- Email: [email protected]
Partial characterization of glutathione S-transferases in pyrethroid-resistant and -susceptible populations of the maize weevil, Sitophilus zeamais
ARTICLE IN PRESS
Journal of Stored Products Research 43 (2007) 167–170 www.elsevier.com/locate/jspr
Partial characterization of glutathione S-transferases in pyrethroidresistant and -susceptible populations of the maize weevil, Sitophilus zeamais Daniel B. Fragosoa,b, Raul Narciso C. Guedesa,, Maria Goreti A. Oliveirac Departamento de Biologia Animal, Universidade Federal de Vic- osa, Vic- osa, MG 36571-000, Brazil Diretoria de Pesquisa Agropecua´ria, Fundac- a˜o Universidade do Tocantins, Palmas, TO 77123-360, Brazil c Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal de Vic- osa, Vic- osa, MG 36571-000, Brazil a
b
Accepted 5 April 2006
Abstract Glutathione S-transferases (GSTs) from a susceptible (Sete Lagoas) and two pyrethroid-resistant populations (Jacarezinho and Juiz de Fora) of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) were characterized through in vitro colorimetric assays. GSTs showed higher activity peaks at pH 9.0 and 30 1C. The Km-values for GSTs were similar among the populations except for the resistant population from Juiz de Fora, which was about two-fold higher than the susceptible population from Sete Lagoas when using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (and a fixed concentration of reduced glutathione—GSH). The Vmax of this same resistant population was also over two-fold higher than that of the pyrethroid-susceptible population when CDNB and GSH were used as substrates. The resistant population from Jacarezinho also had a slightly, but significantly, higher Vmax than the susceptible population when using these two substrates. However, there were no significant differences among the kinetic parameters of GSTs from the maize weevil populations when DCNB and GSH were used as substrates. These results provide evidence of the involvement of enhanced GST activity as an additional pyrethroid-resistant mechanism in at least some maize weevil populations from Brazil. r 2006 Elsevier Ltd. All rights reserved. Keywords: Metabolic detoxification; Enhanced glutathione conjugation; Pyrethroid resistance; Brazil
1. Introduction Glutathione S-transferases (GSTs) are detoxification enzymes frequently associated with insecticide resistance, particularly organophosphate resistance (Soderlund and Bloomquist, 1990; Yu, 1996). The involvement of GSTs in the defense against not only organophosphates, but also organochlorines and cyclodienes, is widely reported and continues to attract attention (Yu, 1996, 2002). In contrast, much less is known about the involvement of GSTs in pyrethroid resistance despite some reports correlating high GSTs in insects with pyrethroid resistance (Grant and Matsumura, 1989; Reidy et al., 1990; Legacid et al., 1993).
Corresponding author. Tel.: +55 31 3899 4008; fax: +55 31 3899 4012.
E-mail address: [email protected] (R.N.C. Guedes). 0022-474X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2006.04.002
Resistance to DDT and pyrethroids was reported in Brazilian populations of the maize weevil by the early 1990s (Guedes et al., 1995). The succession of insecticides used against Sitophilus zeamais Motschulsky in Brazil, which started with DDT and was followed by organophosphates and pyrethroids, suggests the possibility of cross and multiple resistance (Guedes et al., 1995; Ribeiro et al., 2003). Although insecticide resistance studies for this insect species are few and usually not going beyond resistance detection bioassays (Guedes et al., 1995; Subramanyam and Hagstrum, 1996; Perez-Mendoza, 1999), there are some mechanistic studies (Fragoso et al., 2003; Ribeiro et al., 2003). Preliminary investigations of insecticide resistance mechanisms in S. zeamais relied primarily upon in vivo contact bioassays with synergists and suggested the occurrence of altered target site and enhanced detoxification underlying pyrethroid resistance (Fragoso et al., 2003; Ribeiro et al., 2003).
