen, 2001). So far resistance have been reported in several countries for compounds, such as organophosphates (OPs) (Sato et al, 1994; Anazawa et al., 2003), dicofol (Fergusson-Kolmes et al., 1991), organotins (Edge and James, 1986); hexythiazox (Herron and Rophail, 1993), clofentezine (Herron et al., 1993); fenpyroximate (Sato et al., 2004) and abamectin (Beers et al., 1998). ___________________________________________________________________________ Corresponding author: M Ghadamyari Department of Plant Protection College of Agriculture University of Guilan, Rasht, Iran E-mail: ghadamyari@guilan.ac.ir Insensitive AChE causing OP resistance is widespread and has been detected in T. urticae strains from Germany (Matsumura and Voss, 1964; Smissaert et al., 1970), Japan (Anazawa et al., 2003) and New Zealand (Ballantyne and Harrison, 1967) and in a few other tetranychid pest species, including T. cinnabarinus from Israel (Zahavi and Tahori, 1970) and T. kanzawai from Japan (Kuwahara, 1982 and Aiki et al., 2005). Also the insensitivity of AChE to demeton-S-methyl, ethyl paraoxon, chlorpyrifos oxon and carbofuran was identified in a German laboratory strain of T. urticae and a field collected strain from Florida (Stumpf et al., 2001). However, insensitive AChE was not the only mechanism of OP resistance in spider mites described, as some resistant strains of T. urticae showed an enhanced degradation of malathion, malaoxon, and ethyl parathion to nontoxic products (Matsumura and Voss, 1964; Herne and Brown, 1969). OP-resistant strains of T. kanzawai rapidly degraded malathion in vitro and the resistance was obviously attributed to high nonspecific esterase activity (Kuwahara, 1981, 1982). Pilz et al. 97 mailto:ghadamyari@guilan.ac.ir (1978) showed that a German dimethoate- selected laboratory strain of T. urticae possessed multiple mechanisms of OP resistance. In addition to an AChE insensitive to dimethoxon, the toxicity of dimethoate was enhanced by synergists, such as piperonyl butoxide indicating the involvement of cytochrome P-450-mediated oxidative detoxication. Oxydemeton-methyl is currently used in Iran to control some pests, such as aphids and T. urticae in several crops. The intensive use of oxydemeton- methyl to control of T. urticae and aphids in greenhouse facilitates resistance development in some populations of T. urticae in Iran. There is no information about oxydemeton-methyl resistance in this pest in Iran. Resolution of the underlying biochemical mechanisms of resistance can play an important role in circumventing problems associated with pesticide resistance and assist in rational choices of chemicals for pesticide mixtures and rotations. The purpose of this study was to collect information about the presence of esterases, gluthathion s-transferase and insensitive acetylcholinesterases in the resistance of T. urticae by bioassays and biochemical assays. Material and Methods Two spotted spider mite strains The resistant strain was collected from infected been plants grown in the research greenhouse in Plant Pests and Disease Research Institute of Iran, Tehran. A strain from Rasht was considered as a strain susceptible to oxydemeton-methyl which had no previous exposure to pesticides and was collected from Convolvulus sp. in University of Guilan. The mites were reared routinely on been plants (Phaseolus vulgaris) grown under greenhouse conditions [25 ± 4 °C, 60 ± 20 RH (relative humidity)]. Pesticide Oxydemeton-methyl was used as the commercial formulation in the bioassay (EC 25 %) and was purchased from Bayer Crop Science, Germany. Chemicals Acetylthiocholine iodide (ATC), S- butyrylthiocholine iodide (BTC), 5,5 ́-dithiobis-(2- nitrobenzoic acid, DTNB), triton X-100 were purchased from Sigma. Fast blue RR salt, α-naphtyl acetate (α-NA) and β-naphthyl acetate (β-NA) were obtained from Fluka, and oxydemeton-methyl from Accustandard. 