Insect Glutathione S-transferases Research Articles

Characterization of three glutathione S-transferases potentially associated with adaptation of the wheat blossom midge Sitodiplosis mosellana to host plant defense.

Insect glutathione S-transferases (GSTs), a multifunctional protein family, play a crucial role in detoxification of plant defensive compounds. However, they have been rarely investigated in Sitodiplosis mosellana, a destructive pest of wheat worldwide. In this study, we characterized for the first time a delta (SmGSTd1) and two epsilon GST genes (SmGSTe1 and SmGSTe2) and analyzed their expression patterns and functions associated with adaptation to host plant defense in this species. Expression of these SmGST genes greatly increased in S. mosellana larvae feeding on resistant wheat varieties Kenong1006, Shanmai139 and Jinmai47 which contain higher tannin and ferulic acid, the major defensive compounds of wheat against this pest, compared with those feeding on susceptible varieties Xinong822, Xinong88 and Xiaoyan22. Their expression was also tissue-specific, most predominant in larval midgut. Recombinant SmGSTs expressed in Escherichia coli could catalyze the conjugation of 1-chloro-2,4-dinitrobenzene, with activity peak at pH around 7.0 and temperature between 30 and 40 °C. Notably, they could metabolize tannin and ferulic acid, with the strongest metabolic ability by SmGSTe2 against two compounds, followed by SmGSTd1 on tannin, and SmGSTe1 on ferulic acid. The results suggest that these SmGSTs are important in metabolizing wheat defensive chemicals during feeding, which may be related to host plant adaptation of S. mosellana. Our study has provided information for future investigation and development of strategies such as host-induced gene silencing of insect-detoxifying genes for managing pest adaptation. © 2023 Society of Chemical Industry.

Identification and Characterization of Glutathione S-transferase Genes in Spodoptera frugiperda (Lepidoptera: Noctuidae) under Insecticides Stress.

Insect glutathione S-transferases (GSTs) serve critical roles in insecticides and other forms of xenobiotic chemical detoxification. The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a major agricultural pest in several countries, especially Egypt. This is the first study to identify and characterize GST genes in S. frugiperda under insecticidal stress. The present work evaluated the toxicity of emamectin benzoate (EBZ) and chlorantraniliprole (CHP) against the third-instar larvae of S. frugiperda using the leaf disk method. The LC50 values of EBZ and CHP were 0.029 and 1.250 mg/L after 24 h of exposure. Moreover, we identified 31 GST genes, including 28 cytosolic and 3 microsomal SfGSTs from a transcriptome analysis and the genome data of S. frugiperda. Depending on the phylogenetic analysis, sfGSTs were divided into six classes (delta, epsilon, omega, sigma, theta, and microsomal). Furthermore, we investigated the mRNA levels of 28 GST genes using qRT-PCR under EBZ and CHP stress in the third-instar larvae of S. frugiperda. Interestingly, SfGSTe10 and SfGSTe13 stood out with the highest expression after the EBZ and CHP treatments. Finally, a molecular docking model was constructed between EBZ and CHP using the most upregulated genes (SfGSTe10 and SfGSTe13) and the least upregulated genes (SfGSTs1 and SfGSTe2) of S. frugiperda larvae. The molecular docking study showed EBZ and CHP have a high binding affinity with SfGSTe10, with docking energy values of -24.41 and -26.72 kcal/mol, respectively, and sfGSTe13, with docking energy values of -26.85 and -26.78 kcal/mol, respectively. Our findings are important for understanding the role of GSTs in S. frugiperda regarding detoxification processes for EBZ and CHP.

