Molecular characterization of a delta class glutathione S-transferase gene from the black cutworm Agrotis ipsilon

Accepted Manuscript Molecular characterization of a delta class glutathione Stransferase gene from the black cutworm Agrotis ipsilon

Su Liu, Ye Cao, Yu-Xing Zhang, Yue-Min Pan, Shi-Guang Li PII: DOI: Reference:

S1226-8615(17)30384-9 doi: 10.1016/j.aspen.2017.09.004 ASPEN 1055

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

23 June 2017 28 August 2017 5 September 2017

Please cite this article as: Su Liu, Ye Cao, Yu-Xing Zhang, Yue-Min Pan, Shi-Guang Li , Molecular characterization of a delta class glutathione S-transferase gene from the black cutworm Agrotis ipsilon, Journal of Asia-Pacific Entomology (2017), doi: 10.1016/ j.aspen.2017.09.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Molecular characterization of a delta class glutathione S-transferase gene from the black cutworm Agrotis ipsilon Su Liu#, Ye Cao#, Yu-Xing Zhang, Yue-Min Pan, Shi-Guang Li*

These authors contributed equally to this work.

*

Corresponding author:

SC

RI

#

PT

College of Plant Protection, Anhui Agricultural University, Hefei, Anhui 230036, China

NU

Shi-Guang Li

MA

College of Plant Protection, Anhui Agricultural University, 130 West Changjiang Road, Hefei, Anhui 230036, China.

AC

CE

PT E

D

E-mail: [email protected]

1

ACCEPTED MANUSCRIPT

Abstract In insects, glutathione S-transferases (GSTs) play essential roles in the detoxification of xenobiotic toxins and elimination of oxidative stress induced by toxic compounds. In the present study, a delta class GST gene (AiGSTd) was identified and characterized in the

PT

black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). The deduced protein sequence of

RI

AiGSTd contained highly conserved features of GST enzymes and shared high identities

SC

with its orthologs from other lepidopteran species. Recombinant AiGSTD protein was expressed in Escherichia coli and purified. The protein displayed the GSH-dependent

NU

conjugating activity towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB). Moreover,

MA

AiGSTD had the ability to protect DNA from oxidative damage, and the E. coli cells overexpressing AiGSTD showed long-term resistance to oxidative stress. The AiGSTd

D

transcripts were most abundant in the larval midgut. Exposure to chlorpyrifos and

PT E

lambda-cyhalothrin increased lipid peroxidation in larvae and significantly upregulated AiGSTd expression levels. This study is the first report of molecular characterization of a

CE

GST in A. ipsilon, and the present study suggest that AiGSTD might be involved in

AC

protecting against the oxidative stress induced by insecticides.

Keywords

Agrotis ipsilon, antioxidative defense, glutathione S-transferases (GST), insecticide, oxidative stress

2

ACCEPTED MANUSCRIPT

1. Introduction Glutathione S-transferase (GST) is a family of multifunctional enzymes that exist in almost all living organisms (Hayes et al., 2005; Ketterman et al., 2011). The major function of GST enzymes is to catalyze the conjugation of reduced glutathione (GSH) to a wide

PT

range of endogenous and exogenous compounds, making them more soluble and easier to

RI

excrete from the cells (Enayati et al., 2005; Ketterman et al., 2011). In insects, GSTs have

SC

attracted much more attention because they play an essential role in the detoxification of various insecticides (Li et al., 2007). For instance, a GST (CpGSTD1) in the mosquito, pipiens,

is

able

to

metabolize

the

NU

Culex

organochlorine

insecticide,

MA

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), by dehydrochlorination (Samra et al., 2012). Furthermore, metabolization of the organophosphate insecticide, diazinon, by a GST

D

(bmGSTU2) has been observed in the silkworm, Bombyx mori (Yamamoto and Yamada,

PT E

2016).

In addition, insect GSTs display a peroxidation activity that protects against oxidative

CE

stress (Vontas et al., 2001; Corona and Robinson, 2006; Zhang et al., 2013). In living

AC

organisms, the reactive oxygen species (ROS), including peroxides, superoxide, hydroxyl radical, etc., are naturally generated during aerobic metabolism (Dowling and Simmons, 2009). However, exogenous inducers, such as insecticides, plant allelochemicals, toxic heavy metals, microbial infections, ultraviolet (UV) radiation, and thermal stress, will result in excessive accumulation of ROS (Huang et al., 2011; Wang et al., 2012; Yan et al., 2013a; Hamzah and Alias, 2016; Tang et al., 2016). Excessive ROS damages DNA, proteins, and lipids and needs to be eliminated by an antioxidative process. Previous studies have 3

ACCEPTED MANUSCRIPT

reported that some insect GSTs, such as BgGSTD1 from the German cockroach Blattella germanica and AccGSTO1 from the Chinese honey bee Apis cerana cerana, are able to metabolize external prooxidants (Ma and Chang, 2007; Meng et al., 2014). Moreover, in A. cerana cerana and the beet armyworm Spodoptera exigua, purified GST enzymes have the

PT

capability to protect DNA from oxidative damage (Yan et al., 2013b; Wan et al., 2016; Xu

RI

et al., 2016). Furthermore, overexpression of several A. cerana cerana GSTs in Escherichia

SC

coli prevents the cells from long-term oxidative stress (Yu et al., 2012b; Yan et al., 2013a; Zhang et al., 2013). These results strongly suggested that insect GSTs are vitally important

NU

for antioxidative processes.

