Insect Science (2015) 22, 503–511, DOI 10.1111/1744-7917.12142

ORIGINAL ARTICLE

Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura Zhi-Bin Xu, Xiao-Peng Zou, Ni Zhang, Qi-Li Feng and Si-Chun Zheng Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China

Abstract Insect glutathione S-transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up-regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had Vmax (reaction rate of the enzyme saturated with the substrate) and Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and Vmax and Km values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1-chloro-2,4-dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro-diphenyl-trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals. Key words glutathione S-transferase; heavy metal; insecticide; midgut; Spodoptera litura

Introduction Insect digestive tract, especially midgut, is an important organ not only for food digestion, but also for defense against toxicity of xanobiotic compounds and entomopathogens. Glutathione S-transferases (GSTs), a super family of detoxifying enzymes, play an important role in detoxification by catalyzing conjugation of reduced glutathione (GSH) with electrophilic endogenous and xeno-

Correspondence: Si-Chun Zheng, School of Life Sciences, South China Normal University, Guangzhou 510631, China. Tel: +86 20 85210024; fax: +86 20 85215291; email: [email protected]

biotic compounds, including chemosynthetic insecticides and natural phytochemicals, converting them to less toxic water-soluble forms (Grant & Matsumura, 1989; Singh et al., 2001). Two domains of GSTs are responsible for the detoxification function, a GSH-binding domain, which is well conserved in the N-terminal thioredoxin-fold region of different classes of GSTs, and a hydrophobic substrate binding domain, which is structurally diverse in the C-terminal α-helical region of GSTs (Mannervik & Danielson, 1988). Insect GSTs also can defend against oxidative stress caused by toxic chemicals, pathogens and heavy metals through their selenium-independent peroxidase activity. They can metabolize hydroperoxides, such as 4-hydroxynonenal (4-HNE) and malonaldehyde, by 503

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catalyzing conjugation of these lipid peroxidation end products with GSH (Singh et al., 2001; Krishnan & Kodrik, 2006). Many insecticides can cause oxidative stress in insect cells and GSTs induced by the insecticides can function as antioxidant stress agents to protect the insect. For example, organophosphorus insecticide chlorpyrifos increased the amount of both GSTs and malonaldehyde, an indicator of oxidative stress, in Spodoptera litura (Huang et al., 2011), pyrethroid induced lipid peroxidation and protein oxidation in Nilaparvata lugens, while the induced GSTs protect from this oxidative damage (Vontas et al., 2001). Plant allelochemicals also can present their toxicity through the oxidative stress pathway. For example, xanthotoxin, a plant allelochemical from Apiaceae, is a pro-oxidant secondary metabolite and can generate superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, causing deleterious lipid peroxidation and increase in antioxidative activity of some enzymes such as glutathione peroxidase in several insects (Ahmad & Pardini, 1990). In the natural environment, insects have to deal with different heavy metals in their living habits. These heavy metals induce oxidative stress in the insects. For example, oxidative stress and antioxidative enzymes in Propsilocerus akamusi were induced by cadmium (Zheng et al., 2011). High levels of expression of GSTs of delta, epsilon and sigma classes in Chironomus riparius were detected in response to cadmium and silver nanoparticles exposure (Nair & Choi, 2011). Unlike the mechanism of binding to the toxic chemicals, GST probably binds heavy metals in the GSH binding domain at the N-terminal end, rather than in the hydrophobic substrate binding domain at the C-terminal end (Salazar-Medina et al., 2010). There are 40, 35, 13, 23 and 32 GSTs in Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively (Shi et al., 2012). Insect GSTs include microsomal and cytosolic GSTs. Microsomal GSTs have been classified into six classes: MGST1, MGST2, MGST3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP) and prostaglandin E synthase, and insect microsomal members are the most homologous to MGST1 and prostaglandin E synthase (Bresell et al., 2005). Cytosolic GSTs have been assigned into seven classes: delta, epsilon, omega, sigma, theta, zeta and unclassified GSTs (Chelvanayagam et al., 2001; Ranson et al., 2001; Ding et al., 2003). Although other classes of GSTs have also been reported to respond to insecticides (Yamamoto et al., 2011; Zhou et al., 2013), most GSTs that are involved in insecticide resistance or response to insecticides belong to the insect-specific classes of delta and epsilon (Li et al., 2007). However, not all insectspecific GSTs that are increased by insecticide induction  C 2014