A sigma class glutathione S-transferase gene regulated by the CncC pathway is required for phytochemical tolerance in the red flour beetle, Tribolium castaneum

Insect glutathione S-transferases (GSTs) play a crucial role in the detoxification of exogenous compounds, especially insecticides and plant allelochemicals. A sigma class GST gene, TcGSTS7, mediates the response to eugenol in Tribolium castaneum. However, the mechanism underlying this effect remains largely unknown. In this study, TcGSTS7, which exhibits a structural motif and domain organization characteristic of GSTs, was cloned from the T. castaneum genome. Spatiotemporal expression analysis revealed that TcGSTS7 was most highly expressed at the late larva stage and was mainly expressed in the fat body and epidermis of larvae and adults, suggesting that TcGSTS7 may play a potential role in the protection against toxic xenobiotics in T. castaneum. Furthermore, the expression of TcGSTS7 was significantly induced after exposure to eugenol, while RNA interference (RNAi) targeting TcGSTS7 enhanced the sensitivity of the beetle to eugenol, indicating that TcGSTS7 is involved in the tolerance of T. castaneum to this insecticide. Interestingly, the depletion of TcCncC, which encodes a transcription factor of the CncC pathway that has been associated with the regulation of detoxification-related genes in insects, led to a reduction in the TcGSTS7 transcript level following exposure to eugenol, which suggests that TcGSTS7 acts downstream of the CncC pathway. Combined, these results indicated that TcGSTS7 participates in the tolerance of T. castaneum to phytochemicals in a CncC pathway-dependent manner. These findings have implications for the development of novel drugs for use in pest control.

Insecticide resistance associated overexpression of two sigma GST genes assists Nilaparvata lugens to remedy oxidative stress from feeding on resistant rice variety

Insect glutathione S-transferases (GSTs) participate in detoxifying insecticides and plant metabolites in two different ways, metabolizing toxic components and remedying oxidative stress. Here in Nilaparvata lugens, a major insect pest on rice, the roles of cytosolic GSTs in resistance to insecticides and to plant defences were evaluated. The over-expression in four resistant strains indicated that NlGSTs1 and NlGSTs2 were essential to resistances to four test insecticides and H2O2 through an antioxidation mechanism. RNAi verified the antioxidation function of NlGSTs1 and NlGSTs2 in the resistances as a common mechanism, regardless of the structural differences among insecticides and H2O2. NlGSTs1 and NlGSTs2 also provided protection for N. lugens against rice defense by the same mechanism, reducing H2O2 levels when N. lugens were fed on the resistant rice variety Mudogo. The antioxidation activity of recombinant NlGSTs1 and NlGSTs2 is higher than their direct detoxification, which supported the ability of these two GSTs to remedy oxidative stress. For oxidative stress remediation as a common mechanism of NlGSTs1 and NlGSTs2 in both insecticide resistance and host adaptability, the development of insecticide resistance might enhance the ability of insects to remedy oxidative stress from feeding on resistant rice variety and thus to lower the resistance level of rice variety to N. lugens. The results call for careful assessment on N. lugens control when both insecticides and resistant rice variety are applied.

A novel theta-class glutathione S-transferase gene in Rhopalosiphum padi (L.) (Hemiptera: Aphididae): Identification, recombinant protein expression and function analyses of RpGSTT1

Glutathione S-transferases (GSTs) play a major role in the detoxification of insecticides and the protection against oxidative damage caused by ROS (reactive oxygen species). The bird cherry-oat aphid, Rhopalosiphum padi (L.), is a major crop pest worldwide. Chemical insecticides are currently used to control aphids, but insecticide resistance is an evolutionary issue for aphid control. In this research, we identified and cloned the full-length cDNA of a theta-class GST gene (RpGSTT1) from R. padi, which contains 702 bp open reading frame encoding 233 amino acids. RpGSTT1 was clustered together with theta classe of insect GSTs in the phylogenetic tree. Effect of insecticide exposure on the GST enzyme activity was investigated in vivo. After affinity purification, the recombinant RpGSTT1 protein was able to catalyze the conjugation of 1-chloro-2, 4-dinitrobenzene (CDNB). A disc diffusion assay showed that the RpGSTT1 can increase resistance to cumene hydroperoxide-induced oxidative stress. In addition, the purified recombinant RpGSTT1 protected super-coiled DNA from oxidative damage. By quantitative PCR, The relative expression level of RpGSTT1 in the aphid was highly up-regulated after 12 h exposure to β-cypermethrin, isoprocarb, malathion and sulfoxaflor, respectively. To our knowledge, RpGSTT1 is the first theta class of GST gene reported in bird cherry-oat aphid. Our findings indicate that RpGSTT1 may be involved in insecticide detoxification in R. padi.