MA

To date, a great number of GSTs have been identified and functionally annotated in a variety of model insect species (Oakeshott et al., 2010; Zhou et al., 2013; You et al., 2015;

D

Han et al., 2016; Schama et al., 2016). Based on the cellular locations, insect GSTs have

PT E

been divided into two groups: cytosolic and microsomal (Hayes et al., 2005). Most GSTs in insects are cytosolic proteins and classified into six major classes (delta, epsilon, omega,

CE

sigma, theta, and zeta) according to their genomic positions, sequence similarities, and

AC

biochemical properties (Friedman, 2011; Ketterman et al., 2011). Delta and epsilon are insect-specific classes, and many members in the two classes are involved in xenobiotic detoxification and antioxidative defense (Corona and Robinson, 2006; Li et al., 2007). The black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae), is a polyphagous pest insect that causes great economic loss of many crops (Gu et al., 2013; Xue and Hua, 2014). Over the past two decades in China, control of this insect pest has mainly relied on insecticide application. The organophosphate chlorpyrifos and the synthetic pyrethroid 4

ACCEPTED MANUSCRIPT

lambda-cyhalothrin are two insecticides which have been widely used to manage the pest (Wu et al. 2013). However, indiscriminate use of insecticides may result in decreased susceptibility of the pest; a recent report showed that chlorpyrifos and lambda-cyhalothrin have become ineffective even at relatively high doses (Yu et al., 2012a). Considering that

PT

GSTs play vital roles in the detoxification of insecticides and protection against oxidative

RI

stress, the understanding of the biochemical mechanisms of GSTs in A. ipsilon and their

SC

responses to insecticides would provide useful information on the rational use of pesticides and will be beneficial for the effective management of the pest. In the present study, we

NU

identified a delta class GST gene (AiGSTd) from A. ipsilon. The biochemical property and

MA

antioxidant ability of recombinant AiGSTD protein were determined, and the transcriptional patterns of AiGSTd in various larval tissues and in response to chlorpyrifos

PT E

D

and lambda-cyhalothrin were also characterized.

2. Materials and methods

CE

2.1. Insect rearing and tissue collection

AC

The eggs of A. ipsilon were purchased from Genralpest Co., Ltd. (Beijing, China). The hatched first-instar larvae were transferred onto the leaves of the Chinese cabbage (Brassica pekinensis) until they molted to fourth-instar larvae. The rearing conditions were 26 ± 1°C, 65% relative humidity under a 16 h:8 h (L:D) photoperiod. Different tissues, including integument, midgut, Malpighian tubules, and fat body, were dissected from fourth-instar larvae and stored at –80°C before use. The dissections were repeated three times, and 30 larvae were used in each repeat. 5

ACCEPTED MANUSCRIPT

2.2. Insecticide treatment Chlorpyrifos and lambda-cyhalothrin (all ≥ 94% purity) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and diluted with acetone. A total of 1 μl of the

PT

chlorpyrifos (1.8 ng) or lambda-cyhalothrin (0.6 ng) was applied topically to the dorsal part

RI

of the middle abdomen of the fourth-instar larvae. These doses were LD50 doses

SC

(approximately 50% of the test individuals are killed at 24 h), which were determined by probit analysis in a pre-test (data not shown). Control insects were treated with acetone

NU

only. At 1, 2, 4, and 6 h after the treatment, individuals from the insecticide-exposed and

MA

control groups were collected and stored at –80°C. Each treatment was replicated six times, and 30 larvae were used in each replicate. Of these, three replicates were used for

D

investigation of gene expression using quantitative reverse transcription-PCR (qRT-PCR);

PT E

another three replicates were used for determination of malondialdehyde (MDA)

CE

concentration.

AC

2.3. RNA isolation and cDNA synthesis Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA). Each RNA sample was digested with RNase-free DNase I (Takara, Dalian, China) to remove potential contaminants from genomic DNA. First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China).

2.4. Identification of AiGSTd and bioinformatic analyses 6

ACCEPTED MANUSCRIPT

Previously, a transcriptome dataset of A. ipsilon was constructed (Gu et al., 2013). cDNA sequence of AiGSTd was identified by retrieving the dataset. The open reading frame (ORF) was predicted using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The theoretical isoelectric point (pI) and molecular weight (Mw) were calculated using an

PT

ExPASy tool (http://web.expasy.org/compute_pi/). The functional domains and catalytic

RI

residues were predicted by searching the NCBI's conserved domain database

SC

(http://www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml). The amino acid sequences were aligned using Clustal Omega (http://www.ebi.ac.uk/tools/msa/clustalo/). A phylogenetic

NU

tree was constructed by MEGA6 software using the neighbor-joining method with

MA

1000-fold bootstrap resampling (Tamura et al., 2013). The tree was viewed and edited using

D

the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/).

PT E

2.5. Protein expression and purification

The complete ORF of AiGSTd was amplified using gene-specific primers (Table S1),

CE

inserted into the KpnI/BamHI sites of the pET30a vector, and transformed into E. coli

AC

Rosetta (DE3) cells. Recombinant AiGSTD protein (rAiGSTD) was expressed as a fusion protein with a N-terminal 6 × His·tag. E. coli cells were grown in Luria-broth (LB) medium (contained 50 μg/ml kanamycin and 34 μg/ml chloramphenicol) at 37°C while shaken at 200 rpm. Once an OD600 of 0.6 was reached, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM. The culture was grown for another 6 h at 30°C while shaken at 180 rpm. Cells were collected by centrifugation, resuspended in lysis buffer [20 mM 7

ACCEPTED MANUSCRIPT

Tris·HCl, pH 7.4, 500 mM NaCl, 15% glycerol, and 1 mM phenylmethanesulfonyl fluoride (PMSF)], and lysed by sonication on ice using a JY92-IIN probe sonicator (Xinzhi Biotech., Ningbo, China). The recombinant protein was in soluble form. Protein was purified using a HisTrap affinity column (GE Healthcare, Uppsala, Sweden) with a linear

PT

gradient of 0–250 mM imidazole, then desalted using a centrifugal filter device (Centricon

RI

YM-10, Millipore, Ireland). The purity of rAiGSTD was analyzed by 12% sodium dodecyl

SC

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of purified

NU

protein was measured using a BCA protein assay kit (Thermo Scientific, Wilmington, DE).

The

glutathione

S-transferase

MA

2.6. Enzymatic activity assay

activity

of

rAiGSTD

was

measured

using

D

1-chloro-2,4-dinitrobenzene (CDNB, purchased from Sigma-Aldrich, St Louis, MO) as

PT E

substrate, according to a protocol described by (Samra et al., 2012). Briefly, a 200 μl reaction mixture contained 500 ng rAiGSTD, 1 mM CDNB, with or without 5 mM GSH in

CE

0.1 M sodium phosphate buffer (pH 7.0). Increases in absorbance were monitored at 15 s

AC

intervals at 340 nm for 5 min. The assays were biologically repeated three times; the absorbance was recorded on a Multiskan Go microplate reader (Thermo Scientific, Wilmington, DE).