have detoxification activity. Out of 25 GSTs of epsilon and delta classes in A. gambiae, five epsilon GSTs were over-expressed in the dichloro-diphenyl-trichloroethane (DDT)-resistant strain, but only GSTE2 showed the enzyme activity of detoxifying DDT (Ding et al., 2003). Additionally, different GSTs in a species have different detoxification abilities to different toxic compounds. In S. litura, both SlGSTe2 and SlGSTe3 responded to six insecticides, but SlGSTE2 showed much higher activity of detoxification than SlGSTE3 (Deng et al., 2009). Expression of different GST isoforms might be tissue-specific or developmental stage-specific. For example, only 28 GSTs out of total 38 GSTs in D. melanogaster have been found to express in the midgut of 3rd instar larvae and most of these isoforms belong to the delta and epsilon classes (Li et al., 2008). Spodoptera litura is an important agricultural pest and it feeds on more than 90 families of plants. Long-term use of chemical insecticides has resulted in resistance in field populations (Saleem et al., 2008). Searching the GST genes that are involved in detoxifying a wide range of synthetic and natural insecticides in the midgut would help to develop strategies to overcome insecticide resistance and improve the insecticidal efficacy. In our previously study, the expression of eight GSTs in S. litura in response to the organophosphorus insecticide chlorpyrifos and the allelochemical xanthotoxin were analyzed (Huang et al., 2011). One of them is SlGSTe1 (SlGST1 GenBank accession no. AY506545), which only had a trace amount of expression during the life cycle, and its messenger RNA (mRNA) level was greatly increased by chlorpyrifos and xanthotoxin (Huang et al., 2011). In this study, responses of SlGSTE1 cloned from the midgut of S. litura to plant allelochemicals, synthetic insecticides and heavy metals were further studied to better understand the action mechanism of this gene in detoxification of toxicants in S. litura.

Materials and methods Experimental insects and treatments Larvae of Spodoptera litura (Lepidoptera: Noctuidae) were reared on an artificial diet at 26°C, 70% relative humidity and under a photoperiod of 12 h light and 12 h dark, until they reached the pupal stage. For the treatment of chlorpyrifos and cadmium chloride (Cd), 1 μL of 0.35 mg/mL chlorpyrifos or 1% Cd in a small diet plug with 1% sucrose was fed to 1-day-old 6th instar larvae. After consumption of the diet containing chlorpyrifos or Cd, the larvae were transferred onto a Institute of Zoology, Chinese Academy of Sciences, 22, 503–511

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normal diet. The insects were collected at days 1, 3, 5 and 7 for Western blot analysis. The control was the insects treated with the same volume of diet alone. Expression and purification of recombinant SlGSTE1 protein Open reading frame (ORF) of the target complementary DNA (cDNA) was cloned into the pPROEX HTb expression vector (Life Technologies, Burlington, Canada), generating the recombinant expression vector, pPROEXbSlgste1. Escherichia coli cells (DH5α) were transformed with the recombinant plasmid DNA and were grown at 37°C in Luria–Bertani (LB) media containing 100 mg/mL ampicillin. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mmol/L. The cells were cultured for an additional 3 h before collection by centrifugation. The resulting cell pellets were suspended in 20 mmol/L Tris– HCl buffer (pH 8.0) containing 0.5 mol/L NaCl, 4 mg/mL of lysozyme, and 1 mmol/L PMSF (phenymethanesulfonyl fluoride) and then lysed by sonication. The suspension was centrifuged at 10 000 g at 4°C for 5 min. The supernatant and precipitant were collected for protein analysis. The supernatant containing the soluble protein was purified for activity determination, antibody production and Western blot analysis. Protein purification was conducted by using a His taq affinity chromatography system from Amersham Pharmacia Biotech (Piscataway, NJ, USA) following the manufacturer’s instruction. Antibody production The purified recombinant SlGSTE1 protein was used as an antigen. Polyclonal antiserum was made from rabbit by three booster injections, each with 500 ng protein in Freund’s adjuvant. Antiserum was collected after the injections and the pre-immune serum that was collected from the same rabbit prior to immunization was used as a control. Enzyme activity and kinetics analysis of SlGSTE1 Measurement of detoxifying activity of the SlGSTE1 recombinant protein was conducted as described by Habig et al. (1974). Three micrograms of protein was used in a total volume of 300 mL of a reaction mixture. The two GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) were added to the reaction. Change in absorbance of CDNB conjugate for the  C 2014

