CpGSTd3 is a lambda-cyhalothrin metabolizing glutathione S

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Biotechnology and Biological Transformations

CpGSTd3 is a lambda-cyhalothrin metabolizing glutathione S-transferase from Cydia pomonella (L.) Wei Wang, Chao Hu, Xin-Ru Li, Xiao-Qi Wang, and Xue-Qing Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05432 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Journal of Agricultural and Food Chemistry

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CpGSTd3 is a lambda-cyhalothrin metabolizing glutathione S-transferase from

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Cydiapomonella (L.)

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Wei Wang1,2, Chao Hu1,2, Xin-Ru Li1,2, Xiao-Qi Wang1,2, Xue-Qing Yang*1,2

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1. College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, Liaoning, China. 2. Key Laboratory of Economical and Applied Entomology of Liaoning Province, Shenyang 110866, Liaoning, China.

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*Corresponding

16

author. E-mails:[email protected] (X. Yang) Tel.: +86-24-8848-7148; fax: +86-24-8848-7148

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ACS Paragon Plus Environment


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ABSTRACT

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Little is known about the role of specific delta GST genes in the detoxification of

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lambda-cyhalothrin in the global quarantine fruit pest codling moth, Cydia pomonella

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(L.). Real-time quantitative PCR shows that CpGSTd3 was ubiquitously expressed at

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all developmental stages and is most abundant in the larval stage and lowest in the

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egg stage; the mRNA level of CpGSTd3 is higher in the midgut and Malpighian

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tubules of fourth-instar larvae and abdomens of adults than in other tissues. Exposure

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of fourth-instar larvae to an LD10 dosage of lambda-cyhalothrin significantly induced

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the transcript of CpGSTd3 at 3 h, but the mRNA level was down-regulated after 12 h

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of treatment. Recombinant CpGSTd3 expressed in Escherichia coli was able to

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catalyze the conjugation of 1-chloro-2,-4dinitrobenzene (CDNB), and with an IC50

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value of 0.65 mM for lambda-cyhalothrin. Metabolism assays indicate that

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recombinant CpGSTd3 could metabolize lambda-cyhalothrin. These results suggest

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that CpGSTd3 is probably a lambda-cyhalothrin metabolizing GST in C. pomonella.

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KEYWORDS: glutathione S-transferases, Cydia pomonella, delta class, pyrethroid,

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detoxification

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INTRODUCTION

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The codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major

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global quarantine fruit pest infesting the majority of pome fruits and walnuts

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cultivated worldwide.1 In addition, it is also a pest of quarantine concern for apple or

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pear exports to China, Japan and South Korea. Environmentally friendly

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pest-management strategies, such as mating disruption and attract-kill strategy, have

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been recommended for codling moth control.2-4 However, the control of codling moth

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continues to mainly depend on chemical insecticides.5-7 As a consequence, this pest

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has developed serious resistance to most commonly used insecticides including

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organophosphates

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Lambda-cyhalothrin is one of the most commonly used insecticides for C. pomonella

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control worldwide; where it occurs in northeast China, lambda-cyhalothrin was

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sprayed 8-10 times per year. As a result, resistance of lambda-cyhalothrin to chemical

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insecticides was documented.11,15 Among those resistance mechanisms documented,

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glutathione

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lambda-cyhalothrin resistance in field populations of C. pomonella.10,11,16

(OPs),

S-transferases

pyrethroids

(GSTs,

(Pys)

EC

and

2.5.1.18)

carbamates

was

(Carbs).8-14

associated

with

59

As key phase II detoxification enzymes, GSTs are multifunctional enzymes that

60

play crucial roles in detoxification of endogenous and exogenous compounds

61

including insecticides.17-19 They catalyze the conjugation of reduced glutathione (GSH)

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and have been found in both prokaryotic and eukaryotic cells.18,20,21 GSTs are divided

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into three categories: cytosolic, microsomal, and mitochondrial, and only the cytosolic

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and microsomal GSTs have been found in insects.22 In insects, cytosolic GSTs have 3



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been grouped into six major classes: delta, epsilon, omega, sigma, theta, and zeta,17,19

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and the delta- and epsilon-class GSTs are unique to insects and contribute to

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insecticide resistance.22-26 Since Bombyx mori and Plutella xylostella are the model

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insects and main pests in Lepidoptera, their GSTs have been systematically

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characterized based on their genomic sequence and transcriptome data; a total of 4

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delta and 8 epsilon, and 5 delta and 5 epsilon class GST genes were identified,

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respectively.27,28 However, the functional roles of specific GST genes in delta and

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epsilon classes from C. pomonella and their interaction with lambda-cyhalothrin are

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less known. In C. pomonella, a total of 4 delta class GST genes have been identified

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from the C. pomonella transcriptome database (SRX371333). Among these GSTs,

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CpGSTd1 was involved in metabolizing lambda-cyhalothrin,19 whereas the enzymatic

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activity of recombinant CpGSTd2 was found to be inhibited by β-cyfluthrin,

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chlorpyrifos, thimet, and chlorbenzuron in C. pomonella.29

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In this study, we report the identification and functional analysis of the CpGSTd3

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in C. pomonella. It is reported here that CpGSTd3 is probably a lambda-cyhalothrin

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metabolizing GST in C. pomonella.