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Recent efforts using canonical correlation analysis to establish preliminary relationships between insecticide resistance in Brazilian populations of maize weevil and their activity levels of detoxification enzymes indicated the potential effect of GSTs as a pyrethroid-resistance mechanism (Fragoso et al., 2003). The over two-fold increase of GST activity in some pyrethroid-resistant populations of maize weevil (from Jacarezinho, Juiz de Fora and Viyosa) provided further support for this contention, which was reinforced by the robust correlations between pyrethroid resistance and GST activity. Based on these recent findings and the currently unclear involvement of GSTs in pyrethroid resistance, the present study was carried out to initially characterize the activity of this enzyme group in two pyrethroid-resistant populations of maize weevil. 2. Material and methods 2.1. Insects and chemicals Three Brazilian populations of S. zeamais were used in this study. A standard susceptible population was obtained from the National Maize and Sorghum Research Center (EMBRAPA, Sete Lagoas, MG, Brazil), where it has been maintained for over 15 years without insecticide exposure and its susceptibility to pyrethroids and organophosphates is known (Guedes et al., 1994, 1995; Ribeiro et al., 2003). The second population is resistant to DDT and pyrethroids and was originally collected from infested maize in a warehouse in Jacarezinho County (State of Parana˜, Brazil) in the early 1980s; its high resistance levels to pyrethroids (41000-fold) were reported earlier and the inheritance of deltamethrin resistance in this population is sex-linked (Guedes et al., 1994, 1995). The last population was collected in April 1999, in maize grains from a cereal mill in Juiz de Fora County (State of Minas Gerais, Brazil), and shows high pyrethroid resistance resembling the Jacarezinho population and low malathion resistance (Fragoso et al., 2003). Each population was established in the laboratory from at least 500 individuals. All populations were reared in whole maize grains free of insecticides and maintained under controlled conditions (2572 1C and 7075% r.h.). The chemicals used in the present study were all of analytical quality and purchased from SigmaAldrich Quı´ mica Brazil Ltda. (Sa˜o Paulo, SP, Brazil). 2.2. Enzyme preparation Three random samples of 20 non-sexed adult insects from each population were collected, immersed in 1.5% KCl for surface sterilization and ground up in 0.1 M phosphate buffer at pH 9.0 (5.0 ml) containing 3% Triton X-100. The homogenate was filtered through cotton wool and centrifuged at 10,000gmax for 15 min. The pellet was discarded and aliquots of the supernatant were taken for determination of protein content and activity of GSTs (Fragoso et al., 2003).
2.3. Determination of protein content and enzyme assays Three replicates were used in all in vitro colorimetric assays. Protein concentration was determined by the method of Bradford using bovine serum albumin as standard (Bradford, 1976). Activity of GSTs was assayed by measuring the conjugation of two different substrates, CDNB and DCNB, because activity towards these may vary among different GST isozymes (Commandeur et al., 1995; Yu, 2002). Determinations of optimum pH were carried out using buffer solutions with pH varying from 2.0 to 10.0 at 0.5 intervals, while the temperature effect was determined by incubating the samples in a water bath (3 min) at temperatures varying between 15 and 40 1C, with 5 1C intervals. CDNB and reduced glutathione (GSH) were used for these determinations of pH and temperature effects on GST activity at the concentrations of 150 and 15 mM, respectively. Such determinations of optimum pH and temperature were carried out with the insecticidesusceptible population and the pyrethroid-resistant population from Jacarezinho. The determination of the kinetic parameters Km and Vmax for conjugation by GSTs was carried out with CDNB concentrations ranging from 0.75 to 6.0 mM and DCNB concentrations ranging from 0.3 to 10.0 mM with the GSH concentration fixed at 7.35 mM for both determinations; these parameter estimates were also carried out varying GSH from 0.075 to 7.5 mM and fixing CDNB at 1.5 mM or fixing DCNB at 3.0 mM (always at pH 9.0 and after 3 min incubation at 30 1C). The kinetic parameters were obtained by non-linear regression (Michaelis–Menten equation) using the curve-fitting procedure of SigmaPlot (SPSS, 2000). The kinetic parameter estimates from the resistant populations were compared to those of the susceptible population using Dunnett’s test (Po0:05). 3. Results The optimum pH and temperature for GST activity in S. zeamais were 9.0 and 30 1C for both populations investigated, the insecticide-susceptible (Sete Lagoas) and the pyrethroid-resistant from Jacarezinho (data not presented). The Jacarezinho population always showed higher activity levels of GST activity than the susceptible population. All of the three populations investigated showed the Michaelis–Menten hyperbolic type of velocity curve showing that the GSTs of S. zeamais follow the Michaelis– Menten kinetics in the concentration range of the substrates used (Fig. 1). The Michaelis–Menten equation model was derived to account for the kinetic properties of enzymes. The Michaelis constant (Km) and the maximal reaction velocity (Vmax) are the kinetic constants of interest. The first, i.e., Km, is the substrate concentration that results in the filling of one-half of the enzyme’s active sites (leading to an initial velocity of Vmax/2) and in its simplest form it is a (inverted) measure of the affinity of substrate binding to the enzyme active site. The second
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Table 1 Kinetic parameters of glutathione S-transferases of maize weevil populations on two substrates (CDNB, DCNB) combined with the conjugating tripeptide glutathione (GSH)
Fig. 1. Michaelis–Menten plot of the glutathione S-transferase activity from the maize weevil populations using CDNBfixed and GSH as substrates (Po0:001; R2 40:90). Insertion: Lineweaver–Burk plot (double reciprocal) (Po0:001; R2 40:94). Each symbol represents the mean of three independent replicates (n ¼ 3).