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) were purchased from Merck, Germany. Bioassay The toxicities of oxydemeton-methyl to the susceptible and resistant strains of two-spotted spider mite were assayed using the dipping method. The formulated oxydemeton-methyl was diluted with distilled water to generate five serial dilutions. The leaf disc (diameter 3.5 cm) was immersed in the dilutions for 45 s. After drying, adult mites were placed on each treated leaf disk on wet cotton in a petri dish. Up to 10 adults were placed on each leaf disk. Mortality was assessed after the treated mites were maintained at 25 ± 2 °C, 70 ± 10 RH and 16:8 (Light: Dark) for 48 h. Mites that could walk at least one body length after a gentle probe with a fine brush were scored alive. Bioassay data were analyzed for LD50 values and their 95 % confidence intervals (95 % CL) using the POLO-PC computer program (LeOra Software 1987). Resistance factors (RF) were calculated by dividing the LD50 value of the resistant strain by the LD50 value of the susceptible strain. Determination of esterase activity Adults were homogenized in ice-cold 0.2 M phosphate buffer (pH 7.0) containing 0.05 % triton X-100. After the homogenates were centrifuged at 10000 g for 12 min at 4 °C. The esterase activity was measured according to van Asperen’s method (van Asperen, 1962). The substrate was α-NA and β-NA. Fifteen µl of supernatant was added to a microplate containing 35 µl 0.2 M, pH 7.0, phosphate buffer per well. The addition of 100 µl substrate per well (0.65 mM in buffer) initiated a reaction. After incubation for exactly 10 min at room temperature, 50 µl of fast blue RR salt was added and the microplate left in the dark for 30 min. Absorbance at 450 nm (OD450) was then measured in a microplate reader (Awareness Stat Fax® 3200). Determination of glutathione S-transferase (GST) Adults were homogenized in ice-cold 0.2 M phosphate buffer (pH 7.0). After the homogenates were centrifuged at 10,000xg for 12 min at 4 °C. GST activity was measured using 1-chloro-2,4- dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and reduced GSH as substrates with slight modifications according to Habig et al. (1974) in 96- well microplates. The total reaction volume per well of a 96-well microplate was 300 µl, consisting of 100 µl, supernatant, CDNB (or DCNB) and GSH in buffer, giving final concentrations of 0.4 and 4 mM of CDNB (or DCNB) and GSH, respectively. The non-enzymatic reaction of CDNB (or DCNB) with GSH measured without supernatant served as control. The change in absorbance was measured continuously for 10 min at 340 nm in a Thermomax kinetic microplate reader (Awareness Stat Fax® 3200). AChE kinetics Mites were homogenized in ice-cold 0.2 M phosphate buffer (pH 7.0) containing 0.1 % triton X-100. After the homogenates were centrifuged at 10000 g for 15 min at 4 oC. AChE activity was measured according to the methods of Stumpf et al (2001) with some modifications. Fifty microliters of the enzyme source was added to each well of microplate containing 140 µl of 0.2 M phosphate buffer (pH 7.0) and 20 µl DTNB solution. Then 40 µl of ATC was added to each well. The concentrations of the substrate were changed from 0.01 mM to 10 mM to evaluate the Michaels’s constant (Km). Optical density was measured at 415 nm with a microplate reader (Awareness Stat Fax® 3200). 