To estimate the kinetic parameters [Michaelis constant (Km) and maximum velocity (Vmax])] of rAiGSTD, varying concentrations (0.03–1 mM) of CDNB and a fixed concentration (5 mM) of reduced GSH were used. The kinetic parameters were determined by using the Origin 8.0 software (OriginLab Corporation, USA); the double reciprocal plot 8

ACCEPTED MANUSCRIPT

method was used for calculation.

2.7. DNA protection activity assay The ability of rAiGSTD to protect super-coiled DNA from oxidative damage was

PT

assayed using the metal-catalyzed oxidation (MCO) system described previously (Wan et

RI

al., 2014; Wan et al., 2016). Briefly, a 50 μl reaction mixture containing 16.5 μM FeCl3, 3.3

SC

mM dithiothreitol (DTT), and different concentrations (0, 5, 10 and 20 μg) of rAiGSTD was incubated at 37°C for 2 h. Then pUC19 super-coiled plasmid DNA (1000 ng) was

NU

added to the reaction mixture and incubated at 37°C for another 1 h. DNA cleavage was

MA

evaluated by electrophoresis on a 1% (w/v) agarose gel. The bovine serum albumin (BSA,

PT E

2.8. Disc diffusion assay

D

20 μg), instead of rAiGSTD, was used as a control. Experiments were repeated three times.

The disc diffusion assay was performed using cumene hydroperoxide (CHP, purchased

CE

from Sigma-Aldrich, St Louis, MO) as the ROS inducer, based on a previously published

AC

protocol (Zhang et al., 2013). Briefly, 5 × 108 cells of E. coli Rosetta (DE3) (contained pET30a-AiGSTd vector) were spread on LB agar plates (containing 50 μg/ml kanamycin, 34 μg/ml chloramphenicol, and 0.5 mM IPTG) and incubated at 37°C for 1 h. Then, filter discs (6 mm diameter) were soaked with different concentrations of CHP (0, 50, 100, 200, and 300 mM) and were placed on the surface of the agar plate. After incubation at 37°C for 24 h, the inhibition zones around the filter discs were measured. Bacteria transformed with the wildtype pET30a vector were used as negative controls. Experiments were repeated 9

ACCEPTED MANUSCRIPT

three times.

2.9. MDA content determination Insecticide- and acetone-treated larvae were homogenized with ice-cold 10 mM

PT

phosphate-buffered saline (PBS, pH 7.4), and the supernatant was collected by

RI

centrifugation at 15000 × g, 4°C for 10 min. Protein concentration in the supernatant was

SC

measured using a BCA protein assay kit (Thermo Scientific, Wilmington, DE). MDA content was determined using a MDA assay kit (Jiancheng Bioengineering Institute,

NU

Nanjing, China). The kit uses thiobarbituric acid (TBA) as the substrate. TBA combined

D

biological replicates were performed.

MA

with MDA to form red products, and the absorbance was measured at 532 nm. Three

PT E

2.10. qRT-PCR

qRT-PCR was performed using SYBR Premix ExTaq II (Tli RNaseH Plus) (Takara,

CE

Dalian, China). Each reaction (20 μl volume) contained 10 μl SYBR Premix ExTaq, 1 μl

AC

(10 ng) cDNA template, 0.4 μl sense primer, 0.4 μl anti-sense primer (0.2 μM each), and 8.2 μl nuclease-free water. Primers are listed in Table S1; two housekeeping genes β-actin (GenBank: JQ822245) and ribosomal protein S3 (RpS3, GenBank: JQ822246) were used as reference genes to normalize the target gene expression (Gu et al., 2013). qRT-PCR was run on a CFX96 system (Bio-Rad, Hercules, CA) using an amplification protocol that consisted of one cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 25 s. To verify that a single PCR product was being detected by the fluorescent dye, the 10

ACCEPTED MANUSCRIPT

heat-dissociation curves and amplification plots were analyzed at the end of the thermal cycle. In addition, a no-template control and a no transcriptase control were both included in the assay to detect possible contamination. The experiments were biologically repeated three times. Since two housekeeping genes were used, the transcriptional levels of the target

PT

gene were calculated using a modified Pfaffl method (Liu et al., 2015).

RI

2.11. Data statistics

SC

The data were analyzed using Data Processing System (DPS) software v9.5 (Tang and Zhang, 2013). The difference between two samples was compared by student's t-test, and

NU

the differences among different samples were compared by one-way analysis of variance

MA

(ANOVA) with Tukey's post hoc test. The level of significance was set at p < 0.05.

D

3. Results

PT E

3.1. Molecular characterization of AiGSTd A cDNA sequence encoding putative glutathione S-transferase was identified from the

CE

transcriptome of A. ipsilon. The cDNA contained an ORF of 651 bp, which encoded a

AC

protein that consisted of 216 amino acid residues with a predicted pI of 7.3 and Mw of 24230 Dalton. BLASTX search results indicated that the deduced protein sequence of the cDNA had high identity with annotated delta class GSTs from other lepidopteran species, such as GSTD4 in Spodoptera litura (GenBank: AIH07597, 94% identity), GSTD2 in B. mori (BAD60789, 89% identity), GSTD1 in Cydia pomonella (ACG69436, 88% identity), and GSTD2 in Plutella xylostella (AHW45899, 84% identity). Therefore, we named the cDNA AiGSTd (GenBank accession number: MF370558). 11

ACCEPTED MANUSCRIPT

Domain analysis revealed that the deduced protein sequence of AiGSTd presented several putative glutathione binding sites (G-sites) and substrate binding sites (H-sites), and multiple sequence alignment results indicated that the amino acid residues that constitute the G-sites and H-sites were highly conserved among the aligned insect GSTs (Fig. 1).

PT

Additionally, catalytically active residues, including the Ser11, Gln51, His52, Glu66, Ser67,

RI

Arg68, and Phe119, which have been functionally studied in B. mori and Nilaparvata lugens

SC

(Yamamoto et al., 2012; Yamamoto et al., 2015), were also found to be highly conserved in the protein sequence of AiGSTd (Fig. 1).