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MATERIALS AND METHODS

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Insects. The susceptible C. pomonella strain (Ss) was from a colony provided by the

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laboratory of Agricultural Invasive Biological Prevention and Monitoring, Institute of

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Plant Protection, Chinese Academy of Agricultural Sciences. This population was

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originally collected from abandoned apple orchards without management in Wu wei

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Botanical Garden (Wuwei, China) and was reared in the laboratory without exposure 4


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to insecticides for more than 50 generations. Larvae were fed on an artificial diet in

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the laboratory in a growth chamber (MLR-352H-PC, Panasonic) at 26±1℃, 60±5%

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humidity with a photoperiod of 16 : 8 h (L : D) and without exposure to insecticides.

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Moths were supplied with a 10% honey solution.

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Chemicals. The lambda-cyhalothrin (technical grade, purity >98%) was obtained

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from Aladdin Reagent (Shanghai, China). The 2,4-dinitrochlorobenzene (CDNB) and

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the glutathione (GSH) were purchased from Sigma Chemical Corporation (St. Louis,

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MO, USA). All other chemicals and solvents used were reagent grade.

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Developmental stages and tissues. Three groups of 200 eggs, 50 larvae of first- and

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second-instar, 10 larvae of third-instar, 5 larvae of fourth- and fifth-instar, 5 pupae,

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and 5 female and 5 male adults were sampled, respectively. The head, cuticle, fat

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body, midgut and Malpighian tubules were dissected from three groups of 30

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fourth-instar larvae. The heads, thoraxes, abdomens, and legs were dissected from

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three groups of 30 12-h-old male and female adults. All samples were flash-frozen in

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liquid nitrogen and then stored at −80℃ for later transcriptional analysis.

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Bioassay. In order to determine the sublethal dose (LD10) of lambda-cyfluthrin in C.

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pomonella, a stock solution of lambda-cyhalothrin was prepared in acetone and

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diluted to eight concentrations (0, 4, 8, 16, 32, 64, 128 and 256mg L-1). Fourth-instar

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C. pomonella larvae were chosen to measure the LD10 of lambda-cyfluthrin using the

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topical application method with an Eppendorf pipettor (Hamburg, Germany).30 A 2μl

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drop of each concentration of insecticide solution was gently dropped on the thoracic

5



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dorsum of fourth-instar larvae. Control larvae were treated in the same way with

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acetone only. Three replicates of 24 larvae were used in each concentration. After

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treatment, larvae were held individually in a 2×5cm glass tube and fed on artificial

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diet. Mortality was recorded at 12, 24, 48 and 72 hours post exposure. A larva was

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considered dead when it did not respond to stimulation by an ink brush.

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Insecticide exposure. Dose-mortality data were subjected to Probit analysis. The

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dose that would kill 10% (LD10) of the larvae by lambda-cyhalothrin at 24 h was used

115

for the subsequent insecticide exposure experiment. A 2μl drop of LD10 of

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lambda-cyfluthrin was applied on the thoracic dorsum of each fourth-instar larva

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using the same method described above. Larvae treated with acetone were used as a

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control. Three replicates, each consisting of 40 larvae per biological replicates were

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treated to ensure sampling. After treatment, larvae were placed in the same conditions

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as described above. Five larvae were sampled at 3, 6, 12, 24, and 48 h post treatment,

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and flash-frozen in liquid nitrogen and stored at −80℃ for later transcriptional

122

analysis.

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Molecular cloning of CpGSTd3.Total RNA was extracted using Eastep Super Total

124

RNA Extraction Kit (Promega, Shanghai, China) according to the manufacturer’s

125

instructions. RNA purity and concentration was determined on a NanoDrop2000

126

(ThermoFisher Scientific, USA). The first-strand cDNA was synthesized from 1 µg of

127

total RNA using a GoScriptTM Reverse Transcription Mix (Promega, USA), and then

128

stored at -20℃ until use.