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first minute was measured at 340 nm and 25°C using DU800 spectrometer (Beckman Coulter Instruments, Inc., Winooski, VT, USA). Protein concentrations were measured using the Bio-Rad protein reagent and bovine serum albumin as a standard. Enzyme activity is represented as mmol of CDNB conjugated per min per mg protein. The apparent Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) and Vmax (reaction rate of the enzyme saturated with the substrate) were determined using the double reciprocal plot analysis. Each data point represents the average of five measurements. Peroxidase activity analysis of SlGSTE1 Peroxidase activity of the SlGSTE1 recombinant protein toward cumene hydrogen peroxide (4-HNE) was determined spectrophotometrically at 340 nm, according to the procedure described by Alin et al. (1985). After 188 μL reaction solution containing 50 mmol/L sodium phosphate buffer (pH 7.0), 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L GSH, 0.2 mmol/L nicotinamide adenine dinucleotide phosphate (NADPH) and 1 U glutathione reductase was kept at 25°C for 5 min, 10 μL enzyme and 1.2 mmol/L 4-HNE were added. The reaction solution without enzyme and 4-HNE was used as control. One unit of GST peroxidase activity was defined as the amount of the enzyme catalyzing the conjugation of 1 mmol of 4-HNE with GSH per minute. Indirect assay of GST enzymatic activity by insecticides and heavy metals To evaluate responsibility of SlGSTE1 to different insecticides and heavy metals, five insecticides (chlorpyrifos, deltamethrin, malathion, phoxim and DDT) and six metals (zinc, copper, nickel, lead, chromium and cadmium) were selected to assess the reaction of SlGSTE1 with these compounds and metals by in vitro indirect assay. For standard GST activity assay, the two GST substrates CDNB and reduced GSH were added to the reaction system and the change in absorbance of CDNB conjugate for the first minute was measured at 340 nm. There is no specific assay to test binding of GST with other different compounds. Therefore, to test GST binding with a compound or metal, indirect assay was used. When an insecticide or a metal is added to the standard reaction system, it competes with CDNB to bind with GSH. By determining the change of absorbance of CDNB conjugate at 340 nm, the GST activity of binding to the insecticide or metal is determined (Udomsinprasert

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et al., 2005). One hundred μmol/L of each insecticide (dissolved in acetone, then diluted in ddH2 O) and heavy metals (in forms of zinc chloride, copper sulphate, nickel sulfate, basic lead acetate, sodium chromate and cadmium chloride) were used as inhibitors added to the reaction solution described in the GST enzyme activity assay to compete with the substrate CDNB. The reaction solution included 1 mmol/L of CDNB and GSH in 100 mmol/L sodium phosphate buffer (pH 6.5). The control was the same reaction solution without the insecticides or metals. Binding assay of SlGSTE1 with xanthotoxin and chlorpyrifos by HPLC Activity of SlGSTE1 recombinant protein toward xanthotoxin and chlorpyrifos was determined by HPLC (highperformance liquid chromatography) at 297 nm and 289 nm wavelength, respectively. The total reaction volume was 1 mL containing the protein (10 μg for chlorpyrifos, 40 μg for xanthotoxin), 0.1 mol/L Tris/HCl buffer (pH 7.0), 50 μmol/L xanthotoxin or 5 μmol/L chlorpyrifos, 10 mmol/L GSH. The reaction cocktail was mixed gently for 3 h at room temperature, and then filtered by a 0.22 μm filter. Twenty microliters of the reaction liquid was subjected to HPLC. The mobile phase was methanol : water (65 : 35 for xanthotoxin, 80 : 20 for chlorpyrifos) and the flow rate was 1 mL/min. The retention time for xanthotoxin and chlorpyrifos was 8.3 and 19.8 min, respectively. SlGSTE1-specific activity is presented as nmol insecticide per mg protein per hour. Protein isolation and Western blot analysis Larval tissues of S. litura were homogenized in a homogenization buffer (5 mL/g tissue; 50 mmol/L Tris, 10 mmol/L EDTA, 15% glycerol, 0.005% phenylthiourea, pH 7.8) using a motor-driven Teflon pestle in a 1.5 mL polypropylene microcentrifuge tube. The homogenate was centrifuged at 10 000 g for 5 min and the supernatant was re-centrifuged under the same condition. The final supernatant was used for protein analysis. Protein concentration was estimated using the method of Bradford (1976). Absorbance was measured at 595 nm and the concentration was calculated according to a bovine serum albumin (BSA) standard curve. Protein samples were denatured at 100°C for 5 min in an equal volume of 2× protein loading buffer (0.1 mol/L Tris buffer, pH 6.8, 4% sodium dodecyl sulfate [SDS], 0.2% β-mercaptoethanol, 40% glycerol, and 0.002% bromophenol blue). SDS-PAGE (polyacrylamide gel electrophoresis) was performed in 12% acrylamide gels in  C 2014