NU

A phylogenetic tree was constructed based on the protein sequences of annotated GSTs

MA

from different insect orders. The result showed that insect GSTs have been classified into six classes (delta, epsilon, sigma, omega, theta, and zeta) and an 'unclassified' subgroup,

D

and the AiGSTd was clustered into the 'delta' clade together with delta GSTs from B. mori

PT E

(BmGSTd2) and P. xylostella (PxGSTd2) (Fig. 2).

CE

3.2. Enzymatic features of rAiGSTD

AC

To better understand the physiological function of AiGSTd, recombinant AiGSTD protein fused with a N-terminal 6 × His·tag was heterologously expressed in E. coli. SDS-PAGE result showed that a recombinant protein with a Mw of approximately 28 kDa (the 24 kDa protein plus the 4 kDa His·tag) was produced after induction with IPTG (Fig. 3A). The target protein was further purified by affinity chromatography (Fig. 3A). The catalytic activity of rAiGSTD toward the substrate CDNB was 95.8 μmol/min/mg protein (Fig. 3B). However, negligible activity was observed when GSH was absent in the reaction 12

ACCEPTED MANUSCRIPT

mixture (Fig. 3B). The result indicated that the glutathione S-transferase activity of rAiGSTD was GSH-dependent. The kinetic properties of rAiGSTD were further investigated by fixing GSH to a 5 mM final concentration, whereas CDNB was a variable substrate (0.03–1 mM) (Fig. 3C). By

PT

using the double reciprocal plot method, the maximum velocity (Vmax) and the Michaelis

RI

constant (Km) of rAiGSTD were determined as 161.3 μmol/min/mg protein and 0.45 mM,

SC

respectively (Fig. 3C).

NU

3.3. Protective effects of AiGSTD against oxidative stress

MA

A MCO system containing super-coiled pUC19 plasmid DNA was used to evaluate the ability of rAiGSTD against oxidative stress. As shown in the agarose gel (Fig. 4A), the

D

plasmid DNA was converted from the super-coiled form to the nicked form in the absence

PT E

of rAiGSTD, whereas the degree of conversion was alleviated when rAiGSTD was added. This result indicated that rAiGSTD could protect DNA against the ROS produced by the

CE

MCO system. In addition, the protection activity of rAiGSTD seems to be

AC

concentration-dependent (Fig. 4A). Further, E. coli cells overexpressing rAiGSTD were exposed to varying concentrations of CHP for 24 h. The result showed that, when 50, 100, 200, and 300 mM CHP were used, the inhibition zones around the rAiGSTD-overexpressed bacteria were much smaller than those around control bacteria (Fig. 4B), with 34%, 40%, 45%, and 37% halo reductions, respectively (Fig. 4C). Additionally, significant difference in halo diameter was found between rAiGSTD-overexpressed bacteria and controls at each treatment concentration 13

ACCEPTED MANUSCRIPT

(Fig. 4C).

3.4. Tissue-specific transcription of AiGSTd To study the expression pattern of AiGSTd in different larval tissues of A. ipsilon, total

PT

RNA from integument, midgut, Malpighian tubules, and fat body was isolated and used for

RI

qRT-PCR. The result showed that the AiGSTd transcripts could be detected in all the tested

SC

tissues (Fig. 5). The highest AiGSTd mRNA level was found in the midgut, which was significantly higher than in other tissues (Fig. 5). The mRNA level in the midgut was 8.2-,

NU

1.9- and 1.5-fold higher than in the integument, Malpighian tubules, and fat body,

MA

respectively. AiGSTd transcripts were also abundant in the Malpighian tubules and fat body,

D

but relatively scarce in the integument (Fig. 5).

PT E

3.5. MDA content and AiGSTd transcription under insecticide-induced oxidative stress To determine whether insecticide treatment will elevate oxidative stress, MDA

CE

concentrations were measured in chlorpyrifos-treated, lambda-cyhalothrin-treated, and

AC

control larvae. As shown in Fig. 6A, exposure to two insecticides for 1, 2, 4, and 6 h resulted in a significantly higher accumulation of MDA in larvae when compared with those in controls. There were 1.9-, 2.5-, 2.8-, and 2.4-fold increases in MDA contents at 1, 2, 4, and 6 h after the chlorpyrifos exposure, respectively; and 1.8-, 1.5-, 2.4- and 3.0-fold increases in MDA concentrations at these time intervals after the lambda-cyhalothrin exposure, respectively (Fig. 6A). These results suggested that the lipid peroxides could be generated following exposure to these insecticides. 14

ACCEPTED MANUSCRIPT

The expression of AiGSTd was significantly induced after the insecticide treatment (Fig. 6B). At 1, 2, 4, and 6 h after the chlorpyrifos exposure, transcription levels of AiGSTd were upregulated to 2.1-, 2.3-, 4.8- and 3.2-fold, respectively, and at 1, 2, 4 and 6 h after the lambda-cyhalothrin exposure, the AiGSTd mRNA levels were elevated to 1.7-, 2.5-, 3.6-

RI

PT

and 3.7-fold, respectively (Fig. 6B).

SC

4. Discussion

In the present study, a delta class GST gene, AiGSTd, was identified from the black

NU

cutworm. The deduced protein sequence of AiGSTd included conserved functional

MA

domains, including G-sites, which were conserved at the N-terminal region, and H-sites, which were more variable and located at the C-terminal region (Fig. 1). Recently, structural

D

biology and site-directed mutagenesis studies have revealed that several amino acid

PT E

residues in these domains are catalytically active. For instance, the Ser11, Gln51, His52, Ser67, and Arg68 in a delta GST of B. mori, and Ser11, His52, Glu66, and Phe119 in a delta GST from

CE

N. lugens, are important for enzymatic functions (Yamamoto et al., 2012; Yamamoto et al.,

AC

2015). These key residues were also found in the protein sequence of AiGSTd (Ser11, Gln51, His52, Glu66, Ser67, Arg68, and Phe119, Fig. 1), implying these residues might contribute to the catalytic activity of AiGSTD. The general function of GSTs is to catalyze the conjugation of GSH to various endogenous and exogenous compounds (Hayes et al., 2005). To probe the catalytic function of rAiGSTD, the recombinant protein was expressed in E. coli and purified, and the glutathione S-transferase activity was assayed using CDNB as the substrate. CDNB is a 15

ACCEPTED MANUSCRIPT

synthetic substrate, which is commonly used in GST activity assays (Ketterman et al., 2011). We found that rAiGSTD displayed potent CDNB-conjugation activity (95.8 μmol/min/mg), indicating that the protein is a functional enzyme. The activity of rAiGSTD was higher than in A. cerana cerana (55.6 nmol/min/mg) (Yan et al., 2013b), but relatively

PT

lower than in N. lugens (141 μmol/min/mg) and B. germanica (508 μmol/min/mg) (Vontas

RI

et al., 2001; Ma and Chang, 2007).