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Using the delta class GST sequences from B. mori BmGSTd2 (GenBank

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accession number: BAD60789.1), P. xylostella PxGSTd3 (GenBank accession

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number: BAJ10978.1), and Helicoverpa armigera HaGST (GenBank accession

132

number: ABK40535.1)s query sequences and using the local blast-2.2.31+ to BLAST

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against C. pomonella transcriptome database (SRX371333), we confirmed the

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presence of 4 delta class GST genes (Table S1), including CpGSTd1, CpGSTd2,

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CpGSTd3, and CpGSTd4 in C. pomonella. These 4 GST genes were verified by

136

RT-PCR and DNA sequencing. Among these GST genes, CpGSTd1 and CpGSTd2

137

had been previously characterized.19,29 In this study, the CpGSTd3 was characterized.

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Based on the ORF sequence of CpGSTd3 (GenBank accession number KX500030.1),

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two specific primers containing EcoR I and Hind III restriction enzyme sites were

140

designed (Table 1). PCR reaction was conducted in a 50 μl reaction volume

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containing 2 μl cDNA, 2 μl each primer (10 mM), 4 μl dNTP Mixture, 5 μl10×Ex Taq

142

Buffer (Mg2+ Plus) and 0.25 μl Ex Taq® DNA polymerase (TaKaRa, Dalian, China)

143

under the following conditions: 94℃ for 1 min, followed by 35 cycles of 94℃ for 30 s,

144

55℃ for 30 s and 72℃ for 30 s, with a final extension at 72℃ for 2 min. PCR

145

products were cloned into pMD 19-T Vector (TaKaRa, Dalian, China) and

146

transformed into E. coli DH5α (TaKaRa, Dalian, China), and then sequenced (Sangon

147

Biotech, Shanghai, China). Sequence similarity was determined by aligning with

148

DNAMAN (DNAMAN 5.2.2, LynnonBioSoft). The secondary structures of GST

149

protein were predicted by the PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk/psipred/).

150

Real-time quantitative PCR analysis of CpGSTd3. Real-time quantitative RT-PCR 7



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151

(RT-qPCR) was carried out according to Bustin et al. (2009)

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expression level of CpGSTd3 in different developmental stages and tissues, and in

153

larvae exposed to insecticide. Total RNA was extracted and the potential

154

contaminated genomic DNA was removed using the Eastep Super Total RNA

155

Extraction Kit (Promega, Shanghai, China) according to the manufacturer’s

156

instructions. RNA purity and concentration, as well as the synthesization of

157

first-strand cDNA were described above. Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/)

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was used to design primers of CpGSTd3 for RT-qPCR (Table 1). Before RT-qPCR, a

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regular RT-PCR and DNA sequencing was conducted to verify the amplicons.

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RT-qPCR was performed on a Bio-Rad CFX96 (BioRad, USA). A 20 μl reaction

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mixture consisted of 10 μl TB GreenTM Premix Ex TaqTM 2 (TliRNaseH Plus)

162

(TaKaRa, Dalian, China), 1 μl 10× diluted cDNA template, 0.8 μl of 10 μM forward

163

and reverse primers were amplified under the following cycling conditions: 95℃ for

164

30 s, and 40 cycles of 95℃ for 5 s and 60℃ for 30 s. A melting curve was also

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conducted after amplification to determine the specific of PCR products. The β-Actin

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gene of C. pomonella (CpActin) (GenBank accession number: KC832921) was used

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as a reference.19,32 Using a 10-fold linear gradient dilution

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standard curves of CpGSTd3 and CpActin were obtained. The amplification efficiency

169

and correlation coefficient (R2) of target and housekeeping gene was 94.2% and 0.999,

170

and 95% and 0.995, respectively. RNase-free water instead of cDNA templates and

171

RNA without reverse transcription were used as controls to exclude the genomic

172

DNA contamination. Each reaction was carried out in triplicate and each test was 8


to determine the

of cDNA as template, the

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repeated with three independent mRNA samples. The relative expression of CpGSTd3

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was calculated according to the 2−△△CT method.33

175

Expression and purification of CpGSTd3.The recombinant CpGSTd3 was

176

expressed and purified according to Liu et al. (2014)19 with slight modification.

177

Sequencing-verified plasmids were digested with EcoR I and Hind III, and then

178

inserted into the expression vector pET-28a (+), followed by transformation into E.

