purification and biochemical characterization of glutathione s-transferases from four field...

A r t i c l e

PURIFICATION AND BIOCHEMICALCHARACTERIZATION OFGLUTATHIONE S-TRANSFERASESFROM FOUR FIELD POPULATIONSOF Bactrocera dorsalis (HENDEL)(DIPTERA: TEPHRITIDAE)

Fei Hu, Wei Dou, Jing-Jing Wang, Fu-Xian Jia, andJin-Jun WangKey Laboratory of Entomology and Pest Control Engineering, College ofPlant Protection, Southwest University, Chongqing 400715, People’sRepublic of China

Glutathione S-transferases (GSTs) are a group of detoxification enzymesthat catalyze the nucleophilic addition of glutathione to a wide variety ofendogenous and xenobiotic compounds. In this study, GSTs were purifiedfrom four field populations of Bactrocera dorsalis with differentinsecticide susceptibilities by glutathione–agarose affinity chromatography.The populations were collected from Dongguan (DG) and Guangzhou(GZ) of the Guangdong Province, Haikou of the Hainan province (HN),and Kunming of the Yunnan province (YN), China. Differences in GSTcharacteristics among the four populations were studied using purifiedenzyme samples through comparative SDS-PAGE, kinetic, and inhibitionexperiments. The specific activities of the purified enzymes were similar,but the purification yield of the GZ population (31.54%) was the lowest.SDS-PAGE analysis showed only one band at approximately 23 kDa forthese four populations. Kinetic analyses showed that the affinities of the

Grant sponsor: National Basic Research Program of China; Grant numbers: 2009CB125903, 2009CB119200;Grant sponsor: Natural Science Foundation of Chongqing; Grant number: CSTC, 2009BA1042; Grant sponsor:Program for Changjiang Scholars and Innovative Research Team in University; Grant number: IRT0976; Grantsponsors: Earmarked Fund for Modern Agro-industry (Citrus) Technology Research System of China; TheSpecialized Research Fund for the Doctoral Program of Higher Education in China; Grant number:20100182120022; Grant sponsor: The Science and Technology Innovation Foundation for Graduate Studentsof Southwest University; Grant number: kb2010007.Fei Hu and Wei Dou contributed equally to this work.Correspondence to: Jin-Jun Wang, College of Plant Protection, Southwest University, Chongqing 400715,P. R. China. E-mail: [email protected]

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 78, No. 4, 201–215 (2011)

Published online in Wiley Online Library (wileyonlinelibrary.com).

& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20453

purified GSTs from the GZ and YN populations for1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those ofGSTs from the other two populations, whereas the HN population had thehighest catalytic capability in terms of Vmax value. The optimumtemperature for CDNB conjugation was 371C and the optimum pH was7.5 in all four populations. Inhibition kinetics showed that ethacrynicacid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfa-lein, and b-cypermethrin had excellent inhibitory effects on GSTs in thefour populations of B. dorsalis, but the low inhibitory effects of malathionand avermectin did not differ between populations. These results suggestthat GSTs may have a role in detoxification of b-cypermethrin inB. dorsalis. �C 2011 Wiley Periodicals, Inc.

Keywords: Bactrocera dorsalis; glutathione S-transferases; purification;inhibition

INTRODUCTION

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a diverse super family of enzymeswith a range of catalytic functions, including cellular protection from reactive oxygenspecies, reductive maintenance of thiolated proteins, prostaglandin synthesis, andglutathione conjugation of endogenous and exogenous ligands (Sheehan et al., 2001;Li et al., 2007). GSTs have important roles in phase II detoxification of severalchemical insecticide classes, including organophosphates (Wei et al., 2001; Melo-Santoset al., 2010), pyrethroids (David et al., 2005; Lumjuan et al., 2011), and chlorinatedhydrocarbons, such as dichlorodiphenyltrichloroethane (DDT) (Ranson et al., 1997;Low et al., 2010). In addition, GSTs contribute to insecticide resistance in economicallyimportant agricultural pests and disease vectors (Fournier et al., 1992; Yu, 2002; Wuet al., 2009).