Tris-glycine-SDS buffer (10 mmol/L Tris, 50 mmol/L glycine, 0.1% SDS, pH 8.0). The gel was stained with Coomassie Blue R-250. Molecular mass of the proteins was calculated using the Protein Molecular Weight Marker (low) (TaKaRa, Dalian, China). For Western blot analysis, proteins were transferred after electrophoresis from the acrylamide gel to nitrocellulose membrane. The membrane was blocked with 3% BSA in 1× phosphate-buffered saline buffer for 2 h at room temperature, and then incubated with the primary antibody, anti-SlGSTE1 antibody, (1 : 1 000) at room temperature for 1 h. Goat anti-rabbit IgG (Dingguo, Beijing, China) conjugated with alkaline phosphatase was used as the secondary antibody at a dilution of 1 : 1000. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used as substrates for color development. Statistical analysis of data All of the data are presented as mean ± standard deviation (SD) with n = 3. Significance analysis of the experimental data was performed using the analysis of variance (ANOVA) method followed by Duncan’s Multiple Comparison Test. Results Expression of recombinant SlGSTE1 protein and antibody production The ORF of Slgste1 (GenBank accession no.: AAS79891), which encodes a peptide of 218 amino acids with a molecular mass of 24 kDa, was inserted into the expression vector PROEX HTa to express recombinant protein in E. coli DH5α (Fig. 1A). Obtained recombinant SlGSTE1 was a soluble protein and had an apparent molecular mass of 26 kDa including the 6× His-tag. The recombinant protein was purified using His-Tag affinity column and the purified protein was used for antibody production and enzyme activity assay. The generated antibody recognized very well the recombinant SlGSTE1 protein expressed in DH5α cells (Fig. 1B), indicating that the SlGSTE1 antibody has a good immune specificity and can be used in later experiments. Kinetics analysis of SlGSTE1 recombinant protein Enzyme kinetics of the recombinant SlGSTE1 was determined by using double reciprocal plots. The results revealed that Vmax and Km values of the enzyme were Institute of Zoology, Chinese Academy of Sciences, 22, 503–511

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Fig. 1 Expression of recombinant SlGSTE1 in Escherichia coli DHα (A) and its antibody specificity analysis by Western blot (B). M: protein marker; CK: Control; Non-purified: total proteins in E. coli cells expressing the recombinant pPROEX HtaSlGSTE1; Purified: the His-tag affinity purified recombinant SlGSTE1 protein.

