Delta Class Glutathione S-transferases Research Articles

Architecture and potential roles of a delta-class glutathione S-transferase in protecting honey bee from agrochemicals

The European honey bee, Apis mellifera, serves as the principle managed pollinator species globally. In recent decades, honey bee populations have been facing serious health threats from combined biotic and abiotic stressors, including diseases, limited nutrition, and agrochemical exposure. Understanding the molecular mechanisms underlying xenobiotic adaptation of A. mellifera is critical, considering its extensive exposure to phytochemicals and agrochemicals present in the environment. In this study, we conducted a comprehensive structural and functional characterization of AmGSTD1, a delta class glutathione S-transferase (GST), to unravel its roles in agrochemical detoxification and antioxidative stress responses. We determined the 3-dimensional (3D) structure of a honey bee GST using protein crystallography for the first time, providing new insights into its molecular structure. Our investigations revealed that AmGSTD1 metabolizes model substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenyl acetate (PNA), phenylethyl isothiocyanate (PEITC), propyl isothiocyanate (PITC), and the oxidation byproduct 4-hydroxynonenal (HNE). Moreover, we discovered that AmGSTD1 exhibits binding affinity with the fluorophore 8-Anilinonaphthalene-1-sulfonic acid (ANS), which can be inhibited with various herbicides, fungicides, insecticides, and their metabolites. These findings highlight the potential contribution of AmGSTD1 in safeguarding honey bee health against various agrochemicals, while also mitigating oxidative stress resulting from exposure to these substances.

Molecular and functional characterization of an antenna-enriched glutathione S-transferase BminGSTd3 involved in undecanol degradation in the citrus fruit fly, Bactrocera minax (Enderlein) (Diptera Tephritidae)

Bactrocera minax is a disastrous pest of citrus crops in China. Numerous studies focused on the molecular mechanism of odorant perception of B. minax, but the molecular mechanism of odorant degradation remains unclear. Glutathione S-transferases (GSTs) are considered as a class of odorant-degrading enzymes involved in degrading odorant molecules in insects' olfactory system. Here, we identified a delta-class GST gene, BminGSTd3, from B. minax. It was predominantly expressed in adult's olfactory organ antennae. The bacterially expressed recombinant BminGSTd3 was able to catalyze the conjugation of glutathione (GSH) with 2, 4-dinitrochlorobenzene (CDNB). Spectrophotometric analysis showed that undecanol can inhibit catalytic activities of BminGSTd3. Metabolic assays exhibited that undecanol can be depleted by BminGSTd3. Undecanol is believed to be an important B. minax sex pheromone component. The other components of the pheromone remain unclear. To understand how BminGSTd3 specifically recognizes undecanol, a 3D model of BminGSTd3 was constructed by homology modeling. Molecular docking based on this model revealed that E64 and S65 are the key amino acids recognizing undecanol, and this was proven by site-directed mutagenesis and intrinsic fluorescence assays. We suggest that BminGSTd3 is an undecanol metabolizing GST in B.minax, and E64 and S65 may serve as the key binding sites.

Functional characterization of SlGSTD3 and SlGSTD4 associated with phoxim and chlorpyrifos resistance in Spodoptera litura

Glutathione S-transferases (GSTs) is one of the main detoxification enzyme systems in insects and play important roles in insecticide resistance by direct metabolism, sequestration and antioxidant activity. Several GSTs genes in Spodoptera litura, a polyphagous agricultural pest, have been demonstrated to be overexpressed and involved in organophosphates and pyrethroids resistance. Previous studies have indicated the significant overexpression of two delta class GSTs genes (SlGSTd3 and SlGSTd4) in organophosphates and pyrethroids resistant populations. Here, they were heterologous expressed, and their metabolism activity and antioxidant activity were determined. Results indicated that the recombinant protein SlGSTD3 and SlGSTD4 both showed metabolism activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or beta cypermethrin. The metabolism activity of SlGSTD3 to phoxim and chlorpyrifos is higher than that of SlGSTD4. The recombinant vector of SlGSTD3 and SlGSTD4 both showed antioxidant activity after exposure to cumene hydroperoxide. Further modeling and docking analysis indicated that the 3D structure of SlGSTD3 and SlGSTD4 were well shaped for phoxim and chlorpyrifos, and the binding affinity for phoxim was stronger than that of chlorpyrifos. Our work provides evidence that SlGSTd3 and SlGSTd4 both play roles in phoxim and chlorpyrifos resistance in S. litura.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.