The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most economicallyimportant fruit fly pests worldwide. As a polyphagous species, this insect has thepotential to invade new areas and to adapt to new host plants (Clarke et al., 2005). It isan important quarantine pest (Yu et al., 2007). For example, the annual investigationplan for fruit flies in China reported the presence of B. dorsalis in more than 10provinces, across southeast to middle China; it may have spread to this wide area as aconsequence of global climate warming (Hou and Zhang, 2005). Efforts to control thispest often result in development of insecticide resistance. It has been shown to developresistance and cross-resistance to various different classes of insecticides, includingorganophosphates, pyrethroids, and carbamates (Hsu et al., 2004; Jin et al., 2011). Anacetylcholinesterase from B. dorsalis has been biochemically and molecularlycharacterized, as has an altered ace gene found in insecticide-resistant flies (Hsuet al., 2006, 2008, 2011; Jin et al., 2010). However, very little is known about thecharacteristics of GSTs in oriental fruit fly, despite their significance in thedetoxification of xenobiotics and insecticide resistance. In this study, purified GSTsfrom four different field populations of B. dorsalis that showed different levels ofsusceptibilities to malathion, avermectin, and b-cypermethrin were biochemicallycharacterized. These results could help in understanding the role of GSTs indetoxification of the insecticide in B. dorsalis.

202 � Archives of Insect Biochemistry and Physiology December 2011

Archives of Insect Biochemistry and Physiology

MATERIALS AND METHODS

Insects

Four field populations of B. dorsalis were collected as nymphs from Dongguan (DGpopulation) and Guangzhou (GZ population) of the Guangdong Province, Haikou ofthe Hainan province (HN population), and Kunming of the Yunnan province (YNpopulation), China, in 2008. The insects were reared in glass cages and fed on anartificial diet consisting of yeast powder, honey, sugar, vitamin C, and water. Each lifestage was kept in a temperature-controlled room at 27711C, 7075% relativehumidity, and a 14:10 L:D photoperiod. All experiments were conducted under theconditions described above with 3- to 5-day-old adults. The toxicities (LD50) ofmalathion against these four populations were 230.1, 161.8, 111.5, and 73.3 ng/fly forthe DG, GZ, HN, and YN populations, respectively. The LD50s of avermectin were23.7, 20.2, 16.8, and 11.8 ng/fly, and the LD50s of b-cypermethrin were 123.1, 54.9,43.4, and 25.5 ng/fly, for DG, GZ, HN, and YN populations, respectively (Wang et al.,unpublished data).

Chemicals and Reagents

Bovine serum albumin (BSA, Shanghai Bio Life Science and Technology Co. Ltd.,Shanghai, China), 1-chloro-2, 4-dinitrobenzene (CDNB, Shanghai Chem. Ltd.,Shanghai, China), reduced glutathione (GSH, Sigma, St. Louis, MO), and otherbiochemical reagents were of analytical grade. The xenobiotic compounds used for theinhibition bioassays were: ethacrynic acid, 97% diethyl maleate, 97% tetraethylthiuramdisulfide, 94% curcumin (all from Sigma, St. Louis, MO), bromosulfalein (DowAgroSciences LLC, Indianapolis, IN), 95% b-cypermethrin (Jiangsu Fengshan Co.Ltd., Jiangsu, China), 89.1% malathion, and 93% avermectin (Chongqing Pesticide Co.Ltd., Chongqing, China).

Enzyme Preparation and Assay of Protein Contents

One hundred milligrams of adult B. dorsalis were homogenized manually on ice in 4 mlsodium phosphate buffer (20 mM, pH 7.3). The homogenate was centrifuged for5 min at 5,000g and 41C. The pellet was discarded and the supernatant was againcentrifuged for 15 min at 17,500g and 41C. Finally, the supernatants were used as theenzyme source for assays of GST activity. Protein contents of the enzyme homogenatewere determined according to the method of Bradford (1976) using BSA as astandard. The measurement was performed at a wavelength of 595 nm at 251C.