179

coli BL21 (DE3) cells for expression. Expression of recombinant CpGSTd3 protein

180

was induced in a 1 L Luria-Bertani (LB) medium (50 μg ml-1 of kanamycin) with

181

0.2mM of isopropyl β-D-thiogalactoside (IPTG) when A600 reached 0.4-0.6, and this

182

was continued for 12h with shaking at 220 rpm at 25℃. Induced bacteria were

183

harvested by centrifuging at 8,000 g at 4℃ and were re-suspended in 20 ml of 50 mM

184

sodium phosphate buffer (pH 7.2). After being sonicated for 5 min, the collected

185

bacteria were centrifuged for 20 min at 14,000 rmp at 4℃. The soluble fraction of

186

recombinant CpGSTd3 was identified by 12% SDS-PAGE with standard

187

protein-sized markers (Thermo Scientific, USA). Recombinant protein was bound by

188

ProteinlsoTM Ni-NTA Resin (Trans, China) and eluted using an ascending series of

189

imidazole (50 mM, 100 mM, 150 mM, and 200 mM). The purified protein was

190

dialyzed using 50 mM sodium phosphate buffer (pH 7.2). BCA Protein Assay Kit

191

(TaKaRa, Dalian, China) was used to determine the concentration of recombinant

192

CpGSTd3 on a Microplate Reader (VersaMax, Molecular Devices, USA).

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In vitro enzyme kinetics.The kinetic constants were determined as described by Liu

194

et al. (2014)19 with slight modification, using CDNB as a standard substrate. In brief, 9



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4μl of recombinant CpGSTd3 was added to 196 μl of a 50mM sodium phosphate

196

buffer (pH 7.2) containing different concentrations of CDNB (0.1 to 2.0mM) and 1

197

mM of GSH. Changes in absorbance at 340 nm (A340) in one minute was monitored at

198

30℃ using multimode Microplate Reader (VersaMax, Molecular Devices, USA) and

199

converted into mol CDNB conjugated min-1 mg-1 protein using the molar extinction

200

coefficient of the GSH: ε340 =9600 M-1 cm-1. A total of 4 μl 50 mM sodium phosphate

201

buffer (pH 7.2) instead of recombinant CpGSTd3 was used as a negative control.

202

Kinetic parameters were obtained from Michaelis-Menten plots generated using

203

GraphPad Prism 5 (San Diego, USA) with the data produced by varying the

204

concentrations of CDNB. Each determination was repeated three times.

205

Assay of in vitro inhibition of GST activity.To determine the inhibition property of

206

lambda-cyfluthrin against CpGSTd3, the half-inhibitory concentration (IC50) was

207

determined by pre-incubating 4 μl of purified CpGSTd3 with different concentrations

208

of lambda-cyfluthrin (50 to 2000 μM) at 30°C for 5 min before the addition of 2 mM

209

of

210

S-Hexylglutathione (GTx), a potent GST inhibitor (final concentration of 25 to 1200

211

μM) was used as a positive control. Using the software GraphPadPrism 5 (San Diego,

212

USA), IC50 value was calculated.

213

Metabolism assays. The capability of recombinant CpGSTd3 to metabolize

214

lambda-cyhalothrin was determined according to the protocol described by Riveron et

215

al. (2014)24 with slight modification. HPLC was conducted on Agilent 1260 Infinity

216

series equipment (Agilent, USA). In brief, 0.5 mL reaction mixtures containing2.5

CDNB.

The

remaining

activity

was

measured

10


as

described

above.

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mM GSH, 20µg CpGSTd3, and 0.6 mM lambda-cyhalothrin in 50 mM potassium

218

phosphate buffer (pH 7.2) were incubated at 30℃ for 60 min with shaking at 300 rpm.

219

Then 500 μl methanol (HPLC grade) was added and mixed well to stop the reaction

220

for 5min. After that, the samples were centrifuged at 13,000 rpm for 20 min at room

221

temperature, and 500 μl of the resulting supernatant was transferred to a HPLC vial. A

222

total of 100 μl of sample was automatically injected, and the quantity of

223

lambda-cyhalothrin remaining in the samples was determined by reverse-phase HPLC

224

using a C18 column (Agilent Technologies, USA, 4.6 × 150 mm in length, 5 μm

225

particle size) with a monitoring absorbance wavelength of 220 nm at 30℃. Separation

226

of lambda-cyhalothrin was achieved using an isocratic mobile phase of 75%

227

acetonitrile and 25% water with a flow rate of 1 ml min-1. In our previous study, both

228

the heat-inactivated GST and phosphate buffer was proved to be practicable

229

controls19. In this study, the control sample containing the same volume 50 mM

230

potassium phosphate buffer replaced CpGSTd3.

231

Data analysis. Statistical analysis was carried out using SPSS Statistics 20 (IBM,

232

USA). All of the results are presented as mean of triplicates ± standard deviation (SD).

233

Statistical significance was analyzed using the one-way analysis of variance (ANOVA)

234

with Duncan's test or Student's t-test. The level of significance was set at P < 0.05.

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RESULTS

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Sequence analysis of CpGSTd3.The ORF of CpGSTd3 with a 657 bp long mRNA

237

sequence that encodes an enzyme with 218 amino acids was obtained. This GST

11



238

shares 99% identify of both amino acid and nucleotide sequence with the previously

239

submitted one (GenBank accession number ARM39000.1) (Figure S1). A domain

240

analysis revealed that the CpGSTd3 includes 9 α-helices and 4 β-strands (Figure 1).