98 Table 1 Log dose probit-mortality data for oxydemeton-methyl against susceptible and resistant strain of T. urticae Strain Insecticide n LD50 (95 % CI) a Slope ± SE χ2 b RRc Resistant oxydemeton-methyl 245 4675.9 (4473- 4892) 10.79±1.36 0.88 20.47 Susceptible oxydemeton-methyl 250 228.6 (191-268) 2.5 ± 0.27 1.11 aLD50 values and their CI are expressed in ppm formulated pesticide bValues of χ2 smaller than 7.81 (p < 0.05) considered to be represented satisfactory agreement between observed and expected results cResistance ratio, LD50 of resistant strain/LD50 of susceptible strain Inhibition assay The enzyme was preincubated with inhibitor at 37 °C for 15 min. After preincubation, the ATC substrate was added to the mixture (containing 0.2 M phosphate buffer (pH 7.0) and DTNB). The remaining activity was determined at 30 min following preparation of the reaction mixture. Optical density was measured at 415 nm with a microplate reader (Awareness Stat Fax® 3200). I50 values for the AChE of susceptible and resistant strains were estimated by probit analysis using the POLO-PC computer program. Results Resistance levels in bioassay Table 1 summarizes the toxicological data for susceptible and resistant strains exposed to oxydemeton-methyl. The resistance ratio of the resistant strain was 20.47. Fig. 1 Esterase activity in resistant and susceptible strains of T. urticae. The asterisk (*) indicates significant differences between the two strains at P<0.01 (t-test) Activity of esterase The measured esterase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test P < 0.001). The esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-NA and β-NA respectively (Fig. 1). Activity of GST The measured glutathione S-transferase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test P < 0.001). The glutathione s-transferase activity of the resistant strain was 1.75 and 1.27-fold higher than those of the susceptible strain for CDNB and DCNB, respectively (Fig. 2). Kinetic analysis of AChE The effect of substrate concentrations on AChE activity were investigated using ATC and BTC. The different specificities of AChE in resistant and susceptible strains toward two substrates are summarized in Table 2. Km values suggest that AChE in resistant strain was kinetically different from that in susceptible strain, indicating qualitative differences among enzymes in two strains. The kinetic study indicated that AChE from the resistant strain had 1.55 and 2.16- fold lower affinities to substrates ATC and BTC than susceptible strain respectively. AChE of susceptible strain showed significantly higher affinity toward BTC than AChE of resistant strain, suggesting that a modification of the enzyme catalytic site might be present in the AChE from the resistant mite. Inhibition of AChE by oxydemeton-methyl and paraoxon A comparison of the I50 values of the susceptible and resistant strains showed 3.49 and 7.8-fold resistance to oxydemeton-methyl and ethyl paraoxon, respectively (Table 3; Fig. 3). Discussion Metabolic resistance mechanisms seem to be most important in arthropod species exhibiting resistance to organophosphate and carbamate pesticides (Devonshire et al., 1982; Kono and Tomita, 1992; Moores et al., 1994; Ghadamyari et 99 Table 2 Km and Vmax values of AChE in resistant and susceptible strains of T. urticae Vmax (ΔOD/30 min/mite) (±SD) Km (µM) (±SD) Strain Substrate 5 ± 0.4 95± 5.2* resistant ATC 4.33 ± 0.31 61± 4.1 susceptible 3.2±0.27 337±32* resistant BTC 2.9±0.23 156±23 susceptible The asterisk (*) indicates significant differences between the two strains at P<0.01 (t-test) al., 2008a, b). Our results showed that probably glutathione S-transferase was related to oxydemeton-methyl resistance in T. urticae, and there is 1.75- and 1.27-fold increase in glutathione S-transferase activity in the resistant strain, when CDNB and DCNB were used as substrate respectively. GSTs are detoxification enzymes frequently associated with insecticides resistance, particularly OP resistance (Soderlund and Bloomquist, 1990; Yu, 1996). These enzymes may act as binding proteins increasing the activity of other pesticide detoxification enzymes such as esterases (Grant and Matsumura, 1994). Also esterases have a role in resistance of T. urticae to oxydemeton-methyl (Fig.1). These