Functional and Structural Diversity of Insect Glutathione S-transferases in Xenobiotic Adaptation.

As a superfamily of multifunctional enzymes that is mainly associated with xenobiotic adaptation, glutathione S-transferases (GSTs) facilitate insects' survival under chemical stresses in their environment. GSTs confer xenobiotic adaptation through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. In this article, a comprehensive overview of current understanding on the versatile functions of insect GSTs in detoxifying chemical compounds is presented. The diverse structures of different classes of insect GSTs, specifically the spatial localization and composition of their amino acid residues constituted in their active sites are also summarized. Recent availability of whole genome sequences of numerous insect species, accompanied by RNA interference, X-ray crystallography, enzyme kinetics and site-directed mutagenesis techniques have significantly enhanced our understanding of functional and structural diversity of insect GSTs.

Amplification and Characterization of DDT Metabolizing Delta Class GST in Sand Fly, Phlebotomus argentipes (Diptera: Psychodidae) From Bihar, India.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

Characterization of a sigma class GST (GSTS6) required for cellular detoxification and embryogenesis in Tribolium castaneum

The sigma glutathione S-transferases (GSTSs) are a class of cytosolic glutathione S transferases (GSTs) that play important roles in antioxidant defense in insects, but the mechanisms by which GSTSs contribute to antioxidant activity remain unclear. Here, we isolated a GSTS (GSTS6) from Tribolium castaneum and explored its function. Homology and phylogenetic analysis revealed that TcGSTS6 shared high identity with other evolutionarily conserved GSTSs. The recombinant TcGSTS6 protein had strong activity toward cumene hydroperoxide and 4-hydroxynonenal but low activity toward the universal substrate 1-chloro-2,4-dinitrobenzene. Exposure to various types of oxidative stress, including heat, cold, UV and pathogenic microbes, significantly induced TcGSTs6 expression, which indicates that it is involved in antioxidant defense. Knockdown TcGSTs6 by using RNA interference (RNAi) caused reduced antioxidant capacity, which was accomplished by cooperating with other antioxidant genes. Moreover, treatment with various insecticides such as phoxim, lambda-cyhalothrin, dichlorvos and carbofuran revealed that TcGSTS6 plays an important role in insecticide detoxification. The RNAi results showed that TcGSTS6 is essential for embryogenesis in T. castaneum. Our study elucidates the mechanism by which a GSTS contributes to antioxidant activity and enhances our understanding of the functional diversity of GSTSs in insects.

The roles of GSTs in fipronil resistance in Nilaparvata lugens: Over-expression and expression induction

As one of the most important detoxification enzymes in insects, Glutathione S-transferases (GSTs) play key roles in insecticide resistance via direct metabolism and protection against oxidative stress induced by insecticide exposure. Insect GSTs are often considered as the phase II detoxification enzymes, they have potential function to metabolize fipronil as well as its fipronil's metabolites. In the fipronil-resistant Nilaparvata lugens strain G28, GSTs' inhibitor DEM (diethyl maleate) showed the optimal synergistic effects (5.73-fold), indicating the essential roles of GSTs in the resistance to fipronil in this insect species. Four GST genes, NlGSTs1, NlGSTs2, NlGSTe1 and NlGSTd1, were found over-expressed in G28 when compared to its relative susceptible counterpart strain S28. The roles of these four GSTs in fipronil resistance were confirmed via RNAi. The four GST genes were highly over-expressed in the midgut and/or fat body with detoxification action, which might provide more chances for insects to metabolize fipronil and its metabolites. Additionally, the higher induction levels in the GST gene expression by insecticides in the midgut and/or fat body compared to the whole insect also supported the significant roles of the four GSTs in the detoxification. Above all, the results provided evidences to understand the functions of GSTs in fipronil resistance in N. lugens, and gave a reference for other insects in fipronil resistance.