SC

In addition to the glutathione S-transferase activity, insect GSTs display peroxidation activity against oxidative stress (Vontas et al., 2001; Enayati et al., 2005). Insects in their

oxidants,

insecticides,

plant

secondary

metabolites,

pathogenic

MA

environmental

NU

habitat are constantly exposed to many natural and synthesized oxidative inducers, such as

microorganisms, toxic heavy metals, UV radiation, and heat shock. These inducers will lead

D

to a significantly increased content of ROS which is harmful to insects. Therefore, the

PT E

peroxidase function of GSTs is especially important for insect survival because excess ROS could be eliminated rapidly by these enzymes. To verify whether rAiGSTD had the ability

CE

to resist oxidative stress, the MCO system, which can produce hydroxyl radicals (Yan et al.,

AC

2013b; Wan et al., 2016), was used in the assay. The result showed that rAiGSTD could protect super-coiled DNA against cleavage in the MCO system, suggesting that the enzyme has antioxidant activity that protects DNA against oxidative damage. Furthermore, E. coli cells overexpressing rAiGSTD were exposed to different concentrations of CHP. CHP is an extracellular ROS stressor widely used as a model substance to study the mechanism of oxidative stress-induced cell injuries (Burmeister et al., 2008; Yan et al., 2013a; Zhang et al., 2013). The differences of inhibition zones between AiGSTD-overexpressed cells and 16

ACCEPTED MANUSCRIPT

control cells clearly demonstrated that rAiGSTD plays an important role in resistance to oxidative stress. Similar results have been observed for GSTs in other insects, such as A. cerana cerana and S. exigua (Yu et al., 2012b; Wan et al., 2016; Xu et al., 2016). Moreover, delta GSTs in Drosophila melanogaster, N. lugens, B. germanica, and S. litura also

PT

metabolize lipid peroxidation products, such as 4-hydroxynonenal (Vontas et al., 2001;

RI

Sawicki et al., 2003; Ma and Chang, 2007; Huang et al., 2011; Zou et al., 2016). The ability

SC

to metabolize peroxides and lipid peroxidation products suggests that GSTs may play a pivotal role in insect survival under oxidative stress.

NU

The tissue-specific expression of AiGSTd was examined, and the result showed that a

MA

noticeable abundance of AiGSTd transcripts was observed in larval midgut, the major digestive organ. The extensive expression of GST genes in this organ has been found in

D

other insect species, such as Manduca sexta, B. mori, S. exigua and Lymantria dispar

PT E

(Snyder et al., 1995; Huang et al., 2011; Zou et al., 2011; Vlahović et al., 2016; Xu et al., 2016; Zou et al., 2016). Additionally, in D. melanogaster, the expression levels of a number

CE

of GSTs in the midgut increased in response to different concentrations of dietary H2O2 (Li

AC

et al., 2008), and in S. litura, several GSTs in the larval midgut are induced by chlorpyrifos, xanthotoxin, and bacterial infection (Huang et al., 2011). These studies suggest that the midgut-enriched GSTs are involved in the detoxification of a great variety of xenobiotic compounds, including insecticides, plant allelochemicals, and, more importantly, in protecting against oxidative stresses. It is believed that high oxidative stress exists in the larval midgut due to the direct intake of foods that contain toxic oxidative radicals and/or to the production of oxidants through food digestion and metabolism (Krishnan and Kodrík, 17

ACCEPTED MANUSCRIPT

2006). Our result implied that AiGSTd might play important roles in the antioxidative defense and detoxification of dietary toxins. Synthetic insecticides are important abiotic environmental factors that cause physiological changes in insects. It is assumed that the enhanced stress of oxidation is

PT

induced by insecticide exposure and that the expression of GSTs might be elevated to meet

RI

the demand for antioxidant protection (Singh et al., 2001; Vontas et al., 2001; Huang et al.,

SC

2011; Zhang et al., 2016; Zou et al., 2016). Moreover, GSTs in A. cerana cerana, B. mori, N. lugens, and S. litura can respond not only to insecticides but also to other oxidative

NU

inducers including plant allelochemicals, bacteria, heavy metals, H2O2, UV radiation, and

MA

temperature change (Huang et al., 2011; Yamamoto et al., 2011; Yu et al., 2012b; Zhou et al., 2013; Meng et al., 2014). The organophosphate insecticide chlorpyrifos and the

D

synthetic pyrethroid lambda-cyhalothrin are two of the most used insecticides for

PT E

controlling of A. ipsilon (Wu et al. 2013). However, the two pesticides are much less effective to control the pest in recent years (Yu et al., 2012a). It is possible that the

CE

peroxidase function of GST plays an important role in detoxifying the insecticide-induced

AC

ROS in A. ipsilon. In S. exigua and N. lugens, the peroxidase activity of GST proteins are involved in the insecticide detoxification (Vontas et al., 2001; Xu et al., 2016). To determine whether AiGSTd responded to insecticide-induced oxidative stress, MDA concentrations and AiGSTd mRNA levels were investigated in larvae treated with chlorpyrifos and lambda-cyhalothrin. MDA is a product of lipid peroxidation induced by the oxygen radical and is an indicator of oxidative stress (Del Rio et al., 2005). The results showed that the MDA content and AiGSTd transcription were both significantly increased following 18

ACCEPTED MANUSCRIPT

exposure to insecticides. We hypothesized that insecticide exposure may cause ROS generation in A. ipsilon larvae, and the oxidative stress may be eliminated by AiGSTD. However, functional studies, such as knockdown of AiGSTd by RNA interference and metabolism assay, are needed to elucidate the precise roles of AiGSTd in this antioxidative

PT

process.