27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L, respectively, when the CDNB concentration was fixed to 3 mmol/L (Fig. 2). Vmax and Km values of the enzyme were 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L, respectively, when the concentration of GSH was fixed to 3 mmol/L. This result suggests that SlGSTE1 has a higher affinity to CDNB than to GSH and the difference in affinity between these two substrates is not as significant as the other two GSTs (SlGSTE2 and SlGSTE3) reported previously in S. litura (Deng et al., 2009). However, the binding affinity of SlGSTE1 (0.83 ± 0.106 mmol/L) to CDNB is higher than that of SlGSTE2 (0.91 ± 0.09 mmol/L) and SlGSTE3 (1.01 ± 0.1 mmol/L). The Vmax of SlGSTE1 was much slower than that of SlGSTE2 (113 ± 1.98 mmol/mg/min) and SlGSTE3 (111 ± 2.19 mmol/mg/min) (Deng et al., 2009). This implies that the catalysis activity of SlGSTE1 against the standard substrate CDNB is slightly different from that of SlGSTE2 and SlGSTE3. Activity of SlGSTE1 against different toxicants To analyze the insecticide substrate specificity of SlGSTE1, five insecticides of three kinds, including the organophosphorus insecticides (chlopyrifos, malathion and phoxim), the pyrethroid insecticide (deltamethrin) and the insecticide organochlorine (DDT), were tested. Each of the insecticides was used as a competitor to test its inhibition effect on the CDNB binding to SlGSTE1 in the assay. The results showed that all tested insecticides could significantly inhibit the activity of SlGSTE1 binding to CDNB to different degrees from 35% to 69.6% inhibi C 2014

Fig. 2 Enzymatic kinetics analysis of activity of SlGSTE1 with various concentrations of glutathione (GSH) (0.25−3.00 mmol/L) at a fixed concentration (3 mmol/L) of 1-chloro-2, 4dinitrobenzene (CDNB) (A) or CDNB (0.25−3.00 mmol/L) at a fixed concentration (3.00 mmol/L) of GSH (B). Changes in absorbance of CDNB conjugate for the first minute was measured at 340 nm and 25 °C using DU800 spectrometer.

tion (Fig. 3). The order of inhibition activity strength was DDT > malathion > phoxim > deltamethrin > chlopyrifos. These results indicate that SlGSTE1 can bind to insecticides of these different categories. To compare the enzyme activity of SlGSTE1 to synthetic insecticide and natural allelochemicals, two compounds, chlopyrifos (organophosphorus insecticide) and xanthotoxin (furan plant secondary substances) which enhanced Slgste1 expression (Huang et al., 2011), were selected for SlGSTE1 activity comparison by using HPLC. The specific activities of SlGSTE1 to xanthotoxin and chlopyrifos were 2.85 ± 0.08 nmol xanthotoxin/min/mg protein and 1.07 ± 4.16 nmol chlopyrifos/min/mg protein, respectively (Table 1). SlGSTE1 had about a threefold higher binding affinity to xanthotoxin than to chlopyrifos. Analysis of peroxidase activity of SlGSTE1 indicated that SlGSTE1 had a high peroxidase activity to the lipid peroxidative product, cumene hydrogen peroxide

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Fig. 3 Indirect inhibition of the binding activity of SlGSTE1 to 1-chloro-2,4-dinitrobenzene (CDNB) by various insecticides at 100 μmol/L. The reaction solution included 1 mmol/L of each CDNB and glutathione (GSH). The control (CK) was the same reaction solution without the insecticides. Table 1 The binding activity of SlGSTE1 to 4-hydroxynonenal, xanthotoxin and chlorpyrifos. Substrate

Enzyme specific activity

4-hydroxynonenal Xanthotoxin Chlorpyrifos

3.04 ± 0.38 (μmol/min/mg) 2.85 ± 0.08 (nmol/min/mg) 1.07 ± 4.16 (nmol/min/mg)