The determination of Plutella xylostella (L.) GSTs (PxGSTs) involved in the detoxification metabolism of Tolfenpyrad.

Insect glutathione S-transferases (GSTs) play a crucial role in insecticide detoxification. However, there remains a distinct lack of information regarding the role of GSTs in the detoxification of Tolfenpyrad (TFP) in insects. Real-time quantitative PCR showed significant upregulation of PxGSTs after exposure to TFP for 6 h. An in vitro inhibition assay showed that TFP could inhibit PxGSTδ, PxGSTε and PxGSTσ, and the most pronounced inhibitory effect was on PxGSTσ. Metabolism assays displayed that PxGSTσ was superior to other test PxGSTs in metabolizing TFP. The molecular docking of TFP and PxGSTσ revealed that the H-bond provided by the sidechains of Tyr107 and Tyr162 were key to the detoxification of TFP by PxGSTσ. Further tests using mutant PxGSTσ proteins at the sites of Tyr107 (PxGSTσY107A) and Tyr162 (PxGSTσY162A) corroborated that the individual replacement of Tyr107 and Tyr162 could greatly weaken the binding and metabolic abilities to TFP. Metabolic interactions between the Plutella xylostella (L.) GSTs (PxGSTs) and TFP were deciphered. This study illustrates the molecular metabolism mechanism of PxGSTσ towards TFP and provides theoretical underpinnings for the design and optimization of novel TFP-like insecticides. © 2020 Society of Chemical Industry.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B.dorsalis.

Identification and Functional Characterization of Two Sigma Glutathione S-Transferase Genes From Bird Cherry-Oat Aphid (Hemiptera: Aphididae).

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is an insect pest that persistently attacks wheat crops worldwide. Glutathione S-transferases (GSTs) are important detoxification enzymes that play roles in insecticide resistance. In this study, we identified two GST genes (RpGSTS1 and RpGSTS2) from R. padi. Phylogenetic analysis indicated that the genes are associated with the sigma class of insect GSTs. The RpGSTS1 and RpGSTS2 contain nine α-helices and five β-sheets connected by loops, and had 60 and 50% homology with the 3D structure of the Blattella germanica GST5. We tested the toxicity of chlorpyrifos, imidacloprid, isoprocarb, sulfoxaflor, and λ-cyhalothrin to R. padi, and found that the toxicity of five insecticides to the aphid varied. The detoxification activity of GSTs and the expression patterns of RpGSTS1 and RpGSTS2 after insecticide treatments were also analyzed. Compared to the control, the GST activity was increased by 23, 18.5, 13, and 11.5% in aphids treated by LC50 concentrations of chlorpyrifos, isoprocarb, imidacloprid, and sulfoxaflor, respectively. Exposure to different chemical insecticides showed different effects on the expression of RpGSTS1 and RpGSTS2. These results indicate that RpGSTS1 and RpGSTS2 have unique biochemical characteristics and may play roles in resistance to insecticides in R. padi.

Identification of Key Residues Associated with the Interaction between Plutella xylostella Sigma-Class Glutathione S-Transferase and the Inhibitor S-Hexyl Glutathione

Glutathione S-transferases (GSTs) are important detoxification enzymes involved in the development of metabolic resistance in Plutella xylostella. Uncovering the interactions between representative PxGSTs and the inhibitor S-hexyl glutathione (GTX), helps in the development of effective PxGST inhibitors for resistance management. As the PxGST most severely inhibited by GTX, PxGSTσ (sigma-class PxGST) adopts the canonical fold of insect GSTs. The formation of the PxGSTσ-GTX complex is mainly driven by H-bond and hydrophobic interactions derived from the side chains of favorable residues. Of the residues composing the active site of PxGSTσ, Lys43 and Arg99 are two hot spots, first reported in the binding of GSH derivatives to GSTs. Such differences indicate the metabolism discrimination of different insect GSTs. Unfavorable interactions between the PxGSTσ active site and GTX are depicted as well. The research guides the discovery and optimization of PxGSTσ inhibitors.

Identification, Expression, and Regulation of an Omega Class Glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) Under Insecticide Stress.