RI

In conclusion, a delta class GST gene, AiGSTd, was identified and characterized from

SC

A. ipsilon. Recombinant AiGSTD protein displayed glutathione S-transferase activity and could protect DNA and E. coli cells from oxidative damage. The mRNA level of AiGSTd

NU

was significantly upregulated in response to insecticide challenge, and this upregulation

MA

may be associated with the increased oxidative stress caused by insecticide exposure. Our findings provided useful information for better understanding the antioxidative mechanisms

Acknowledgements

PT E

D

in A. ipsilon.

CE

This work was supported by the National Key Research and Development Program of

AC

China (grant numbers 2017YFD0201708 and 2016YFD0200205-7) and the Anhui Provincial Natural Science Foundation (grant number 1708085QC50).

19

ACCEPTED MANUSCRIPT

Conflict of interest statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other

PT

personal interest of any nature or kind in any product, service and/or company that could be

RI

construed as influencing the position presented in the manuscript entitled "Molecular

SC

characterization of a delta class glutathione S-transferase gene from the black cutworm

AC

CE

PT E

D

MA

NU

Agrotis ipsilon".

20

ACCEPTED MANUSCRIPT

References Burmeister, C., Lüersen, K., Heinick, A., Hussein, A., Domagalski, M., Walter, R.D., Liebau, E., 2008. Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1). FASEB J. 22, 343–354. Corona, M., Robinson, G.E., 2006. Genes of the antioxidant system of the honey bee: annotation and

PT

phylogeny. Insect Mol. Biol. 15, 687–701. Del Rio, D., Stewart, A.J., Pellegrini, N., 2005. A review of recent studies on malondialdehyde as toxic

RI

molecule and biological marker of oxidative stress. Nutr. Metab. Cardiovasc. Dis. 15, 316–328.

evolution. Proc. Roy. Soc. B 276, 1737–1745.

SC

Dowling, D.K., Simmons, L.W., 2009. Reactive oxygen species as universal constraints in life-history

NU

Enayati, A.A., Ranson, H., Hemingway, J., 2005. Insect glutathione transferases and insecticide resistance. Insect Mol. Biol. 14, 3–8.

Phylogenet. Evol. 61, 924–932.

MA

Friedman, R., 2011. Genomic organization of the glutathione S-transferase family in insects. Mol.

Gu, S.-H., Wu, K.-M., Guo, Y.-Y., Pickett, J., Field, L., Zhou, J.-J., Zhang, Y.-J., 2013. Identification of

D

genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative

PT E

roles in sex pheromone biosynthesis and transport. BMC Genomics 14, 636. Hamzah, S.N., Alias, Z., 2016. Developmental expression and oxidative stress induction of proteome of

869–875.

CE

glutathione S-transferases in Aedes albopictus (Diptera: Culicidae). J. Asia Pac. Entomol. 19,

AC

Han, J.-B., Li, G.-Q., Wan, P.-J., Zhu, T.-T., Meng, Q.-W., 2016. Identification of glutathione S-transferase genes in Leptinotarsa decemlineata and their expression patterns under stress of three insecticides. Pestic. Biochem. Physiol. 133, 26–34. Hayes, J.D., Flanagan, J.U., Jowsey, I.R., 2005. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51–88. Huang, Y., Xu, Z., Lin, X., Feng, Q., Zheng, S., 2011. Structure and expression of glutathione S-transferase genes from the midgut of the Common cutworm, Spodoptera litura (Noctuidae) and their response to xenobiotic compounds and bacteria. J. Insect Physiol. 57, 1033–1044. Ketterman, A.J., Saisawang, C., Wongsantichon, J., 2011. Insect glutathione transferases. Drug Metab.

21

ACCEPTED MANUSCRIPT

Rev. 43, 253–265. Krishnan, N., Kodrík, D., 2006. Antioxidant enzymes in Spodoptera littoralis (Boisduval): Are they enhanced to protect gut tissues during oxidative stress? J. Insect Physiol. 52, 11–20. Li, H.M., Buczkowski, G., Mittapalli, O., Xie, J., Wu, J., Westerman, R., Schemerhorn, B.J., Murdock, L.L., Pittendrigh, B.R., 2008. Transcriptomic profiles of Drosophila melanogaster third instar

PT

larval midgut and responses to oxidative stress. Insect Mol. Biol. 17, 325–339. Li, X., Schuler, M.A., Berenbaum, M.R., 2007. Molecular mechanisms of metabolic resistance to

RI

synthetic and natural xenobiotics. Annu. Rev. Entomol. 52, 231–253.

SC

Liu, S., Gong, Z.-J., Rao, X.-J., Li, M.-Y., Li, S.-G., 2015. Identification of putative carboxylesterase and glutathione S-transferase genes from the antennae of the Chilo suppressalis (Lepidoptera:

NU

Pyralidae). J. Insect Sci. 15, 103.

Ma, B., Chang, F.N., 2007. Purification and cloning of a Delta class glutathione S-transferase displaying

MA

high peroxidase activity isolated from the German cockroach Blattella germanica. FEBS J. 274, 1793–1803.

Meng, F., Zhang, Y., Liu, F., Guo, X., Xu, B., 2014. Characterization and mutational analysis of

PT E

stress. PLoS One 9, e93100.

D

omega-class GST (GSTO1) from Apis cerana cerana, a gene involved in response to oxidative

Oakeshott, J.G., Johnson, R.M., Berenbaum, M.R., Ranson, H., Cristino, A.S., Claudianos, C., 2010. Metabolic enzymes associated with xenobiotic and chemosensory responses in Nasonia

CE

vitripennis. Insect Mol. Biol. 19, 147–163. Samra, A.I., Kamita, S.G., Yao, H.-W., Cornel, A.J., Hammock, B.D., 2012. Cloning and characterization

AC

of two glutathione S-transferases from pyrethroid-resistant Culex pipiens. Pest Manag. Sci. 68, 764–772.