(3.04 ± 0.38 μmol 4-HNE/mg protein/min, Table 1), even much higher than to xanthotoxin and chlopyrifos. Heavy metals can cause toxic stress in insects. To understand the role of SlGSTE1 in detoxifying heavy metals in S. litura, six heavy metals (zinc, copper, nickel, lead, chromium and cadmium) were tested for their affinity with the enzyme in the in vitro replacement assay, where each heavy metal was used as an competitor of CDNB and GSH to bind with SlGSTE1. The result showed that the inhibition efficiency of these heavy metals was 17.3%, 17.7%, 33.4%, 45.7%, 60.7% and 68.8% for zinc, copper, nickel, lead, chromium and cadmium, respectively (Fig. 4). Cadmium was the most effective competitor for SlGSTE1. Induced expression of SlGSTE1 by chlorpyrifo and cadmium chloride To examine whether or not the expression of SlGSTE1 was induced by toxicants, S. litura larvae were fed with the organophosphorus insecticide chlopyrifos and heavy metal cadmium. After feeding with different doses of these two toxicants, the guts of the larvae were dissected and the protein level of SlGSTE1 was detected by using Western blot analysis. Chlopyrifos at 5 μg/g and 10 μg/g induced the expression of SlGSTE1 in the midgut, foregut  C 2014

Fig. 4 Indirect inhibition of the binding activity of SlGSTE1 to 1-chloro-2,4-dinitrobenzene (CDNB) by various heavy metal ions at 100 μmol/L. The reaction solution included 1 mmol/L of each CDNB and glutathione (GSH). The control was the same reaction solution without the metals.

Fig. 5 Analysis of SlGSTE1 protein expression in the gut of 1-day-old 6th instar larvae of Spodopera litura reared on the artificial diet containing different concentrations of chlorpyrifos for 5 days. Upper panel: SDS-PAGE analysis; lower panel: Western blot analysis. M: protein marker; 0: artificial diet without chlorpyrifos; 5 and 10: reared on the diet containing 5 and 10 mg/kg chlorpyrifos, respectively. MG: midgut; FG: foregut; HG: hindgut. The arrows show the band of SlGSTE1.

and hindgut, but no difference was found between these two concentrations (Fig. 5). Expression level of SlGSTE1 was also induced by cadmium in the midgut of S. litura and increased with feeding days (Fig. 6), implying that SlGSTE1 plays a role in binding the toxic heavy metal in the midgut. Discussion Insects can survive in complex and severe environments through their special immune and enzyme detoxification systems. The insect detoxification enzyme system includes the superfamilies of P450, GST and esterase. Finding out the key detoxification enzyme that is responsible for detoxifying a specific insecticide would Institute of Zoology, Chinese Academy of Sciences, 22, 503–511

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Fig. 6 Analysis of SlGSTE1 protein expression in 1-day-old 6th instar larvae of Spodopera litura reared on the artificial diet containing heavy metal cadmium (chromic chloride) at 15 mg/kg. (A): Time course analysis (days 1 to 7) of SlGSTE1 expression in the midgut of 1-day-old 6th instar larvae; (B): analysis of SlGSTE1 expression in the gut of 1-day-old 6th instar larvae after 7-day exposure to chromic chloride. MG: midgut; FG: foregut; HG: hindgut; Other: rest tissues. Upper panel: SDS-PAGE analysis; lower panel: Western blot analysis. The arrows show the band of SlGSTE1.

be helpful for developing efficient pest control strategies targeted at that enzyme and overcoming insecticide resistance. However, several factors make it difficult to identify which detoxifying enzyme is responsible for a specific toxic compound. First, each member of these superfamilies might respond to different toxicants, or work together to detoxify the toxicants, therefore, it is difficult to find out which gene or enzyme is mainly responsible for detoxifying a specific toxic compound. Second, generalists usually have a more complex detoxification enzyme system than specialists. For example, CYP6B8 and CYP321A1 from the generalist Helicoverpa zea metabolize a variety of linear and angular furanocoumarins and other allelochemicals, such as flavone, α-naphthoflavone, chlorogenic acid, indole-3-carbinol, quercetin and rutin (Li et al., 2004; Rupasinghe et al., 2007), while CYP6B1 from the specialist Papilio polyxenes was induced only by xanthotoxin (Cohen et al., 1992) and can metabolize several linear and angular furanocoumarins only when supplemented with additional P450 reductase (Wen et al., 2003). Third, there is a close relationship among an insect species, host plant that it feeds on and insecticide resistance. In many cases detoxification enzymes can respond to both toxic plant secondary metabolites and synthetic insecticides. Once the enzymes are induced in an insect by toxic secondary metabolites after it feeds on its host, the insect becomes less susceptible or more resistant to insecticides applied to it later on. In this study, function of SlGSTE1 in detoxification of synthetic insecticides, allelochemicals and heavy metals was investigated. S. litura is a generalist which can feed on more than 290 species of plants belonging to 99 families, causing considerable field and economic lose (Wu et al., 2004). In C 2014