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Sequence and domain analysis confirmed that we obtained the whole ORF sequence

242

of the CpGSTd3 gene. The amino acid sequence of CpGSTd3 has a high identity with

243

delta class GSTs from other insect species such as BmGSTd2 (58% identity),

244

PxGSTd3 (56% identity) and HaGST (54% identity), confirming CpGSTd3 belongs

245

to the delta class. The amino acid sequence identity among 4 delta class GSTs

246

(CpGSTd1, CpGSTd2, CpGSTd3, and CpGSTd4) in C. pomonella was also

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determined (Figure S2). The amino acid sequence of CpGSTd3 shares 59% identity

248

with CpGSTd1, and 49% identity with CpGSTd2 and CpGSTd4 (Table S2).

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Toxicity of lambda-cyfluthrin against C. pomonella.The linear regression of dose–

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mortality relationship was fitted for lambda-cyfluthrin, and the Probit analyses are

251

listed in Table 2. The LD10, LD50, and LD90 values were 19.80, 95.89, and 464.45 ng

252

larvae-1 at 48 h, respectively (Table 2). The mortality in the controls was lower than

253

10%, suggesting that the bioassay was valid.

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Expression patterns of CpGSTd3.RT-qPCR shows that CpGSTd3 was ubiquitously

255

expressed at all developmental stages and was the most abundant in the larval stage

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and the lowest in the egg stage. In larval stages, the highest mRNA expression was

257

found in second- and fourth-instars (Figure2A).

258

Tissue-specific expression analysis showed that the mRNA level of CpGSTd3

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was detected in all tested tissues except the fat body of fourth-instar larvae. The

260

abundance of CpGSTd3 transcript was higher in the midgut and Malpighian tubules

261

than in other tissues (Figure2B). In adults, mRNA level of CpGSTd3 was detected in

262

thoraxes, abdomens, and legs of both male and female except the heads, and was

263

higher in male abdomens than in female abdomens (Figure 2C).

264

The results indicate that exposure of fourth-instar larvae to an LD10 of

265

lambda-cyhalothrin resulted in 1.84-fold up-regulation of CpGSTd3 expression at 3 h,

266

and 0.59-, 0.51 and 0.73-fold down-regulation of CpGSTd3 expression at 12h, 24h,

267

and 48h, respectively (Figure2D).

268

Expression and kinetic properties of recombinant CpGSTd3.The CpGSTd3 was

269

mainly expressed as soluble protein in E. coli BL 21 (DE3), with an expected

270

molecular mass of approximately 28 kDa (Figure3A), that the result of adding the

271

predicted molecular weight of CpGSTd3 (25 kDa) and tags in expression vector

272

pET-28a (+). It is estimated that more than 100 mg recombinant CpGSTd3 was

273

obtained from 1 L bacterial cultures after induced expression and purification,

274

suggesting a good protein yield.

275

According the Michaelis-Menten plot, the kinetic parameters (Km and Vmax) for

276

the CDNB conjugating reaction were determined. The Km was 0.629 ± 0.087 mM and

277

the Vmax was 13.1 ± 0.678 µmol mg-1 min-1 (Figure3B), and the enzyme’s catalytic

278

constant kcat and catalytic efficiency (kcat / Km) were 1.8×103 min-1 and 2.87×103 min-1

279

mM-1, respectively.

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In vitro inhibition of GST activities by lambda-cyhalothrin.The IC50 value of

281

lambda-cyhalothrin against was 653 μM, suggesting that lambda-cyhalothrin could

282

significantly inhibit the CDNB conjugating activity of CpGSTd3. The positive control

283

GTx strongly inhibits the CpGSTd3 activity, with a IC50 value of 116 μM (Figure4).

284

Lambda-cyhalothrin metabolism.To determine the effect of purified CpGSTd3

285

protein to metabolize lambda-cyhalothrin, an HPLC assay was conducted. Results

286

indicate that CpGSTd3 was able to metabolize 32.10% of the lambda-cyhalothrin in a

287

1 h reaction (Table 3). The ratio of peak areas of lambda-cyhalothrin was different

288

when using 20 μg CpGSTd3 (Figure S3).

289

DISCUSSION

290

The great interest in insect delta and epsilon GSTsis due to their roles in

291

detoxification

292

insecticides.17-19 In this study, a delta class GST, CpGSTd3, was cloned and

293

functional

294

lambda-cyhalothrin.

of

both

analysis

endogenous

revealed

that

and

xenobiotic

CpGSTd3

compounds

functions

to

including

metabolize

295

Lambda-cyhalothrin is widely used in the control of C. pomonella. In this study,

296

the toxicity of lambda-cyhalothrin against fourth-instar larvae of susceptible C.