Purification of Enzymes

GSTs from B. dorsalis were purified using prepacked GSH SepharoseTM 4B (GEHealthcare Biosciences, Uppsala, Sweden), according to the protocol supplied byAmersham Biosciences and as described by Wu et al. (2009). The column wasprewashed with 10–20 ml sodium phosphate buffer (pH 7.3). Thereafter, the gel bedwas equilibrated with 6 ml PBS11% Triton X-100. The homogenate from 100 mg adultflies was prepared, as described above. The supernatant was filtered through a0.45 mm Millex-HV filter (Millipore, Billerica, MA) and loaded on the GSH–agaroseaffinity gel column with a bed volume of 4 ml. The column was equilibrated with

GSTs of the Oriental Fruit Fly � 203


20 mM sodium phosphate buffer and washed with the same buffer. Finally, the boundGSTs enzyme was eluted with 10 ml buffer (5 mM GSH in 50 mM Tris-HCl, pH 8.0)and 1 ml fractions were collected. Fractions containing GST activity were pooled forfurther analysis.

Enzyme Activity, Kinetics, Temperature, and Optimum pH

GST activity was determined using CDNB and reduced GSH as substrates, accordingto Habig et al. (1974). The total reaction volume per well of a 96-well microplate was300 ml, consisting of 100 ml supernatant, CDNB [containing 2% (v/v) ethanol], and GSHin 0.05 M, Tris-HCl, pH 7.5, giving a final concentration of 0.6 mM CDNB and 6 mMGSH. The nonenzymatic reaction of CDNB with GSH measured without supernatantserved as the control. The change in absorbance was measured at 30 sec intervals for5 min at 340 nm and 371C in a Thermomax kinetic microplate reader (TECAN,Sunrise Remote, Austria). Changes in absorbance per minute were converted intonmol CDNB conjugated min�1 mg�1 protein using the extinction coefficient of theresulting 2, 4-dinitrophenyl-glutathione: e340 nm 5 9.6 mM�1 cm�1 (Habig et al., 1974).

The Michaelis constant (Km) and maximum velocity (Vmax) for GSTs fromfour field populations were determined for GSH (1.2–6 mM) or CDNB (0.06–0.6 mM)as substrates, respectively. Km and Vmax values were calculated using theMichaelis–Menten equation (Dowd and Riggs, 1965).

Temperature dependence of the purified GSTs was assayed at a series of reactiontemperatures (25, 28, 31, 34, 37, and 411C) for CDNB as substrate. The pH optimumwas determined for CDNB conjugation activity. The purified fruit fly GSTs wereincubated at 371C for 5 min in 0.05 M sodium phosphate buffer (pH 6.0, 6.5, and 7.0),and Tris-HCl buffer (pH 7.5, 8.0, and 8.5). Conjugation activity was determined, asdescribed above. The experiment was replicated at least three times for eachtreatment.

Electrophoresis

Electrophoresis was performed, according to the method of Laemmli (1970) using aBio-Rad Mini Protean 3 cell and a Bio-Rad PowerPacTM HV (Bio-Rad Laboratories,Hercules, CA). The enzyme samples were diluted 1:4 with a solubilizer (5% SDS,0.006% bromophenol blue and 1.25% b-mercaptoethanol in running buffer) andboiled for 5 min before electrophoresis. Separation gels were 12.5% acrylamide in0.375 M Tris-HCl (pH 8.9). Stacking gels contained 4% acrylamide in 0.125 M Tris-HCl(pH 6.8). The samples were then run into the stacking gel at 15 mA for 30 min and intothe resolving gel at 30 mA for 90 min. Silver staining was performed according to themanufacturer’s instructions (Bio-Rad). The molecular weight of GSTs was calculatedby the Gel DocTM XR System and Quantity One 1-D Analysis Software (Bio-RadLaboratories, Hercules, CA) after scanning the stained gel.

In vitro Enzyme Inhibition

The inhibition study was performed using the GST assay conditions in the absence orpresence of various concentrations of inhibitors. Stock solutions of the inhibitors,including ethacrynic acid, bromosulfalein, diethyl maleate, tetraethylthiuram disulfide,curcumin, b-cypermethrin, malathion, and avermectin were prepared in ethanol anddiluted with Tris-HCl (50 mM, pH 7.5). The highest acetone concentration was 1% inthe test solutions. Twenty-five microliters of enzyme source and the same volume of



inhibitor solutions with appropriate concentrations were incubated for 10 min at 371Cand then added to the CDNB/GSH substrate mixture, as described above. Reactionswithout the inhibitor were included as controls. Acetone with an appropriateconcentration (up to 1%) was also added in control reactions to exclude the potentialinhibition against GSTs by this solvent. The median inhibition concentration (I50) foreach inhibitor was determined from a log concentration vs. probit (% inhibition)regression analysis.