secticide resistance has been developed in this species because of over-use of insecticides for a long time (Saleem, et al., 2008; Tong et al., 2013). Several lines of evidence indicate that SlGSTE1 plays an important role in detoxification of toxic compounds in S. litura. First, in our previous studies, Slgste1 was found to be induced by chlorpyrifos and xanthotoxin, whereas Slgste2 was not (Huang et al., 2011). Although both isoenzymes can bind to DDT and deltamethrin (Deng et al., 2009; Fig. 3), SlGSTE1 has higher activities than SlGSTE2 for DDT and deltamethrin. Second, out of a total 37 GSTs in S. litura, only eight GSTs are responsible to chlorpyrifos exposure and Slgste1 is one of them (Si-Chun Zheng, unpublished data). This protein is significantly increased after S. litura feeds on several host plants (Si-Chun Zheng, unpublished data). Third, in this study, it was further demonstrated that SlGSTE1 can bind to xanthotoxin and several insecticides, organophosphorus, pyrethroid and organochlorine (Fig. 3). These results together suggest that SlGSTE1 is a critical player in detoxifying toxic compounds in S. litura. In insects, detoxification usually takes place in the fat body, midgut and hemolymph (Enayati et al., 2005; Dubovskiy et al., 2011). In this study, it was found that SlGSTE1 only had a trace expression during the life cycle of S. litura when they were reared on artificial diet, but its expression was significantly increased when the larvae were fed with chlorpyrifos and the protein was expressed in the intestinal tract (Fig. 5), indicating that SlGSTE1 may function in the gut of S. litura. GSTs are phase II detoxification enzymes and can detoxify insecticides themselves and the oxidative products of the insecticides by P450, such as chlorpyrifos and its products chlorpyrifos oxon and TCP (3, 5,

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6-trichloro-2-pyridinol) (Fujioka & Casida, 2007), which usually are more toxic than the insecticides themselves. In addition, GSTs have peroxidase activity and they can defend against oxidative stress induced by insecticides (Jakobsson et al., 1997; Yamamoto et al., 2013). In this study, SlGSTE1 was found to have a high level of peroxidase activity toward the lipid peroxidative product, cumene hydrogen peroxide, indicating that SlGSTE1 may play its detoxification role not only by directly binding to the toxic compounds but also by its peroxidase activity against oxidative stress caused by the toxic compounds. Another finding of this study is that besides organic toxic compounds, SlGSTE1 might also play a role in detoxifying heavy metals. In some insect species, it was reported that heavy metals enhanced lipid peroxidation, leading to oxidative stress (Felton & Summers, 1995), and increased the antioxidative activity of the detoxifying enzymes such as GSTs in the haemolymph of Galleria mellonella larvae (Dubovskiy et al., 2011). Binding of SlGSTE1 with six heavy metals was tested in this study. The results indicated that SlGSTE1 had a higher binding activity to the toxic chromium and cadmium (60.7% and 68.8%, respectively) than to the essential elements that are necessary for insect growth, such as zinc and copper (17.3% and 17.7%, respectively). The expression of SlGSTE1 was increased in the midgut of larvae fed with cadmium (Fig. 6). Thus, SlGSTE1 probably detoxifies these toxic metals in vivo by directly binding with them. Acknowledgments This research was supported by National Natural Science Foundation of China (Grant No. 31071981) and National Natural Science Foundation of China (Grant No. 31272381). Disclosure The authors declare no conflicts of interest. References Ahmad, S. and Pardini, R.S. (1990) Mechanisms for regulating oxygen toxicity in phytophagous insects. Free Radical Biology and Medicine, 8, 401−413. Alin, P., Danielson, U.H. and Mannervik, B. (1985) 4-Hydroxy2-enals are substrates for glutathione transferase. FEBS Letters, 179, 267−270. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the  C 2014

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Accepted May 13, 2014

Institute of Zoology, Chinese Academy of Sciences, 22, 503–511