Sawicki, R., Singh, S.P., Mondal, A.K., Benes, H., Zimniak, P., 2003. Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. Biochem. J. 370, 661–669. Schama, R., Pedrini, N., Juárez, M.P., Nelson, D.R., Torres, A.Q., Valle, D., Mesquita, R.D., 2016. Rhodnius prolixus supergene families of enzymes potentially associated with insecticide

22

ACCEPTED MANUSCRIPT

resistance. Insect Biochem. Mol. Biol. 69, 91–104. Singh, S.P., Coronella, J.A., Beneš, H., Cochrane, B.J., Zimniak, P., 2001. Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products. Eur. J. Biochem. 268, 2912–2923. Snyder, M.J., Walding, J.K., Feyereisen, R., 1995. Glutathione S-transferases from larval Manduca sexta

PT

midgut: Sequence of two cdnas and enzyme induction. Insect Biochem. Mol, Biol. 25, 455–465. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary

RI

Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

SC

Tang, Q.-Y., Zhang, C.-X., 2013. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci. 20,

NU

254–260.

Tang, X., Li, N., Wang, W., Yu, J., Xu, L., Shen, Z., 2016. DGE analysis of changes in gene expression

MA

in response to temperature and deltamethrin stress in the silkworm (Bombyx mori). J.Asia Pac. Entomol. 19, 45–50.

Vlahović, M., Ilijin, L., Mrdaković, M., Todorović, D., Matić, D., Lazarević, J., Mataruga, V.P., 2016.

D

Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars

PT E

exposed to dietary cadmium. Environ. Toxicol. Phar. 44, 13–17. Vontas, J.G., Small, G.J., Hemingway, J., 2001. Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens. Biochem. J. 357, 65–72.

CE

Wan, H., Kang, T., Zhan, S., You, H., Zhu, F., Lee, K.S., Zhao, H., Jin, B.R., Li, J., 2014. Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme. Comp.

AC

Biochem. Physiol. B 175, 53–61. Wan, H., Zhan, S., Xia, X., Xu, P., You, H., Jin, B.R., Li, J., 2016. Identification and functional characterization of an epsilon glutathione S-transferase from the beet armyworm (Spodoptera exigua). Pestic. Biochem. Physiol. 132, 81–88. Wang, Y., Wang, L., Zhu, Z., Ma, W., Lei, C., 2012. The molecular characterization of antioxidant enzyme genes in Helicoverpa armigera adults and their involvement in response to ultraviolet-A stress. J. Insect Physiol. 58, 1250–1258. Wu, H.-B., Fan, K., Zhang, K.-P., Gong, Q.-T., Sun, R.-H., 2013. Biological activity of 13 insecticides to

23

ACCEPTED MANUSCRIPT

eggs and larva of Agrotis ypsilon Rottemberg. J. Environ. Entomol. 35, 409–414. Xu, P., Han, N., Kang, T., Zhan, S., Lee, K.S., Jin, B.R., Li, J., Wan, H., 2016. SeGSTo, a novel glutathione S-transferase from the beet armyworm (Spodoptera exigua), involved in detoxification and oxidative stress. Cell Stress Chaperon. 21, 805–816. Xue, S., Hua, B.-z., 2014. Proboscis sensilla of the black cutworm Agrotis ypsilon (Rottemberg)

PT

(Lepidoptera: Noctuidae). J. Asia Pac. Entomol. 17, 295–301. Yamamoto, K., Higashiura, A., Hossain, M.D.T., Yamada, N., Shiotsuki, T., Nakagawa, A., 2015.

RI

Structural characterization of the catalytic site of a Nilaparvata lugens delta-class glutathione

SC

transferase. Arch. Biochem. Biophys. 566, 36–42.

Yamamoto, K., Teshiba, S., Shigeoka, Y., Aso, Y., Banno, Y., Fujiki, T., Katakura, Y., 2011.

NU

Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth. Insect Mol. Biol. 20, 379–386.

MA

Yamamoto, K., Usuda, K., Kakuta, Y., Kimura, M., Higashiura, A., Nakagawa, A., Aso, Y., Suzuki, M., 2012. Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase. Biochim. Biophys. Acta 1820, 1469–1474.

D

Yamamoto, K., Yamada, N., 2016. Identification of a diazinon-metabolizing glutathione S-transferase in

PT E

the silkworm, Bombyx mori. Sci. Rep. 6, 30073. Yan, H., Jia, H., Gao, H., Guo, X., Xu, B., 2013a. Identification, genomic organization, and oxidative stress response of a sigma class glutathione S-transferase gene (AccGSTS1) in the honey bee, Apis

CE

cerana cerana. Cell Stress Chaperon. 18, 415–426. Yan, H., Jia, H., Wang, X., Gao, H., Guo, X., Xu, B., 2013b. Identification and characterization of an

AC

Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress. Naturwissenschaften 100, 153–163. You, Y., Xie, M., Ren, N., Cheng, X., Li, J., Ma, X., Zou, M., Vasseur, L., Gurr, G., You, M., 2015. Characterization and expression profiling of glutathione S-transferases in the diamondback moth, Plutella xylostella (L.). BMC Genomics 16, 152. Yu, W., Du, J., Hu, Y., Shen, R., Wei, M., 2012a. Toxicity of six insecticides to black cutworm Agrotis ypsilon (Rottemberg) and safety evaluation to oil organisms. Acta Phytophy. Sin. 39, 277–282. Yu, X., Sun, R., Yan, H., Guo, X., Xu, B., 2012b. Characterization of a sigma class glutathione

24

ACCEPTED MANUSCRIPT

S-transferase gene in the larvae of the honeybee (Apis cerana cerana) on exposure to mercury. Comp. Biochem. Physiol. B 161, 356–364. Zhang, N., Liu, J., Chen, S.-N., Huang, L.-H., Feng, Q.-L., Zheng, S.-C., 2016. Expression profiles of glutathione S-transferase superfamily in Spodoptera litura tolerated to sublethal doses of chlorpyrifos. Insect Sci. 23, 675–687.

PT

Zhang, Y., Yan, H., Lu, W., Li, Y., Guo, X., Xu, B., 2013. A novel Omega-class glutathione S-transferase gene in Apis cerana cerana: molecular characterisation of GSTO2 and its protective effects in

RI

oxidative stress. Cell Stress Chaperon. 18, 503–516.