kinetic constant, Vmax, is attained when all the enzyme’s active sites are filled with substrate molecules and its importance lies in allowing the estimation of the number of substrate molecules converted into product by an enzyme in a unit of time, when the enzyme is fully saturated with substrate. By taking the reciprocal of both sides of the Michaelis–Menten equation, the disadvantages of nonlinear kinetic analysis are avoided converting it into the Lineweaver–Burk relationship, which is linear. The kinetic parameters Km and Vmax were therefore determined using the Lineweaver–Burk (double reciprocal) transformation and are presented in Table 1. The GST Km-value for the susceptible population using CDNB as substrate and fixing GSH was similar to the estimated Km-value of the pyrethroid-resistant population of Jacarezinho and half the estimated Km-value of Juiz de Fora (Table 1). The Vmax for Juiz de Fora was approximately double that from the other populations (Table 1). The kinetic determinations fixing the CDNB concentration and varying GSH produced similar Kmvalues, but the Vmax-values were significantly higher for the resistant populations, particularly in the case of the Juiz de Fora population (Table 1). When DCNB was used as substrate fixing GSH and vice versa, there were no significant differences between the kinetic parameters from pyrethroid-susceptible and resistant populations (Table 1). 4. Discussion Maize weevil resistance to pyrethroids was correlated with enhanced GST activity in a previous study (Fragoso et al., 2003), and here we tried to further explore this possibility by trying to provide preliminary characterization of GST activity in the pyrethroid-susceptible and resistant populations investigated earlier. GST activity
Vmax (mmol/min/mg protein)
Populations
Km (mM)
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
CDNB GSHfixed 3.6571.84 15.0174.56 3.8571.49 21.7274.91 7.9072.75* 32.6877.90*
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
CDNBfixed GSH 0.8770.22 7.6370.28 1.7570.56 9.5271.02* 1.1170.21 13.0070.67*
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
DCNB GSHfixed 1.6370.45 1.4870.73 1.1470.34
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
DCNBfixed GSH 4.4071.44 1.3570.23 3.7871.04 1.4770.10 2.0970.63 1.1570.12
0.3070.04 0.4270.10 0.2870.08
Letters (S) and (R) indicate which population is susceptible (S) or resistant (R) to pyrethroid insecticides. Results are reported as means7standard error (n ¼ 3). The asterisks indicate that the kinetic parameter of the resistant population is significantly different from the kinetic parameter of the susceptible population from Sete Lagoas by Dunnett’s test (Po0:05).