297

pomonella was determined by the topical application method. As time goes on, the

298

LD10 and LD50 values gradually decreased. In other words, insects treated by

299

lambda-cyhalothrin at the same dose will increase mortality over time. In our

300

previous studies, fourth-instar larvae of the C. pomonella strain originally collected

301

from an abandoned apple orchard in Gansu province, China, and reared in laboratory 14


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for 8 generations exhibited an LC50 of lambda-cyhalothrin of 0.59 mg L-1 after 36 h of

303

treatment using an artificial mixed insecticide method.14 The results of the two studies

304

are similar, although bioassay methods of lambda-cyhalothrin for these two strains are

305

different. It is noteworthy that it is best to determine the toxicity of an insecticide

306

against eggs or neonate larvae because they are the main target of insecticides on C.

307

pomonella.10 However, in this experiment the fourth-instar larvae were selected as the

308

test specimens. The main reason is that technical grade lambda-cyhalothrin was used

309

in this study, and the most suitable organic solvent acetone is toxic to neonate larvae,

310

with mortality higher than 90% in the controls (data not published). Another reason

311

for using fourth-instar larvae is that we found a relatively high expression level of

312

CpGSTd3 in fourth-instar larvae; such a selection is more favorable to determining

313

the induction expression of lambda-cyhalothrin. The third reason is that the third- to

314

fourth-instar larvae may be exposed to insecticides during turn fruit harm in orchards.

315

The expression pattern of a specific GST gene in insects may imply their

316

biological function. It has been demonstrated that the developmental-dependent

317

expression patterns of insect GST genes are highly diverse among species.28,34 For

318

example, the GSTd1 and GSTd3 are mainly expressed in the fourth-instar larval, pupal

319

and adult stages, while the expression level of GSTd2 in the larval stage is lower than

320

in pupal and adult stages in Pieris rapae.34 The GSTd4 and GSTd5 have higher

321

expression levels at larval stages than other stages of P. xylostella.28 In C. pomonella,

322

CpGSTd1 was up-regulated with aging and reached the peak at the pupal and adult

323

stage.19 In this study, CpGSTd3 is mainly expressed in the larval stage, the main 15



324

feeding stage, which is congruent with previous study of delta class GST genes

325

GSTd4 and GSTd5 in P. xylostella.28 These results suggest that CpGSTd3 might be

326

involved in metabolizing plant secondary metabolites and insecticides in C.

327

pomonella. However, topical exposure was chosen instead of the feeding test in this

328

study. Beyond LD50 the administration route can modify the pharmacokinetics of the

329

insecticide and consequently influence the gene profile expression. Moreover, the

330

CpGSTd3 transcript level varied among different life stages, this suggested that

331

CpGSTd3 might have also a role in larvae development. Similar observation was

332

reported by Zou et al. (2011) who found that midgut hormones have an important role

333

in the regulation of basal GST expression 35.

334

To better understand the function of CpGSTd3, the tissue-specific expression

335

profile was also examined. It has been proposed that the distribution of GST genes in

336

the tissues of an insect might imply some biological and physiological functions.19

337

GST genes are prominently expressed in the Malpighian tubule, midgut and fat bodies,

338

the main detoxification tissues in insects.34, 36, 37. In our study, CpGSTd3 transcript is

339

expressed in many tissues in fourth-instar larvae, the highest expression levels of

340

CpGSTd3 were observed in midgut, followed by the Malpighian tubule. Our results

341

are in line with epsilon class GST genes BmGSTe2, BmGSTe3, BmGSTe4, BmGSTe5,

342

BmGSTe6, and BmGSTe7 in B. mori.27 It is well known that midgut and Malpighian

343

tubule are important tissues involved in detoxification of insecticides.38 Therefore, the

344

high expression level of CpGSTd3 in midgut and Malpighian tubule could support

345

CpGSTd3 playing an important role in detoxification of insecticides. However, we did 16


Page 16 of 39

Page 17 of 39


346

not find similar expression in the fat body, which is thought to be the main

347

detoxification organ. Fat body plays multiple metabolic functions especially lipid

348

metabolism in insects. The adipocytes in fat body can store a great amount of lipid

349

and provide energy the insect needs during non-feeding periods.39 These results imply

350

that CpGSTd3 is likely not involved in lipid metabolism in C. pomonella. Our finding

351

is not congruent with CpGSTd1 in C. pomonella.19 CpGSTd3 is also expressed in

352

thoraxes, abdomens, and legs of both male and female adults except the heads, and

353

was significant higher in male abdomens than female abdomens by RT-qPCR,

354

suggesting a sexually biased expression pattern for CpGSTd3 in adults and probably

355

this gene has robust detoxification function in male than female; however, in a

356

previous study, CpGSTd3 was only found to be expressed in the female abdomens by

357

endpoint PCR

358

higher in antennae than other tissues of male and female adult.