Molecular identification and biochemical characteristics of a delta class glutathione S-transferase gene (FcδGST) from Chinese shrimp Fenneropenaeus chinensis

Molecular identification and biochemical characteristics of a delta class glutathione S-transferase gene (FcδGST) from Chinese shrimp Fenneropenaeus chinensis

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at −440 bp to +54 bp, and that of PcGSTD2 promoter was between −1609∼−1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.

Glutathione S-transferase gene diversity and their regulation by Nrf2 in Chinese mitten crab (Eriocheir sinensis) during nitrite stress

Eriocheir sinensis is one of the most important economic aquatic products in China. However, nitrite pollution has become a serious threat to the healthy culture of E. sinensis. Glutathione S-transferase (GST) is an important phase II detoxification enzyme, which plays a leading role in the cellular detoxification of exogenous substances. In this study, we obtained 15 GST genes (designated as EsGST1-15) from E. sinensis, and their expression and regulation in E. sinensis under nitrite stress were studied. EsGST1-15 belonged to different GST subclasses. EsGST1, EsGST2, EsGST3, EsGST4, and EsGST5 belonged to Delta-class GSTs; EsGST6 and EsGST7 are Theta-class GSTs; EsGST8 is a mGST-3-class GST; EsGST9 belonged to mGST-1-class GSTs; EsGST10 and EsGST11 belonged to Sigma-class GSTs; EsGST12, EsGST13, and EsGST14 are Mu-class GSTs; EsGST15 is a Kappa-class GST. Tissue distribution experiments showed that EsGSTs were widely distributed in all detected tissues. The expression level of EsGST1-15 was significantly increased in the hepatopancreas under nitrite stress, indicating that EsGSTs were involved in the detoxification of E. sinensis under nitrite stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcription factor that can activate the expression of detoxification enzyme. We detected the expression of EsGST1-15 after interfering with EsNrf2 in the hepatopancreas of E. sinensis with or without nitrite stress. Results showed that EsGST1-15 were all regulated by EsNrf2 with or without nitrite stress. Our study provides new information about the diversity, expression, and regulation of GSTs in E. sinensis under nitrite stress.

Analysis of glutathione-S-transferases from larvae of Galleria mellonella (Lepidoptera, Pyralidae) with potential alkaloid detoxification function.

The greater wax moth, Galleria mellonella, is a global pest for beehives, doing damage in the larval stage. Although a significant number of studies have reported on larvae and adults, to date no effective pest control has been implemented. In this study, we tested larval resistance to alkaloids from Berberis microphylla, and the objective was to identify enzymes that participate in alkaloid detoxification through enzymatic assays, bioinformatics analysis and qRT-PCR. Findings suggest glutathione-S-transferases (GSTs), from an increased metabolic mechanism, are responsible for alkaloid detoxification rather than cytochrome P450 (CYP), carboxylesterases (CarE). A bioinformatics analysis from transcriptome data revealed 22 GSTs present in both G. mellonella larvae and adults. The qRT-PCR experiments corroborated the presence of the 22 GSTs in larvae, where GST8 and GST20 stood out with the highest expression after berberine treatment. Structural information around GST8 and GST20 suggests that GST8 could bind berberine stronger than GST20. These findings represent an important advance in the study of detoxification enzymes in G. mellonella, expanding the role of delta-class GSTs towards alkaloids. Likewise, GST inhibition by alkaloid analogs is proposed in the framework of integrated pest management strategies.