levels towards the substrate CDNB were always higher, but not always significantly, in the resistant populations from Jacarezinho and Juiz de Fora when compared with the susceptible population. The usually higher GST activity of the resistant population from Juiz de Fora compared with that from the Jacarezinho population is a likely consequence of their distinct selection history (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003), which also seems to lead to differences in fitness cost associated with insecticide resistance in these populations (Fragoso et al., 2005; Guedes et al., 2006). The optimum pH value observed for GST activity in the present study was higher than the pH values more frequently used for GST purification and characterization in insects, which are around 7.0 (Grant and Matsumura, 1989; Reidy et al., 1990; Legacid et al., 1993; Yu, 1996), but it is still within the range reported for this group of enzymes (Commandeur et al., 1995; Yu, 2002). Colorimetric assays for GST are frequently carried out at ambient temperature, but S. zeamais showed higher GST activity at 30 1C for both populations studied. These differences are no surprise since GSTs are a large family of detoxification enzymes with wide species distribution and a multiplicity of forms so each isoform may require specific conditions of substrate concentrations, pH, temperature, etc., for maximum activity (Commandeur et al., 1995; Yu, 2002). The higher catalytic activity of GSTs, particularly from the pyrethroid-resistant population from Juiz de Fora, provides support for the hypothesis of their involvement in the resistance to this insecticide group in some maize weevil
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populations. GSTs may act as binding proteins increasing the activity of other pyrethroid detoxification enzymes such as esterases (Grant and Matsumura, 1989; Kostaropoulos et al., 2001). However this does not seem to be the case for pyrethroid-resistant populations of the maize weevil, where the modified enzymes from the resistant populations did not show significant differences in affinity based on the Km-values obtained with the substrates used; the population from Juiz de Fora actually showed an apparently lower affinity (high Km) towards CDNB (fixed GST). An alternative explanation for the GST role as a binding protein is that the higher GST activity levels in pyrethroidresistant populations of maize weevil, as reported here, may be favoring their direct catalytic activity over pyrethroids as earlier recognized (Yu and Ngyen, 1996), or their activity as antioxidant agents decreasing the oxidative stress initiated by pyrethroids as more recently suggested (Vontas et al., 2001). Either way, there seems to be an involvement of enhanced GST activity in pyrethroid resistance in Brazilian populations of maize weevil, but this resistance mechanism is apparently secondary in importance to altered target site (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003), and is not as stable based on demographic and physiological studies with these same populations of S. zeamais (Fragoso et al., 2005; Guedes et al., 2006). Acknowledgements Appreciation is expressed to J.P. Santos, S.T. Rezende, and B.L. Damasceno for the assistance and suggestions provided. Financial support provided by the CAPES, CNPq, and FAPEMIG was also greatly appreciated. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Commandeur, J.N.M., Stijntjes, G.J., Vermeulen, N.P.E., 1995. Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Pharmacology Review 47, 271–330. Fragoso, D.B., Guedes, R.N.C., Rezende, S.B., 2003. Glutathione Stransferase detoxification as a potential pyrethroid resistance mechanism in the maize weevil, Sitophilus zeamais. Entomologia Experimentalis et Applicata 109, 21–29. Fragoso, D.B., Guedes, R.N.C., Peternelli, L.A., 2005. Developmental rates and population growth of insecticide-resistant and susceptible
populations of Sitophilus zeamais. Journal of Stored Products Research 41, 271–281. Grant, D.F., Matsumura, F., 1989. Glutathione S-transferases 1 and 2 in susceptible and insecticide resistant Aedes aegypti. Pesticide Biochemistry and Physiology 33, 132–143. Guedes, R.N.C., Lima, J.O.G., Santos, J.P., Cruz, C.D., 1994. Inheritance of deltamethrin resistance in a Brazilian strain of maize weevil (Sitophilus zeamais Mots.). International Journal of Pest Management 40, 103–106. Guedes, R.N.C., Lima, J.O.G., Santos, J.P., Cruz, C.D., 1995. Resistance to DDT and pyrethroids in Brazilian populations of Sitophilus zeamais Motsch. (Coleoptera: Curculionidae). Journal of Stored Products Research 31, 145–150. Guedes, R.N.C., Oliveira, E.E., Guedes, N.M.P., Ribeiro, B., Serra˜o, J.E., 2006. Cost and mitigation of insecticide resistance in the maize weevil, Sitophilus zeamais. Physiological Entomology 31, 