SC

Zhou, W.-W., Liang, Q.-M., Xu, Y., Gurr, G.M., Bao, Y.-Y., Zhou, X.-P., Zhang, C.-X., Cheng, J., Zhu, Z.-R., 2013. Genomic insights into the glutathione S-transferase gene family of two rice

NU

planthoppers, Nilaparvata lugens (Stål) and Sogatella furcifera (Horváth) (Hemiptera: Delphacidae). PLoS One 8, e56604.

MA

Zou, F.-M., Lou, D.-S., Zhu, Y.-H., Wang, S.-P., Jin, B.-R., Gui, Z.-Z., 2011. Expression profiles of glutathione S-transferase genes in larval midgut of Bombyx mori exposed to insect hormones. Mol. Biol. Rep. 38, 639–647.

D

Zou, X., Xu, Z., Zou, H., Liu, J., Chen, S., Feng, Q., Zheng, S., 2016. Glutathione S-transferase

PT E

SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host plants. Insect

AC

CE

Biochem. Mol. Biol. 70, 32–43.

25

ACCEPTED MANUSCRIPT

Figure captions Fig. 1. Alignment of the deduced amino acid sequences of AiGSTd and delta class GSTs from other insect species, including Bombyx mori (BmGSTd2, GenBank: AB176691), Nilaparvata lugens (NlGSTd2, JQ917467), Tribolium castaneum (TcGSTd1, XM_969180), Drosophila melanogaster (DmGSTd1, NP_524326), and Apis cerana cerana (AccGSTd1, JF798573). Asterisks indicate

PT

conserved amino acid residues, whereas colons and dots indicate similar residues. Predicted GSH binding sites (G-sites) and substrate binding sites (H-sites) are shaded yellow and green,

RI

respectively. Catalytically active residues, which have been functionally studied in B. mori and N.

SC

lugens, were also found in the protein sequence of AiGSTd and indicated by solid triangles.

NU

Fig. 2. Phylogenetic relationships of AiGSTd with GSTs from other insect species, including Drosophila melanogaster (Dm-prefix), Anopheles gambiae (Ag), Bombyx mori (Bm), Tribolium

MA

castaneum (Tc), Nilaparvata lugens (Nl), and Plutella xylostella (Px). Insect GSTs are classified into six classes (delta, epsilon, omega, sigma, theta and zeta) and an 'unclassified' subgroup. Bootstrap values on each node are indicated by colors from green (0) to red (100). AiGSTd is in red

PT E

D

font.

Fig. 3. Purification and enzymatic features of rAiGSTD. (A) Expression and purification of rAiGSTD. Lane 1, molecular-mass marker; lane 2, crude extracts from the bacterial pellets before

CE

induction with isopropyl β-D-1-thiogalactopyranoside (IPTG); lane 3, crude extracts from the bacterial pellets after induction with IPTG; lane 4, purified rAiGSTD. (B) CDNB-conjugating

AC

activity of rAiGSTD in the presence or absence of GSH. Data were presented as means (n = 3) ± standard error (SE). (C) Kinetics of rAiGSTD determined by fixing GSH at 5 mM against varying concentrations of CDNB (0.03–1 mM). The regression line was fitted using the double-reciprocal plot method.

Fig. 4. Antioxidant activity of recombinant AiGSTD (rAiGSTD) protein. (A) Protection of DNA from oxidative damage by rAiGSTD in a metal-catalyzed oxidation (MCO) system. Lane 1, pUC19 plasmid DNA only; lane 2, pUC19 plasmid DNA + DTT; lane 3, pUC19 plasmid DNA + DTT +

26

ACCEPTED MANUSCRIPT FeCl3; lane 4, pUC19 plasmid DNA + DTT + FeCl3 + BSA (20 μg); lanes 5–7, pUC19 plasmid DNA + DTT + FeCl3 + rAiGSTD (5, 10, and 20 μg, respectively). NF, nicked form; SF, super-coiled form. (B) Protective effects of rAiGSTD against oxidative stress based on a disc diffusion assay. A total of 5×108 E. coli cells overexpressing rAiGSTD were spread on the LB agar plates, filter discs soaked with different concentrations (labels 1–5: 0, 50, 100, 200, and 300 mM,

PT

respectively) of cumene hydroperoxide (CHP) were placed on the surface of the plate. Bacteria transformed with the wildtype pET30a vector were used as controls. (C) Statistical analysis of the

RI

halo diameter of the inhibition zones. Data were presented as means (n = 3) ± SE. '*' denotes a

SC

significant difference in halo diameter between rAiGSTD-overexpressed bacteria and controls (p <

NU

0.05, student's t-test), whereas 'ns' represents 'not significant' (p > 0.05, student's t-test).

Fig. 5. Relative expression levels of AiGSTd in various larval tissues. IN, integument; MG, midgut;

MA

MT, Malpighian tubes; FB, fat body. The expression levels in different tissues were normalized relative to that of the integument. Data were presented as means (n = 3) ± SE. Different lowercase

PT E

Tukey's post hoc test, p < 0.05).

D

letters indicate significant variation in transcription among different tissues (one-way ANOVA with

Fig. 6. Relative MDA content (A) and AiGSTd transcription level (B) in larvae treated with sublethal concentrations of chlorpyrifos and lambda-cyhalothrin at different time intervals (1, 2, 4,

CE

and 6 h). The MDA content and AiGSTd mRNA level at each time point in insecticide-treated individuals were normalized relative to that of acetone-treated (control) individuals. Data were

AC

presented as means (n = 3) ± SE. '*' denotes a significant difference in MDA contents or AiGSTd expression levels between treated and control insects (p < 0.05, student's t-test).

27

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

Fig. 1

28

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

Fig. 2

29

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 3

30

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

Fig. 4

31

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 5

32

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 6

33

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Graphical abstract

34

ACCEPTED MANUSCRIPT

Highlights A delta class GST gene (AiGSTd) was identified in Agrotis ipsilon AiGSTD protein can protect cells from ROS damage AiGSTd is significantly upregulated by insecticides

AC

CE

PT E

D

MA

NU

SC

RI

PT

AiGSTd is potentially involved in antioxidative defense

35