enzymes probably sequester or degrade insecticide esters before they reach their target sites in the nervous system. This mechanism seems to be important in the insecticide resistance of Culex mosquitoes (Mouches et al, 1986; Kono and Tomita, 1992; Tomita et al., 1996) and Aphis gossypii (Suzuki et al., 1993). The relationship between the enzymes which catalyze hydrolysis of β-NA and degradation of malathion was studied in resistance Fig. 2 GST activity in resistant and susceptible strains of T. urticae. The asterisk (*) indicates significant differences between the two strains at P<0.01 (t-test) and susceptible strains of T. kanzawai Kishida by Kuwahara (1981). Their results showed that resistance to malathion was associated with increased esterase activity at E3 and E4 bonds on which the main peak of malathion degradation was detected. Further experimental data are required to evaluate the importance of these two degradation pathways and to clarify the existence of general esterase and glutathione transferase for oxydemeton-methyl resistance in T. urticae. Although metabolic detoxification mechanisms are implicated, insensitive AChE is considered one of the mechanisms of resistance to oxydemeton- methyl in T. urticae. The occurrence of pesticide- insensitive AChE in spider mite was first demonstrated by Smissaert (1964). The present study indicates that the resistant strain possesses an altered AChE with decreased sensitivity to inhibition by oxydemeton-methyl and paraoxon and decreased affinity to ATC and BTC substrates. The Km values for ATC determined in our study were 95 and 61 µM for the insensitive and sensitive forms of AChE, respectively (Table 2). The maximum velocities of AChE from resistant and susceptible strains were equal and only differed in terms of the affinity toward ATC and BTC (Table 2). Our results agree well with those reported by Anazawa et al. (2003) with respect to the involvement of insensitive AChE in conferring OP resistance in T. urticae. Because AChE from the resistant strain had reduced affinity to ATC and BTC (i.e., increased Km values) and reduced sensitivity to inhibition by oxydemetn-methyl and paraoxon (i.e., increased I50 values) compared with AChE from susceptible strain, it is clear that the resistant strain possesses qualitatively altered AChE. Recent molecular investigations suggest that some amino acid substitutions in the AChE of T. urticae may result in different responses of the altered AChEs to different substrates and inhibitors (Anazawa et al., 2003). Therefore the amino acid sequences of AChE in Iranian strains need to be analyzed. The mechanisms of oxydemeton-methyl resistance in T. urticae vary and belong to different classes of biochemical mechanisms and both detoxification and target alteration are involved in resistance of T. urticae to oxydemeton-methyl. Zhu and Gao (1999) showed that the modified AChE alone was not sufficient to cause a high degree of resistance in insects. It seems that these two mechanisms (esterase, GST and AChE insensitivity) have an additive interaction in T. urticae. On the basis of these data, it can not be 100 Table 3 I50 values of oxydemeton-methyl and paraoxon on AChE from susceptible and resistant strains of T. urticae I50 (M) (95 % CI) Inhibitor Resistant Susceptible IR (95 % CI)a 2.68×10-6 (2.3×10-6- 3.15×10-6) 7.79×10-7 (6.6×10-7- 9×10-7) 3.49 (2.82-4.37)* Oxydemeton-methyl Paraoxon 6.5×10-6 (5.4×10-6- 7.8×10-6) 8×10-7 (5.2×10-7-12.2×10-7) 7.8(5.2-11.8)* aInsensitivity ratio = I50 for resistant strain/susceptible strain and confidence interval (CI) The asterisk (*) indicates significant differences between the two strains at P<0.05 (t-test) decided which mechanism is the dominant factor in the oxydemeton-methyl resistance. Resistant strain has high potential to develop cross-resistance to parathion since the