A near-chromosome level genome assembly of the European hoverfly, Sphaerophoria rueppellii (Diptera: Syrphidae), provides comparative insights into insecticide resistance-related gene family evolution

BackgroundSphaerophoria rueppellii, a European species of hoverfly, is a highly effective beneficial predator of hemipteran crop pests including aphids, thrips and coleopteran/lepidopteran larvae in integrated pest management (IPM) programmes. It is also a key pollinator of a wide variety of important agricultural crops. No genomic information is currently available for S. rueppellii. Without genomic information for such beneficial predator species, we are unable to perform comparative analyses of insecticide target-sites and genes encoding metabolic enzymes potentially responsible for insecticide resistance, between crop pests and their predators. These metabolic mechanisms include several gene families - cytochrome P450 monooxygenases (P450s), ATP binding cassette transporters (ABCs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs) and carboxyl/choline esterases (CCEs).Methods and findingsIn this study, a high-quality near-chromosome level de novo genome assembly (as well as a mitochondrial genome assembly) for S. rueppellii has been generated using a hybrid approach with PacBio long-read and Illumina short-read data, followed by super scaffolding using Hi-C data. The final assembly achieved a scaffold N50 of 87Mb, a total genome size of 537.6Mb and a level of completeness of 96% using a set of 1,658 core insect genes present as full-length genes. The assembly was annotated with 14,249 protein-coding genes. Comparative analysis revealed gene expansions of CYP6Zx P450s, epsilon-class GSTs, dietary CCEs and multiple UGT families (UGT37/302/308/430/431). Conversely, ABCs, delta-class GSTs and non-CYP6Zx P450s showed limited expansion. Differences were seen in the distributions of resistance-associated gene families across subfamilies between S. rueppellii and some hemipteran crop pests. Additionally, S. rueppellii had larger numbers of detoxification genes than other pollinator species.Conclusion and significanceThis assembly is the first published genome for a predatory member of the Syrphidae family and will serve as a useful resource for further research into selectivity and potential tolerance of insecticides by beneficial predators. Furthermore, the expansion of some gene families often linked to insecticide resistance and selectivity may be an indicator of the capacity of this predator to detoxify IPM selective insecticides. These findings could be exploited by targeted insecticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably and effectively controlling pests without impacting beneficial predator populations.

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.

Delta class glutathione S-transferase (TuGSTd01) from the two-spotted spider mite Tetranychus urticae is inhibited by abamectin

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 μM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

Target-Site Mutations and Glutathione S-Transferases Are Associated with Acequinocyl and Pyridaben Resistance in the Two-Spotted Spider Mite Tetranychus urticae (Acari: Tetranychidae).

Simple SummaryThe two-spotted spider mite Tetranychus urticae is a difficult-to-control pest due to its short life cycle and rapid resistance development. In this study, we characterized field strains collected in 2001 and 2003 that have been selected for acequinocyl resistance and pyridaben resistance, respectively. These strains displayed resistance ratios of 1798.6 and 5555.6, respectively, and were screened for cross-resistance against several currently used acaricides. The acequinocyl resistant strain exhibited pyridaben cross-resistance, but the pyridaben resistant strain showed no cross-resistance. The acequinocyl resistant strain exhibited point mutations in cytb (I256V and N321S) and PSST (H92R). In contrast, the pyridaben resistant strain exhibited the H92R but not the I256V and N321S point mutations. In addition, the increased GST metabolism and GST delta expression might be related to acequinocyl resistance in Tetranychus urticae. We hope that the data and patterns described here can now be exploited in the continued quest for rational resistance management strategies.The two-spotted spider mite Tetranychus urticae is a difficult-to-control pest due to its short life cycle and rapid resistance development. In this study, we characterized field strains collected in 2001 and 2003 that were selected for acequinocyl resistance (AR) and pyridaben resistance (PR), respectively. These strains displayed resistance ratios of 1798.6 (susceptible vs. AR) and 5555.6 (susceptible vs. PR), respectively, and were screened for cross-resistance against several currently used acaricides. The AR strain exhibited pyridaben cross-resistance, but the PR strain showed no cross-resistance. The AR strain exhibited point mutations in cytb (I256V, N321S) and PSST (H92R). In contrast, the PR strain exhibited the H92R but not the I256V and N321S point mutations. In some cases increased glutathione S-transferase (GST) activity has previously been linked to enhanced detoxification. The AR strain exhibited approximately 2.3-, 1.8-, and 2.2-fold increased GST activity against 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrobenzyl chloride (NBC), respectively. Among the five GST subclass genes (delta, omega, mu, zeta, and kappa), the relative expression of delta class GSTs in the AR strain were significantly higher than the PR and susceptible strain. These results suggest that the I256V and N321S mutations and the increased GST metabolism and GST delta overexpression might be related to acequinocyl resistance in T. urticae.