359

expression profiles of delta class GSTs imply that GSTs may have multiple functions

360

in C. pomonella.

29.

Furthermore, in a previous study, the expression of CpGSTd2 was 29

Such diversified

361

The induction of expression levels of GST genes in response to xenobiotics has

362

been documented in insects.19,23,40 In this study, the expression level of CpGSTd3 was

363

significantly induced by LD10 lambda-cyhalothrin after 3 h of treatment, and the

364

expression level was then decreased gradually, and was significantly lower than that

365

of the control group after 12 h of treatment (Figure 2C). This result is in accordance

366

with the expression pattern of GSTs in P. rapae after treatment with a sublethal dose

367

of lambda-cyhalothrin,34 but not in line with the expression level of CpGSTd1 in C. 17



368

pomonella treated with a low dosage of lambda-cyhalothrin over time and delta class

369

GST genes in Leptinotarsa decemlineata 41. As mentioned above, the decreasing LD10

370

value from 12 h to 72 h (Table 2) as well as the observed down-regulation of

371

CpGSTd3 after treatment with LD10 of lambda-cyhalothrin for 12 h to 48 h suggests

372

that the expression of CpGSTd3 may play a role in survival from lambda-cyhalothrin

373

exposure. However, the induction is only 1.84 fold compared to the control measured

374

on whole-body larvae. Therefore, a more informative analysis of the CpGSTd3

375

expression in midgut and Malpighian tubules should be determined. Whatever, these

376

results indicate that CpGSTd3 plays a role in detoxification of lambda-cyhalothrin.

377

The most important function of GSTs is to catalyze various endogenous and

378

exogenous compounds with the conjugation of GSH.18,20,21 The substrate CDNB is

379

commonly used in GST activity assays.42 Recombinant CpGSTd3 expressed in E. coli

380

exhibited enzymatic activity with the substrate CDNB, which is similar to the GSTs

381

from Culex pipiens (GSTd2), Anopheles dirus (GST1-1), Anopheles gambiae

382

(GSTd2), Tetranychusurticae (GSTm09), P. xylostella (GSTe3 and GSTe4) and S.

383

exigua (GSTo).43-48 Several studies have documented the kinetic properties of

384

CpGSTd1 and CpGSTd2 expressed from E. coli.19,29 In this study, the recombinant

385

CpGSTd3 protein exhibited kinetic properties similar to CpGSTd2,29 with a Vmax

386

value of 13.1 μmol min−1 mg−1and Km value of 0.629 mM for CpGSTd3 towards

387

CDNB. This observation was not consistent with CpGSTd1.19 Moreover, the kcat and

388

kcat / Km values of CpGSTd3 protein were lower than that of CpGSTd119, suggesting a

389

lower catalytic efficiency of CpGSTd3 than CpGSTd1 in C. pomonella. Difference in 18


Page 18 of 39

Page 19 of 39


390

catalytic efficiency between CpGSTd3 and CpGSTd1was further explained by

391

comparison of the residues possibly formation of the G-site and H-site between

392

CpGSTd1 and CpGSTd3 were aligned. Results indicate that 5 of 8 residues formation

393

of the G-site were conserved, and 6 of 12 residues possibly contribute to H-site

394

formation divergence were observed (Figure S4).

395

GSTs are well known multifunctional enzymes that play important roles in

396

detoxification insecticides including pyrethroids in insects.17-19 The binding affinity of

397

a GST enzyme with insecticide has been shown to associate with the metabolic

398

capability in insect.19 GST is a receptor for GTx but not for insecticides including

399

lambda-cyhalothrin, however, GST interacts with insecticides through the binding

400

and sequestration45. Therefore, the IC50 value of an insecticide against GST could

401

represent the binding affinity of insecticide with GST.

402

and lambda-cyhalothrin exhibited inhibition property against CpGSTd3, but the IC50

403

values were higher compared to CpGSTd119, suggesting that lambda-cyhalothrin has

404

a weaker binding affinity with CpGSTd3 than CpGSTd1 in C. pomonella. Previous

405

studies have shown that insect GSTs could not directly metabolize pyrethroid

406

insecticides, but these enzymes facilitate the binding of metabolized proteins (such as

407

P450) with insecticides in a sequestration mechanism.49 In a challenge to the previous

408

finding, recent studies reported that some insect GSTs could directly metabolize

409

pyrethroid molecules. For example, GST-8 from H. armigera can effectively

410

eliminate OP and partially metabolize cypermethrin.50 GSTD1 and GSTD2 from C.