AChE from resistant strain showed 7.8-fold insensitivity to ethyl paraoxon and this strain had no previous exposure to parathion. It may be inferred that probably altered AChE due to extensive use of oxydemeton methyl might have caused insensitivity to paraoxon. In conclusion, oxydemeton-methyl is the most commonly used insecticide and acaricide for controlling T. urticae and aphids in Iran and therefore the present findings may be regarded as a future strategy for controlling T. urticae. Fig. 3 Inhibition of AChE from T. urticae by oxydemeton-methyl and ethyl paraoxon Acknowledgments This work was supported by University of Guilan (Rasht, Iran). References Anazawa Y, Tomita T, Aiki Y, Kozaki T, Kono Y. Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, Tetranychus urticae, Insect Biochem. Mol. Biol. 33: 509-514, 2003. Aiki Y, Kozaki T, Mizuno H, Kono Y. Amino acid substitution in Ace paralogous acetylcholinesterase accompanied by organophosphate resistance in spider mite Tetranychus kanzawai. Pestic. Biochem. Physiol. 82: 154-161, 2005. Ballantyne GH, Harrison RA. Genetic and biochemical comparisons of organophosphate resistance between strains of spider mites (Tetranychus species: Acari), Entomol. Exp. Appl. 10: 231-239, 1967. Beers EH, Riedl H, Dunley JE. Resistance to abamectin and reversion to susceptibility to fenbutatin oxide in spider mite (Acari: Tetranychidae) populations in the Pacific Northwest. J. Econ. Entomol. 91: 352-360, 1998. Cranham JE, Helle W. Pesticide resistance in Tetranychidae. In: Helle W, Sabelis MW (eds), Spider mites: Their biology, natural enemies, and control. Vol. 1B, Amsterdam, Elsevier, pp 405-421, 1985. Devine GJ, Barber M, Denholm I. Incidence and inheritance of resistance to METI-acaricides in European strains of the two-spotted spider mite (Tetranychus urticae) (Acari: Tetranychidae). Pest Manag. Sci. 57: 443-448, 2001. Devonshire AL, Moores GD. A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate, pyrethroid resistance in peach potato aphid (Myzus persicae). Pestic. Biochem. Physiol. 18: 235- 246, 1982. Edge VE, James, DG. Organotin resistance in Tetranychus urticae (Acari: Tetranychidae) in Australia. J. Econ. Entomol. 79: 1477-1483, 1986. Fergusson-Kolmes LA, Scott JG, Dennehy TJ. Dicofol resistance in Tetranychus urticae (Acari: Tetranychidae): Cross-resistance and pharmacokinetics. J. Econ. Entomol. 84: 41-48, 1991. 101 Ghadamyari M, Mizuno H, Oh S, Talebi K, Kono Y. Studies on pirimicarb resistance mechanisms in Iranian populations of the peach-potato aphid, Myzus persicae. Appl. Entomol. Zool. 43: 149- 157, 2008a. Ghadamyari M, Talebi K, Mizuno H, Kono Y. Oxydemeton-methyl resistance, mechanisms and associated fitness cost in green peach potato aphids (Homopotera: Aphididae). J. Econ. Entomol. [in press], 2008b. Grant DF, Matsumura F. Glutathione S-transferase 1 and 2 in susceptible and insecticide resistant Aedes aegypti. Pestic. Biochem. Physiol. 33: 132-143, 1994. Habig WH, Pabst MJ, Jakoby WB. Glutathione S- transferases, the first step in mercapturic acid formation. J. Biol. Chem. 249: 7130-7139, 1974. Herne DHC, Brown AWA. Inheritance and biochemistry of OP-resistance in a New York strain of the two-spotted spider mite. J. Econ. Entomol. 62: 205-209, 1969. Herron GA, Edge V, Rophail J. Clofentezine and hexythiazox resistance in Tetranychus urticae Koch in Australia. Exp. Appl. Acarol. 17: 433- 440, 1993. Herron GA, Rophail J. Genetics of hexythiazox resistance in two spotted spider mite, Tetranychus urticae Koch. Exp. Appl. Acarol. 17: 423-431, 1993. Keena MA, Granett J. Genetic analysis of propargite resistance in pacific spider mites and two spotted spider mites (Acari: Tetranychidae). J. Econ. Entomol. 