30–38. Kostaropoulos, I., Papadopoulos, A.I., Metaxakis, A., Boukouvala, E., Papadopoulou-Mourkidou, E., 2001. Glutathione S-transferases in the defense against pyrethroids in insects. Pesticide Biochemistry and Physiology 31, 313–319. Legacid, L., Cuany, A., Berge´, J.B., Echaubard, M., 1993. Purification and partial characterization of glutathione S-transferases from insecticideresistant and lindane-induced susceptible Spodoptera littoralis (Boisd.) larvae. Insect Biochemistry and Molecular Biology 23, 467–474. Perez-Mendoza, J., 1999. Survey of insecticide resistance in Mexican populations of maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Journal of Stored Products Research 35, 107–115. Reidy, G.F., Rose, H.A., Visetson, S., Murray, M., 1990. Increased glutathione S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum (Herbst.). Pesticide Biochemistry and Physiology 36, 269–276. Ribeiro, B.M., Guedes, R.N.C., Oliveira, E.E., Santos, J.P., 2003. Insecticide resistance and synergism in Brazilian populations of Sitophilus zeamais (Coleoptera: Curculionidae). Journal of Stored Products Research 35, 21–31. Soderlund, D.M., Bloomquist, J.R., 1990. Molecular mechanisms of insecticide resistance. In: Roush, R.T., Tabashnik, B.E. (Eds.), Pesticide Resistance in Arthropods. Chapman & Hall, New York, pp. 58–96. SPSS, 2000. SigmaPlot 2000 User’s Guide. SPSS Inc., Chicago. Subramanyam, Bh., Hagstrum, D.W., 1996. Resistance measurement and management. In: Subramanyam, Bh., Hagstrum, D.W. (Eds.), Integrated Management of Insects in Stored Products. Marcel Dekker, New York, pp. 331–397. Vontas, J.G., Small, G.J., Hemingway, J., 2001. Glutathione S-transferases as antioxidant defense agents confer pyrethroid resistance in Nilaparvata lugens. Biochemical Journal 357, 65–72. Yu, S.J., 1996. Insect glutathione S-transferases. Zoological Studies 35, 9–19. Yu, S.J., 2002. Substrate specificity of glutathione S-transferases from the fall armyworm. Pesticide Biochemistry and Physiology 74, 41–51. Yu, S.J., Ngyen, S.N., 1996. Insecticide susceptibility and detoxification enzyme activities in permethrin-selected diamondback moths. Pesticide Biochemistry and Physiology 56, 69–77.
Journal of Stored Products Research 43 (2007) 167–170 www.elsevier.com/locate/jspr
Partial characterization of glutathione S-transferases in pyrethroidresistant and -susceptible populations of the maize weevil, Sitophilus zeamais Daniel B. Fragosoa,b, Raul Narciso C. Guedesa,, Maria Goreti A. Oliveirac Departamento de Biologia Animal, Universidade Federal de Vic- osa, Vic- osa, MG 36571-000, Brazil Diretoria de Pesquisa Agropecua´ria, Fundac- a˜o Universidade do Tocantins, Palmas, TO 77123-360, Brazil c Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal de Vic- osa, Vic- osa, MG 36571-000, Brazil a
b
Accepted 5 April 2006
Abstract Glutathione S-transferases (GSTs) from a susceptible (Sete Lagoas) and two pyrethroid-resistant populations (Jacarezinho and Juiz de Fora) of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) were characterized through in vitro colorimetric assays. GSTs showed higher activity peaks at pH 9.0 and 30 1C. The Km-values for GSTs were similar among the populations except for the resistant population from Juiz de Fora, which was about two-fold higher than the susceptible population from Sete Lagoas when using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (and a fixed concentration of reduced glutathione—GSH). The Vmax of this same resistant population was also over two-fold higher than that of the pyrethroid-susceptible population when CDNB and GSH were used as substrates. The resistant population from Jacarezinho also had a slightly, but significantly, higher Vmax than the susceptible population when using these two substrates. However, there were no significant differences among the kinetic parameters of GSTs from the maize weevil populations when DCNB and GSH were used as substrates. These results provide evidence of the involvement of enhanced GST activity as an additional pyrethroid-resistant mechanism in at least some maize weevil populations from Brazil. r 2006 Elsevier Ltd. All rights reserved. Keywords: Metabolic detoxification; Enhanced glutathione conjugation; Pyrethroid resistance; Brazil
1. Introduction Glutathione S-transferases (GSTs) are detoxification enzymes frequently associated with insecticide resistance, particularly organophosphate resistance (Soderlund and Bloomquist, 1990; Yu, 1996). The involvement of GSTs in the defense against not only organophosphates, but also organochlorines and cyclodienes, is widely reported and continues to attract attention (Yu, 1996, 2002). In contrast, much less is known about the involvement of GSTs in pyrethroid resistance despite some reports correlating high GSTs in insects with pyrethroid resistance (Grant and Matsumura, 1989; Reidy et al., 1990; Legacid et al., 1993).