Functional diversification of three delta-class glutathione S-transferases involved in development and detoxification in Tribolium castaneum.

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme superfamily. Forty-one GSTs have been identified in Tribolium castaneum; however, none of the 41 GSTs has been functionally characterized. Here, three delta-class GSTs, TcGSTd1, TcGSTd2 and TcGSTd3, of T. castaneum were successfully cloned and expressed in Escherichia coli. All of the studied GSTs catalysed the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene. Insecticide treatment showed that the expression levels of TcGSTd3 and TcGSTd2 were significantly increased after exposure to phoxim and lambda-cyhalothrin, whereas TcGSTd1 was slightly upregulated only in response to phoxim. A disc diffusion assay showed that overexpression of TcGSTD3, but not TcGSTD1 or TcGSTD2, in E. coli increased resistance to paraquat-induced oxidative stress. RNA interference knockdown of TcGSTd1 caused metamorphosis deficiencies and reduced fecundity by regulating insulin/target-of-rapamycin signalling pathway-mediated ecdysteroid biosynthesis, and knockdown of TcGSTd3 led to reduced fertility and a decreased hatch rate of the offspring, probably caused by the reduced antioxidative activity in the reproductive organs. These results indicate that TcGSTd3 and TcGSTd2 may play vital roles in cellular detoxification, whereas TcGSTd1 may play essential roles in normal development of T. castaneum. These delta-class GSTs in T. castaneum have obtained different functions during the evolution.

Combined transcriptomic and proteomic analysis of harmine on Spodoptera frugiperda Sf9 cells to reveal the potential resistance mechanism

The fall armyworm, Spodoptera frugiperda, is an important invasive pest and exhibits resistance to many insecticides. Harmine, a remarkable, natural β-carboline alkaloid, exhibits a variety of bioactivities and induces programmed cell death in Sf9 cells. In the present study, transcriptomic and proteomic analyses were combined to investigate the effects of harmine on Sf9 cells. A sublethal dose, 0.05 mM, was selected and the transcriptomic analysis revealed 2463 upregulated and 689 downregulated genes after harmine treatment. The most frequently enriched pathways of differentially expressed genes (DEGs) were mainly involved in drug and xenobiotic metabolism. The proteomics analysis revealed 36 upregulated and 77 downregulated proteins, and the results showed a nonlinear relationship with mRNA expression. All the genes related to detoxification and resistance in the transcriptome and DEGs were identified and annotated. Complete open reading frames of 27 cytochrome P450s (CYPs), 27 glutathione S-transferases (GSTs), 11 carboxylesterases (CarEs), 10 UDP-glucuronosyltransferases (UGTs) and 29 heat shock proteins (HSPs) were assembled and verified using qRT-PCR. Harmine exhibited a completely different detoxification mechanism from normal pesticides. The Sigma and Delta class GSTs and UGTs might play important roles, rather than CYP6 and CYP9 clans, CarEs or HSPs. Biological significanceHarmine, a natural β-carboline alkaloid, inhibits the cell proliferation and induces programmed cell death of Sf9 cells, which derived from Spodoptera frugiperda, an important world invasive pest. In the article, the combined transcriptomic and proteomic analysis is used to explore the potential solution for its resistance management. These results supporting that harmine can be applied as a novel adjuvant or pesticide. In addition, the systematically identified resistance-related genes in fall armyworm provide the foundation for potential resistance monitoring and management.