Statistical Analysis

Data were analyzed using the analysis of variance (ANOVA) and the means wereseparated by Duncan’s Multiple Range Test or LSD Test for significance (P 5 0.05)with SPSS 12.0 for Windows. Regression analyses were also performed to calculatekinetic parameters of GSTs, purification profiles, and I50 values of various inhibitors.

RESULTS

Assay of Crude GST Activity, Kinetic Parameters, and Inhibition

The specific activities of crude GSTs from the four populations of B. dorsalis are similar(Table 1). The kinetic parameters of crude GSTs were determined using Line-weaver–Burk plots and are presented in Table 1. The statistical analyses showed thatGSTs from the four populations of B. dorsalis had similar Km and Vmax values towardeither CDNB or GSH.

The inhibitory actions of five xenobiotic compounds (ethacrynic acid, bromosul-falein, diethyl maleate, disulfiram, and curcumin) against the crude GSTs from thefour populations of B. dorsalis are presented in Table 2. The HN population had thehighest I50 values toward ethacrynic acid and disulfiram, whereas the YN populationhad the lowest values. The I50 value of diethyl maleate against the YN population wassignificantly lower than that of the other three populations. The I50 values ofbromosulfalein and curcumin were not significantly different among the fourpopulations of B. dorsalis investigated here.

Table 1. Comparison of the Specific Activities Toward CDNB and Kinetic Parameters of Crude GSTsFrom Four Populations of B. dorsalis

Specific activityCDNB GSH

Population (nmol min�1 mg�1) Km (mM) Vmax (nmol min�1) Km (mM) Vmax (nmol min�1)

DG 0.2570.02 0.1770.06 0.8370.21 0.3670.05 0.3970.07GZ 0.2670.02 0.0870.01 0.8370.28 0.4370.05 0.5970.18HN 0.2370.01 0.1070.01 0.6470.17 0.3370.05 0.4170.22YN 0.2570.03 0.1170.03 0.7370.10 0.3370.02 0.4270.03

DG, GZ, HN, and YN represent the B. dorsalis population from Dongguan city and Guangzhou city of theGuangdong province, Haikou city of the Hainan province, and Kunming city of the Yunnan province, China,respectively. Each value represents the mean (M7SE) of three replications.



GST Purification

GSTs were purified from the four field populations of B. dorsalis with GSH affinitychromatography. The chromatography elution profiles are shown in Figure 1. In total,10 fractions were eluted through the chromatography column (1 ml eluted solutionper collection tube) for use in assays. The third fraction from GZ and YN populationsshowed the highest specific activity of GSTs, whereas that of DG and HN populationswas the fourth fraction (Fig. 1). The protein, total activity, specific activity, andpurification fold of the purified GSTs did not differ among the four populations ofB. dorsalis (Table 3). However, the yield of the purified GSTs from GZ population(31.54%) was significantly lower than those of the other three field populations(Table 3).

The purity of the final enzyme preparation from the four populations of B. dorsaliswas confirmed by SDS-PAGE electrophoresis. The purified enzyme of four popula-tions of B. dorsalis appeared as one GST band after silver staining on SDS-PAGE(Fig. 2). The molecular mass of the purified GSTs was estimated at approximately23 kDa for all four populations of B. dorsalis.

GST Characterization

The kinetic parameters of purified GSTs are presented in Table 5. For the catalyticactivity of GSTs toward CDNB, the Km values for the DG (0.15 mM) and HN (0.18 mM)populations were significantly higher than those of the GZ and YN populations; theVmax values of GSTs from the HN population (52.29 nmol min�1) were higher thanthose of the other three populations. For the catalytic activity of GSTs toward GSH, theKm and Vmax values from the four field populations did not differ (Table 4).

The temperature dependence of the purified GSTs from the four populations ofB. dorsalis is presented in Figure 3. The specific activity of GSTs was broadly optimal at371C for the four populations. The activities varied slightly at lower temperatures inthe GZ population, but varied more in the other three populations. The activitiesdecreased sharply at 401C in the four populations. The pH dependence of the purifiedGSTs from the four populations is presented in Figure 3. The optimal pH for GSTactivity was 7.5. The activities varied slightly at pH 6.0–7.5 and decreased abruptly atpH 8.0–8.5 for each population.