411

pipiens were unable to metabolize permethrin, but GSTD1 was able to metabolize 19


19, 24

In this study, both GTx


412

both cis- and trans-isomers fluorescent permethrin-like substrates.42 Our previous

413

study reported that in C. pomonella, CpGSTd1 was able to metabolize

414

lambda-cyhalothrin.19 In this study, another delta class GST gene CpGSTd3 was

415

found to directly metabolize lambda-cyhalothrin. The CpGSTd1 is mainly expressed

416

in adulthood whereas CpGSTd3 has a high expression level in larval stage of C.

417

pomonella. We speculate that CpGSTd3 might have a higher contribution to

418

lambda-cyhalothrin metabolism than CpGSTd1. However, the weaker binding affinity

419

of lambda-cyhalothrin with CpGSTd3 compared to CpGSTd1 in C. pomonella

420

indicates that in addition to directly metabolize, a sequestration probably plays a

421

greater role in detoxification of lambda-cyhalothrin in CpGSTd1 than CpGSTd3 does

422

in C. pomonella. This finding enriches the growing body of literature indicating that a

423

delta class GST can directly metabolize an insecticide.

424 425

Taken together, these findings suggest that delta class GST gene CpGSTd3 plays a crucial role in detoxification of lambda-cyhalothrin.

426 427

■ASSOCIATED CONTENT

428

Supporting Information Available: Supplements to the amino acid sequence

429

alignment of CpGSTd3 (Figure S1), amino acid sequence alignment of 4 delta class

430

GST genes (Figure S2), HPCL analysis of the metabolic ability of CpGSTd3 against

431

lambda-cyhalothrin in vitro (Figure S3) , and sequence alignment of G-site and H-site

432

residues between CpGSTd1 and CpGSTd3 (Figure S4). The Supporting Information

433

is available free of charge via the Internet at http://pubs.acs.org. 20


Page 20 of 39

Page 21 of 39


434

AUTHOR INFORMATION

435

Corresponding Author

436

*Tel.:+86

437

E-mail:[email protected](X.Q. Yang).

438

Notes

439

The authors declare no competing financial interest.

440

ACKNOWLEDGMENTS

441

We thank John Richard Schrock from Emporia State University (USA) for

442

proofreading an earlier version of this manuscript. This research was supported, in

443

part, by the National Natural Science Foundation of China (Grant 31501666), the

444

Young Elite Scientists Sponsorship Program by CAST (YESS20160085), the China

445

Postdoctoral Science Foundation (2017M621160), and the Scientific Research

446

Foundation of Talent Introduction of Shenyang Agricultural University (20153011).

02488487148.

Fax:

447 448 449 450 451 452 453 454 455 456

21


+86

02488487148.


457

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625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 29



641

Figure legends

642

Figure 1. Amino acid sequence alignment of CpGSTd3 with insect GSTs. BmGSTd2

643

from B. mori, BAD60789.1; PxGSTd3 from P. xylostella, BAJ10978.1; and HaGST

644

from H. armigera, ABK40535.1. The position of β-sheets (β1-β4) and α-helices

645

(H1-H9) in the CpGSTd3 protein sequence was derived from the secondary structures

646

of the CpGSTd3 predicted by the PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk/psipred/)

647

and is shown on top of the alignment. The putative glutathione binding region (G-site)

648

is boxed with dotted line, and the substrate binding region (H-site) is boxed with solid

649

line. The positions of highly conserved G-site residues are indicated with a black

650

rectangle, and the H-site related residues are indicated by a triangle.

651

Figure 2.The expression level of CpGSTd3 at developmental stages (A), tissues of

652

fourth-instar larvae (B) and 12-h-old adults (C), and exposure to LD10

653

lambda-cyhalothrin (D). The increased fold was calculated from the expression level

654

of CpGSTd3 in insecticide-treated group divided by acetone-treated control, and the

655

ratio was used for expression analysis. P-F Pupa-female, P-M Pupa-male, A-F

656

Adult-female, A-M Adult-male, F-A: female abdomens, M-A: male abdomens, F-T:

657

female thoraxes, M-T: male thoraxes, F-L: female legs, M-L: female legs, F-H:

658

female heads, M-H: male heads. HE head, CU cuticle, FB fat body, MD midgut, MT

659

Malpighian tubules. n.d.: not detected. The results are shown as the mean ± SD. The

660

error bars represent the standard errors calculated from three replicates. Letters on the

661

error bars indicate significant differences analyzed by the one-way analysis of

662

variance (ANOVA) with Duncan's test (P

CpGSTd3 is a lambda-cyhalothrin metabolizing glutathione S

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