Glutathione S-transferases (GSTs) play an essential role in the detoxification of xenobiotic toxins in insects, including insecticides. However, few data are available for the bird cherry-oat aphid, Rhopalosiphum padi (L.). In this study, we cloned and sequenced the full-length cDNA of an omega GST gene (RpGSTO1) from R. padi, which contains 720 bp in length and encodes 239 amino acids. A phylogenetic analysis revealed that RpGSTO1 belongs to the omega class of insect GSTs. RpGSTO1 gene was highly expressed in transformed Escherichia coli and the protein was purified by affinity chromatography. The recombinant RpGSTO1 displayed reduced glutathione (GSH)-dependent conjugating activity toward the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) substrate. The recombinant RpGSTO1 protein exhibited optimal activity at pH 7.0 and 30°C. In addition, a disk diffusion assay showed that E. coli overexpressing RpGSTO1 increased resistance to cumene hydroperoxide-induced oxidative stress. Real-time quantitative PCR analysis showed that the relative expression level of RpGSTO1 was different in response to different insecticides, suggesting that the enzyme could contribute to insecticide metabolism in R. padi. These findings indicate that RpGSTO1 may play a crucial role in counteracting oxidative stress and detoxifying the insecticides. The results of our study contribute to a better understanding the mechanisms of insecticide detoxification and resistance in R. padi.

Glutathione S-transferase (GST) of American Cockroach, Periplaneta americana: Classes, Isoforms, and Allergenicity

Insect glutathione S-transferases (GSTs) play important roles in insecticide/drug resistance and stress response. Medically, GSTs of house dust mites (Dermatophagoides pteronyssinus and Blomia tropicalis) and German cockroach (Blattella germanica) are human allergens. In this study, classes, isoforms and B-cell and allergenic epitopes of GST of American cockroach, Periplaneta americana, the predominant species in the tropics and subtropics were investigated for the first time. Enzymatically active native and recombinant P. americana-GSTs bound to IgE in sera of all P. americana allergic patients that were tested. By gel-based proteomics and multiple sequence alignments, the native GST comprises three isoforms of delta and sigma classes. All isoforms interacted with serum IgE of the cockroach allergic subjects. Molecularly, the protein contains six B-cell epitopes; two epitopes located at β1-α1 and β4-α3 regions bound to patients’ serum IgE, indicating that they are allergenic. P. americana are ubiquitous and their GST can sensitize humans to allergic diseases; thus, the protein should be included in the allergen array for component resolved diagnosis (CRD) of allergic patients, either by skin prick test or specific IgE determination. The GST is suitable also as a target of environmental allergen detection and quantification for intervention of cockroach sensitization and allergic morbidity.

Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae

Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1–PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides.

Evolution of GSTD1 in Cactophilic Drosophila.

GSTD1 is an insect glutathione S-transferase that has received considerable attention because of its role in detoxification of xenobiotic compounds, specifically pesticides and plant allelochemicals involved in detoxification, or in the use of some substrates as a nutritional source. GSTD1 has been implicated in the adaptation to a new cactus host in Drosophila mojavensis and thus constitutes an interesting candidate to study ecological genetics of adaptation in Drosophila. We conducted population genetic and molecular evolution analyses of the GstD1 gene in the context of association with different cactus hosts (Opuntia sp. vs. Columnar) in nine Drosophila species from the repleta group. We observed strong evidence of selection in GstD1 from D. hexastigma. This species is associated with a diverse set of columnar cacti with very complex chemistries. GstD1 sequences from D. hexastigma show evidence of a recent selective sweep, and positive selection at one residue just outside of the active site of the enzyme. The substitution (Q116T) at the site under selection leads to a conformational change in the enzyme that could have important consequences for substrate binding efficiency. Our results suggest that GSTD1 from D. hexastigma may have evolved improved substrate binding in order to adapt to the diverse chemical environments that this species encounters in the wild.

A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana.

Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions.

Molecular cloning, characterization and positively selected sites of the glutathione S-transferase family from Locusta migratoria.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily.

Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura.

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±0.028mmol/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.