Corresponding author. Tel.: +55 31 3899 4008; fax: +55 31 3899 4012.
E-mail address: [email protected] (R.N.C. Guedes). 0022-474X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2006.04.002
Resistance to DDT and pyrethroids was reported in Brazilian populations of the maize weevil by the early 1990s (Guedes et al., 1995). The succession of insecticides used against Sitophilus zeamais Motschulsky in Brazil, which started with DDT and was followed by organophosphates and pyrethroids, suggests the possibility of cross and multiple resistance (Guedes et al., 1995; Ribeiro et al., 2003). Although insecticide resistance studies for this insect species are few and usually not going beyond resistance detection bioassays (Guedes et al., 1995; Subramanyam and Hagstrum, 1996; Perez-Mendoza, 1999), there are some mechanistic studies (Fragoso et al., 2003; Ribeiro et al., 2003). Preliminary investigations of insecticide resistance mechanisms in S. zeamais relied primarily upon in vivo contact bioassays with synergists and suggested the occurrence of altered target site and enhanced detoxification underlying pyrethroid resistance (Fragoso et al., 2003; Ribeiro et al., 2003).
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Recent efforts using canonical correlation analysis to establish preliminary relationships between insecticide resistance in Brazilian populations of maize weevil and their activity levels of detoxification enzymes indicated the potential effect of GSTs as a pyrethroid-resistance mechanism (Fragoso et al., 2003). The over two-fold increase of GST activity in some pyrethroid-resistant populations of maize weevil (from Jacarezinho, Juiz de Fora and Viyosa) provided further support for this contention, which was reinforced by the robust correlations between pyrethroid resistance and GST activity. Based on these recent findings and the currently unclear involvement of GSTs in pyrethroid resistance, the present study was carried out to initially characterize the activity of this enzyme group in two pyrethroid-resistant populations of maize weevil. 2. Material and methods 2.1. Insects and chemicals Three Brazilian populations of S. zeamais were used in this study. A standard susceptible population was obtained from the National Maize and Sorghum Research Center (EMBRAPA, Sete Lagoas, MG, Brazil), where it has been maintained for over 15 years without insecticide exposure and its susceptibility to pyrethroids and organophosphates is known (Guedes et al., 1994, 1995; Ribeiro et al., 2003). The second population is resistant to DDT and pyrethroids and was originally collected from infested maize in a warehouse in Jacarezinho County (State of Parana˜, Brazil) in the early 1980s; its high resistance levels to pyrethroids (41000-fold) were reported earlier and the inheritance of deltamethrin resistance in this population is sex-linked (Guedes et al., 1994, 1995). The last population was collected in April 1999, in maize grains from a cereal mill in Juiz de Fora County (State of Minas Gerais, Brazil), and shows high pyrethroid resistance resembling the Jacarezinho population and low malathion resistance (Fragoso et al., 2003). Each population was established in the laboratory from at least 500 individuals. All populations were reared in whole maize grains free of insecticides and maintained under controlled conditions (2572 1C and 7075% r.h.). The chemicals used in the present study were all of analytical quality and purchased from SigmaAldrich Quı´ mica Brazil Ltda. (Sa˜o Paulo, SP, Brazil). 2.2. Enzyme preparation Three random samples of 20 non-sexed adult insects from each population were collected, immersed in 1.5% KCl for surface sterilization and ground up in 0.1 M phosphate buffer at pH 9.0 (5.0 ml) containing 3% Triton X-100. The homogenate was filtered through cotton wool and centrifuged at 10,000gmax for 15 min. The pellet was discarded and aliquots of the supernatant were taken for determination of protein content and activity of GSTs (Fragoso et al., 2003).