Table 2. I50 Inhibition Constants of Five Xenobiotic Compounds on Crude GSTs From FourPopulations of B. dorsalis

Population

Effector (I50) DG GZ HN YN

Ethacrynic acid (mM) 17.7776.23ab 26.9275.02ab 56.92721.86b 9.7871.01aBromosulfalein (mM) 11.8473.64a 16.6876.40a 80.23740.85a 11.2373.46aDiethyl maleate (mM) 9.3071.70a 8.1371.03a 6.7270.29a 1.9770.19bDisulfiram (mM) 10.2773.08a 8.1871.26a 170.11713.14b 12.6073.63aCurcumin (mM) 241.24737.07a 259.36715.61a 208.00713.45a 278.3075.98a

Each value represents the mean (M7SE) of three replications. Means within the same row followed by differentletters are significantly different (Po0.05).



In vitro Inhibition of GSTs

The inhibitory kinetics of several compounds against purified GSTs from fourpopulations of B. dorsalis are presented in Table 5. The efficiencies of the testedinhibitors were compared using their I50 values (the concentration required to inhibit50% of GST activity). The I50 values of ethacrynic acid, bromosulfalein, and disulfiram

Figure 1. Elution profiles for GST activities from four populations of B. dorsalis for GSH affinitychromatography. DG, GZ, HN, and YN refer to the populations from Dongguan and Guangzhou of theGuangdong Province, Haikou of the Hainan Province, and Kunming of the Yunnan Province, China,respectively. Each value represents the mean7SE of at three independent experiments.

Table 3. Comparison of the Activities of Purified GSTs Toward CDNB From Four Populations ofB. dorsalis

GSTs activity

PopulationProtein

(mg ml�1)Total activity(nmol min�1)

Specific activity(nmol min�1 mg�1)

Purification(-fold) Yield (%)

DG 2.7170.13 379.76719.22 14.1571.31 57.3772.27 37.5471.66aGZ 2.5370.58 387.78734.39 16.4472.41 64.19711.11 31.5472.02bHN 2.1970.03 370.7674.43 16.9270.01 73.9975.22 39.6071.75aYN 1.9770.22 381.88712.46 20.0873.16 80.5576.94 38.3371.79a

DG, GZ, HN, and YN represent the B. dorsalis population from Dongguan and Guangzhou of the Guangdongprovince, Haikou of the Hainan province, and Kunming of the Yunnan province, China, respectively. Each valuerepresents the mean (M7SE) of three replications. Means within the same column followed by different letters aresignificantly different (Po0.05).



did not differ significantly among the four populations. The I50 value of diethylmaleate against the HN population (0.60 mM) was significantly lower than those of theGZ and YN populations. The I50 value of curcumin against the HN population(101.78 mM) was significantly higher than that for the other three populations. The I50

value of b-cypermethrin was significantly lower than that for the other threepopulations. However, both malathion and avermectin showed poor inhibitory effectson GSTs for four populations of B. dorsalis (Fig. 4), and no significant differences wereobserved among the various concentrations.

DISCUSSION

GSTs are present in almost all eukaryotes, where they occur in multiple isoenzymicforms and provide an important intracellular mechanism of detoxification. GSTs arephase-II detoxification proteins that often facilitate the excretion of primary oxidativeand hydrolytic insecticide metabolites resulting from phase-I detoxification (Li et al.,2007). Recently, rapid development of resistance to insecticide in 25 field populationsof oriental fruit fly in mainland China has been reported (Jin et al., 2011). Because ofthe increasing interest in the fruit flies, coupled with the widespread use of variousdifferent chemical classes of insecticides, we biochemically characterized of purified

Figure 2. SDS-PAGE of purified GSTs from four populations of B. dorsalis. M, molecular weight markers;DG, GZ, HN, and YN indicate purified GSTs from the four populations of B. dorsalis, as in Figure 1.

Table 4. Kinetic Parameters of GSTs Purified From Four Populations of B. dorsalis

CDNB GSH

Population Km (mM) Vmax (nmol min�1) Km (mM) Vmax (nmol min�1)

DG 0.1570.01a 25.8174.23a 0.2070.01a 19.3074.16aGZ 0.0670.01b 24.4473.94a 0.1770.02a 21.3674.71aHN 0.1870.04a 52.29713.76b 0.1870.02a 28.3079.12aYN 0.0770.01b 23.2072.78a 0.1870.03a 19.1972.69a

DG, GZ, HN, and YN represent the B. dorsalis population from Dongguan city and Guangzhou city of theGuangdong province, Haikou city of the Hainan province, and Kunming city of the Yunnan province, China,respectively. Each value represents the mean (M7SE) of three replications. Means within the same column followedby different letters are significantly different (Po0.05).