83: 655-661, 1990. Kono Y, Tomita T. Characteristics of highly active carboxylesterases in insecticide-resistant Culex pipiens quinquefasciatus. Jpn. J. Sanit. Zool. 43: 297-305, 1992. Kuwahara M. Insensitivity of the acetylcholinesterase from the organophosphate-resistant kanzawa spider mite, Tetranychus kanzawai Kishida (Acarina: Tetranychidae), to organophosphorus and carbamate insecticide. Appl. Entomol. Zool. 17: 486-493, 1982. Kuwahara M, Miyata T, Saito T, Eto M. Relationship between high esterase activity and in vitro degradation of 14C-Malathion by organophosphate-resistant and susceptible strains of the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acarina: Tetranychidae), and their inhibition with specific synergists. Appl. Entomol. Zool. 16: 297 -305, 1981. LeOra Software. POLO-PC: A user guide to probit or logit analysis. LeOra Software, Berkeley, CA, 1987. Matsumura T, Voss G. Mechanism of malathion and ethyl parathion resistance in the two-spotted spider mite, Tetranychus urticae. J. Econ. Entomol. 57: 911-916, 1964. Moores GD, Devine J, Devonshire AL. Insecticide- acetylcholinesterase can enhance esterase- based resistance in Myzus persicae and Myzus nicotianae. Pestic. Biochem. Physiol. 49: 114- 120, 1994. Mouches C, Pasteur N, Berge JM, Hyrien O, Raymond M, De Saint Vincent BR, De Silvestri M, Georgihiou GP. Amplification of an esterase gene is responsible to insecticide resistance in a California Culex mosquito. Science 233: 778- 780, 1986. Pilz R, Pfeiffer G, Otto D. Untersuchungen zum Resistenz mechanismus eines Dimethoat- resistenten Spinnmilbenstammes (Tetranychus urticae Koch). Arch. Phytopathol. 14: 383- 391, 1978. Sato ME, Miyata T, da Silva M, Raga A de Souza Filho MF. Selections for fenpyroximate resistance and susceptibility, and inheritance, crossresistance and stability of fenpyroximate resistance in Tetranychus urticae Koch (Acari: Tetranychidae). Appl. Entomol. Zool. 39: 293- 302, 2004. Sato ME, Suplicy Filho N, de Souza Filho MF, Takematsu AP. Resistência do ácaro rajado Tetranychus urticae (Koch, 1836) (Acari: Tetranychidae) a diversos acaricidas em morangueiro (Fragaria sp.) nos municípios de Atibaia-SP e Piedade- SP. Ecossistema 19: 40- 46, 1994. Smissaert HR. Cholinesterase inhibition in spider mites susceptible and resistant to organophosphates, Science 143: 129-131, 1964. Smissaert HR, Voerman S, Oostenbrugge L, Renooy N. Acetylcholinesterase of organophosphate susceptible and resistant spider mites. J. Agric. Food Chem. 18: 65-75, 1970. Soderlund DM, Bloomquist JR. Molecular mechanisms of insecticide resistance. In: Roush RT, Tabashnik BE (eds), Pesticide resistance in arthropods, Chapman & Hall, New York, pp.58-96, 1990. Stumpf N, Nauen R. Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari: Tetranychidae). J. Econ. Entomol. 94: 1577-1583, 2001. Stumpf N, Zebitz CPW, Kraus W, Moores, GD, Nauen R. Resistance to organophosphates and biochemical genotyping of acetylcholinesterases in Tetranychus urticae (Acari: Tetranychidae). Pestic. Biochem. Physiol. 69: 131-142, 2001. Suzuki K, Hama H, Konno Y. Carboxylesterase of the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), responsible for fenitrothion resistance as a sequestering protein. Appl. Entomol. Zool. 28: 439-450, 1993. Tomita T, Kono Y, Shimada T. Chromosomal localization of amplified esterase genes in insecticide resistant Culex mosquitoes. Insect Biochem. Molec. Biol. 26: 853-857, 1996. van Asperen K. A study of housefly esterases by means of a sensitive colorimetric method. J. Insect Physiol. 8: 401-416, 1962. Yu, SJ. Insect glutathione S-transferases. Zoological Studies. 35: 9-19, 1996. Zahavi M, Tahori AS. Sensitivity of acetylcholinesterase in spider mites to organophosphorous compounds. Biochem. Pharmacol. 19: 219-225, 1970. Zhu YK, Gao JR. Increased activity associated with reduced sensitivity of acetylcholinesterase in organophosphate-resistant greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Sci. 55: 11-17, 1999. 102