2.3. Determination of protein content and enzyme assays Three replicates were used in all in vitro colorimetric assays. Protein concentration was determined by the method of Bradford using bovine serum albumin as standard (Bradford, 1976). Activity of GSTs was assayed by measuring the conjugation of two different substrates, CDNB and DCNB, because activity towards these may vary among different GST isozymes (Commandeur et al., 1995; Yu, 2002). Determinations of optimum pH were carried out using buffer solutions with pH varying from 2.0 to 10.0 at 0.5 intervals, while the temperature effect was determined by incubating the samples in a water bath (3 min) at temperatures varying between 15 and 40 1C, with 5 1C intervals. CDNB and reduced glutathione (GSH) were used for these determinations of pH and temperature effects on GST activity at the concentrations of 150 and 15 mM, respectively. Such determinations of optimum pH and temperature were carried out with the insecticidesusceptible population and the pyrethroid-resistant population from Jacarezinho. The determination of the kinetic parameters Km and Vmax for conjugation by GSTs was carried out with CDNB concentrations ranging from 0.75 to 6.0 mM and DCNB concentrations ranging from 0.3 to 10.0 mM with the GSH concentration fixed at 7.35 mM for both determinations; these parameter estimates were also carried out varying GSH from 0.075 to 7.5 mM and fixing CDNB at 1.5 mM or fixing DCNB at 3.0 mM (always at pH 9.0 and after 3 min incubation at 30 1C). The kinetic parameters were obtained by non-linear regression (Michaelis–Menten equation) using the curve-fitting procedure of SigmaPlot (SPSS, 2000). The kinetic parameter estimates from the resistant populations were compared to those of the susceptible population using Dunnett’s test (Po0:05). 3. Results The optimum pH and temperature for GST activity in S. zeamais were 9.0 and 30 1C for both populations investigated, the insecticide-susceptible (Sete Lagoas) and the pyrethroid-resistant from Jacarezinho (data not presented). The Jacarezinho population always showed higher activity levels of GST activity than the susceptible population. All of the three populations investigated showed the Michaelis–Menten hyperbolic type of velocity curve showing that the GSTs of S. zeamais follow the Michaelis– Menten kinetics in the concentration range of the substrates used (Fig. 1). The Michaelis–Menten equation model was derived to account for the kinetic properties of enzymes. The Michaelis constant (Km) and the maximal reaction velocity (Vmax) are the kinetic constants of interest. The first, i.e., Km, is the substrate concentration that results in the filling of one-half of the enzyme’s active sites (leading to an initial velocity of Vmax/2) and in its simplest form it is a (inverted) measure of the affinity of substrate binding to the enzyme active site. The second
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Table 1 Kinetic parameters of glutathione S-transferases of maize weevil populations on two substrates (CDNB, DCNB) combined with the conjugating tripeptide glutathione (GSH)
Fig. 1. Michaelis–Menten plot of the glutathione S-transferase activity from the maize weevil populations using CDNBfixed and GSH as substrates (Po0:001; R2 40:90). Insertion: Lineweaver–Burk plot (double reciprocal) (Po0:001; R2 40:94). Each symbol represents the mean of three independent replicates (n ¼ 3).
kinetic constant, Vmax, is attained when all the enzyme’s active sites are filled with substrate molecules and its importance lies in allowing the estimation of the number of substrate molecules converted into product by an enzyme in a unit of time, when the enzyme is fully saturated with substrate. By taking the reciprocal of both sides of the Michaelis–Menten equation, the disadvantages of nonlinear kinetic analysis are avoided converting it into the Lineweaver–Burk relationship, which is linear. The kinetic parameters Km and Vmax were therefore determined using the Lineweaver–Burk (double reciprocal) transformation and are presented in Table 1. The GST Km-value for the susceptible population using CDNB as substrate and fixing GSH was similar to the estimated Km-value of the pyrethroid-resistant population of Jacarezinho and half the estimated Km-value of Juiz de Fora (Table 1). The Vmax for Juiz de Fora was approximately double that from the other populations (Table 1). The kinetic determinations fixing the CDNB concentration and varying GSH produced similar Kmvalues, but the Vmax-values were significantly higher for the resistant populations, particularly in the case of the Juiz de Fora population (Table 1). When DCNB was used as substrate fixing GSH and vice versa, there were no significant differences between the kinetic parameters from pyrethroid-susceptible and resistant populations (Table 1). 4. Discussion Maize weevil resistance to pyrethroids was correlated with enhanced GST activity in a previous study (Fragoso et al., 2003), and here we tried to further explore this possibility by trying to provide preliminary characterization of GST activity in the pyrethroid-susceptible and resistant populations investigated earlier. GST activity
Vmax (mmol/min/mg protein)
Populations
Km (mM)
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
CDNB GSHfixed 3.6571.84 15.0174.56 3.8571.49 21.7274.91 7.9072.75* 32.6877.90*
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
CDNBfixed GSH 0.8770.22 7.6370.28 1.7570.56 9.5271.02* 1.1170.21 13.0070.67*
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
DCNB GSHfixed 1.6370.45 1.4870.73 1.1470.34
Sete Lagoas (S) Jacarezinho (R) Juiz de Fora (R)
DCNBfixed GSH 4.4071.44 1.3570.23 3.7871.04 1.4770.10 2.0970.63 1.1570.12
0.3070.04 0.4270.10 0.2870.08
Letters (S) and (R) indicate which population is susceptible (S) or resistant (R) to pyrethroid insecticides. Results are reported as means7standard error (n ¼ 3). The asterisks indicate that the kinetic parameter of the resistant population is significantly different from the kinetic parameter of the susceptible population from Sete Lagoas by Dunnett’s test (Po0:05).