Figure 3. Effect of temperature and pH on specific activities of purified GSTs from four populationsof B. dorsalis. Each value represents the mean7SE of three independent experiments.



GSTs in B. dorsalis from four different field populations in this study. The molecularmass of the purified GSTs was estimated at 23 kDa, which is in the same range as thoseof GSTs from the two-spot ladybird, Adalia bipunctata (Francis et al., 2002), the fallwebworm, Hyphantria cunea (Yamamoto et al., 2007), the red imported fire ant,Solenopsis invicta (Valles et al., 2003), and psocids (Dou et al., 2009, 2010; Wu et al.,2009). Previous studies have shown that in insects GST isoform composition canlargely depend on the feeding behavior of the species or population (Adewale andAfolayan 2006). This suggests that the GST enzymes from the four field populations ofB. dorsalis studied here were the same isoenzymes.

GSTs make up a superfamily of multifunctional proteins. Twenty-three,twenty-eight, and thirty-seven GST genes from Bombyx mori, Anopheles gambiae, andDrosophila melanogaster have been sequenced from their genomes (Enayati et al., 2005;Tu and Akgul, 2005; Yu et al., 2008). The molecular mass of the GST protein isapproximately 23 kDa, as determined by the one-dimensional SDS-PAGE in manyinsect species. The use of two-dimensional gel electrophoresis for GST identificationhas also been reported (Alias and Clark, 2007, 2010). In order to conclusively identifythe GSTs from the four populations of B. dorsalis, two-dimensional gel and molecularcharacteristics should be investigated in the future.

The purification yields of the four field populations of B. dorsalis measured in thisstudy were much lower than those of Anopheles dirus (76%) (Prapanthadara et al., 1996)and of the two syrphid flies Syrphus ribesii (65%) and Myathropa florae (77%) (Vanhaelenet al., 2004). In contrast, the yields were much higher than those of S. invicta (3.3%)(Valles et al., 2003). In insects, multiplicity of GST largely depends on the feedingbehavior of the insects (Adewale and Afolayan, 2006). It may be that the difference inpurification yields might be related to the living conditions and feeding behavior of thefour field populations. The purification folds were similar to those for various fieldpopulations of Liposcelis paeta (Wu et al., 2009) and S. invicta (Valles et al., 2003).

The GST kinetic constants for the four field populations of B. dorsalis towardCDNB and GSH were comparable to values reported from several insect species(Valles et al., 2003; Yamamoto et al., 2008; Dou et al., 2009; Wu et al., 2009). WithCDNB as the substrate, the Km values of the purified GSTs in the DG and HNpopulations are significantly higher than those for the GZ and YN populations,suggesting a lower affinity of GSTs toward the substrates in the DG and HNpopulations of B. dorsalis. The Vmax value of the purified GST from the HN population

Table 5. I50 Inhibition Constants of Xenobiotic Compounds on Purified GSTs From Four Populationsof B. dorsalis

Population

Effector (I50) DG GZ HN YN

Ethacrynic acid (mM) 1.8570.49a 1.5070.35a 1.1570.18a 1.6970.29aBromosulfalein (mM) 25.0176.06a 24.9973.68a 23.6475.55a 20.2872.99aDiethyl maleate (mM) 1.8070.27ab 2.2370.46b 0.6070.14a 2.4070.67bDisulfiram (mM) 186.81712.7a 140.92740.17a 193.23737.48a 217.68787.91aCurcumin (mM) 37.0973.27a 9.0873.16a 101.78714.14b 43.66717.67ab-cypermethrin (mM) 0.9570.12a 0.7570.09a 0.8370.11a 0.5270.08b

Each value represents the mean (M7SE) of three replications. Means within the same row followed by differentletters are significantly different (Po0.05).