levels towards the substrate CDNB were always higher, but not always significantly, in the resistant populations from Jacarezinho and Juiz de Fora when compared with the susceptible population. The usually higher GST activity of the resistant population from Juiz de Fora compared with that from the Jacarezinho population is a likely consequence of their distinct selection history (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003), which also seems to lead to differences in fitness cost associated with insecticide resistance in these populations (Fragoso et al., 2005; Guedes et al., 2006). The optimum pH value observed for GST activity in the present study was higher than the pH values more frequently used for GST purification and characterization in insects, which are around 7.0 (Grant and Matsumura, 1989; Reidy et al., 1990; Legacid et al., 1993; Yu, 1996), but it is still within the range reported for this group of enzymes (Commandeur et al., 1995; Yu, 2002). Colorimetric assays for GST are frequently carried out at ambient temperature, but S. zeamais showed higher GST activity at 30 1C for both populations studied. These differences are no surprise since GSTs are a large family of detoxification enzymes with wide species distribution and a multiplicity of forms so each isoform may require specific conditions of substrate concentrations, pH, temperature, etc., for maximum activity (Commandeur et al., 1995; Yu, 2002). The higher catalytic activity of GSTs, particularly from the pyrethroid-resistant population from Juiz de Fora, provides support for the hypothesis of their involvement in the resistance to this insecticide group in some maize weevil
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populations. GSTs may act as binding proteins increasing the activity of other pyrethroid detoxification enzymes such as esterases (Grant and Matsumura, 1989; Kostaropoulos et al., 2001). However this does not seem to be the case for pyrethroid-resistant populations of the maize weevil, where the modified enzymes from the resistant populations did not show significant differences in affinity based on the Km-values obtained with the substrates used; the population from Juiz de Fora actually showed an apparently lower affinity (high Km) towards CDNB (fixed GST). An alternative explanation for the GST role as a binding protein is that the higher GST activity levels in pyrethroidresistant populations of maize weevil, as reported here, may be favoring their direct catalytic activity over pyrethroids as earlier recognized (Yu and Ngyen, 1996), or their activity as antioxidant agents decreasing the oxidative stress initiated by pyrethroids as more recently suggested (Vontas et al., 2001). Either way, there seems to be an involvement of enhanced GST activity in pyrethroid resistance in Brazilian populations of maize weevil, but this resistance mechanism is apparently secondary in importance to altered target site (Guedes et al., 1995; Fragoso et al., 2003; Ribeiro et al., 2003), and is not as stable based on demographic and physiological studies with these same populations of S. zeamais (Fragoso et al., 2005; Guedes et al., 2006). Acknowledgements Appreciation is expressed to J.P. Santos, S.T. Rezende, and B.L. Damasceno for the assistance and suggestions provided. Financial support provided by the CAPES, CNPq, and FAPEMIG was also greatly appreciated. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Commandeur, J.N.M., Stijntjes, G.J., Vermeulen, N.P.E., 1995. Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Pharmacology Review 47, 271–330. Fragoso, D.B., Guedes, R.N.C., Rezende, S.B., 2003. Glutathione Stransferase detoxification as a potential pyrethroid resistance mechanism in the maize weevil, Sitophilus zeamais. Entomologia Experimentalis et Applicata 109, 21–29. Fragoso, D.B., Guedes, R.N.C., Peternelli, L.A., 2005. Developmental rates and population growth of insecticide-resistant and susceptible
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