Figure 4. Inhibition of malathion and avermectin against purified GSTs from four populations ofB. dorsalis. Each value represents the mean7SE of three independent experiments.



was the highest of the four populations tested, indicating higher catalytic capability of GSTsin this population. Interestingly, for GSH as the substrate, the Km values were similar forcrude and purified GSTs; however, the Vmax values were higher after purification. It can beconcluded that the intrinsic characteristics of the enzyme GST are the same according tothe similar Km value in crude and purified populations of B. dorsalis.

To examine the environmental adaptability of four populations of B. dorsalis, theeffects of different temperature and pH on specific activities of the GSTs weresubsequently investigated. The activities of GSTs from each population were highest at371C, which is congruent with reports for Cnaphalocrocis medinalis (Yamamoto et al.,2008) and S. invicta (Valles et al., 2003). Each population tested was characterized by apH optimum of 7.5, similar to that quantified for H. cunea (Yamamoto et al., 2007) andvarious field populations of L. paeta (Wu et al., 2009). GSTs are a large family ofdetoxification enzymes with wide species distribution and a multiplicity of forms; thus,each isoform may require specific conditions of substrate concentrations, temperature,pH, and so on for optimal activity (Yu, 2002).

Ethacrynic acid, bromosulfalein, diethyl maleate, disulfiram, and curcuminshowed the highest inhibitory effects, with I50 values at micromolar levels on GSTsin B. dorsalis. The I50 values of these compounds (except disulfiram) against the crudeGSTs from four field populations were significantly higher than those of purifiedGSTs. These results are similar to previous investigations of Liposcelis bostrychophila andL. entomophila (Dou et al., 2010). Ethacrynic acid is considered to be one of the specificinhibitors of GSTs (Wu et al., 2009). In previous studies, some insect GSTs showedlower I50 values toward ethacrynic acid, e.g., Blattella germanica (350 nM) (Yu andHuang, 2000) and Spodoptera frugiperda (150 nM) (Yu, 2002). Based on our bioassay ofthe toxicity of b-cypermethrin, malathion, and avermectin against the four populationsof B. dorsalis, the oriental fruit flies were very susceptible to avermectin, but insensitiveto malathion (Wang et al., unpublished data). Resistance was the highest in the DGpopulation and lower in the YN population for these three insecticides. It wasreported that there was a positive correlation between GST activities and the toxicitiesof pyridaben against nine field populations of Panonychus citri from Chinese citrusorchards (Niu et al., 2011). In Aedes aegypti, an Epsilon GST, GSTE2, is very efficient atmetabolizing DDT, and the expression of this enzyme is elevated in a DDT andpyrethroid-resistant population from Thailand (Lumjuan et al., 2005). Although wedid not find a positive correlation between enzyme activities with susceptibility levels,the I50 value of b-cypermethrin against the purified GSTs from the YN population wassignificantly lower than those of the other three populations. The toxicities ofb-cypermethrin were positively correlated with the inhibition efficiency values. Theseresults suggest that GSTs may have a role in detoxification of the pyrethroid,b-cypermethrin in B. dorsalis. GSTs are known to detoxify various pyrethroids in manyspecies, including H. cunea, D. melanogaster, and A. aegypti (Yamamoto et al., 2007; Lowet al., 2010; Lumjuan et al., 2011). On the other hand, the inhibition efficiency did notreach 50% on GSTs with malathion and avermectin. It should be noted that a liveinsect bioassay and an in vitro enzyme activity assay are two different systems. Theenzyme activity assay only measured GST activity with a specific substrate, whereasin vivo conditions in the bioassay system are more complicated. Several detoxifyingenzymes are involved in insecticide resistance, including cytochrome P450s,carboxylesterase, GSTs, and other potential proteins (Li et al., 2007).

In conclusion, this study has provided evidence of GST variation in associationwith insecticide susceptibilities in oriental fruit fly populations from China. Our results



suggest that GSTs may have a role in detoxification of b-cypermethrin in B. dorsalis.Undoubtedly, genomic and proteomic techniques would help in the identification andclassification of oriental fruit fly GSTs. Transcriptome analyses will be applied toexplorations of molecular genetic mechanisms responsible for resistance of orientalfruit fly GSTs in further studies. Experiments concerning the identification of GSTs areunderway and will provide more detailed information in the future regardingcomparative properties, such as the expression, activities, and substrate specificitiesamong GSTs and related enzymes of B. dorsalis.

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