Identification and Functional Analysis of a Delta Class Glutathione S-Transferase Gene Associated with Insecticide Detoxification in Bradysia odoriphaga.

A delta class glutathione S-transferase gene (BoGSTd2) is identified from Bradysia odoriphaga for the first time. Developmental expression analysis showed that expression of BoGSTd2 is significantly higher in the fourth instar larval stage and the adult stage. Tissue-specific expression analysis found that BoGSTd2 was expressed predominantly in the midgut and Malpighian tubules in the fourth instar larvae and the abdomen of adults. Expression of BoGSTd2 was significantly upregulated following exposure to chlorpyrifos and clothianidin. In vitro inhibition and metabolic assays indicated that recombinant BoGSTd2 could not directly metabolize chlorpyrifos and clothianidin. Nevertheless, disk diffusion assays indicated that BoGSTd2 plays an important role in protection against oxidative stress. RNAi assays showed that BoGSTd2 participates in the elimination of reactive oxygen species induced by chlorpyrifos and clothianidin. These results strongly suggest that BoGSTd2 plays an important role in chlorpyrifos and clothianidin detoxification in B. odoriphaga by protecting tissues from oxidative stress induced by these insecticides.

Two delta class glutathione S-transferases involved in the detoxification of malathion in Bactrocera dorsalis (Hendel).

The oriental fruit fly Bactrocera dorsalis (Hendel), a widespread agricultural pest, has evolved resistance to many insecticides, including organophosphorus compounds. Glutathione S-transferases (GSTs) are involved in xenobiotic detoxification and insecticide resistance in many insects. However, the role of delta class GSTs in detoxifying malathion in B. dorsalis is unknown. Here, we evaluated the roles of two delta class GSTs in malathion detoxification in this species. Two delta class GSTs genes, BdGSTd1 and BdGSTd10, were characterized in B. dorsalis. They were highly expressed in 5-day-old adults, as well as in midgut and Malpighian tubules. Upon malathion exposure, the two genes were upregulated by 2.63- and 2.85-fold, respectively. Injection of double-stranded RNA targeting BdGSTd1 or BdGSTd10 significantly reduced their mRNA levels in adults and also significantly increased adult susceptibility to malathion. The expression of these two GSTs in Escherichia coli helped the host to endure malathion stress at a concentration of 10µg mL-1 according to a Cell Counting Kit-8 assay. High-performance liquid chromatography analyses indicated that malathion could be significantly depleted by the two delta GSTs. The role of BdGSTd10 in malathion sequestration was also discussed. BdGSTd1 and BdGSTd10 play important roles in the detoxification of malathion in B. dorsalis. © 2019 Society of Chemical Industry.

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

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 black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). The deduced protein sequence of AiGSTd contained highly conserved features of GST enzymes and shared high identities with its orthologs from other lepidopteran species. Recombinant AiGSTD protein was expressed in Escherichia coli and purified. The protein displayed the GSH-dependent conjugating activity toward the substrate 1-chloro-2,4-dinitrobenzene (CDNB). Moreover, 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 transcripts were most abundant in the larval midgut. Exposure to chlorpyrifos and lambda-cyhalothrin increased lipid peroxidation in larvae and significantly upregulated AiGSTd expression levels. This study is the first report of molecular characterization of a GST in A. ipsilon, and the present study suggest that AiGSTD might be involved in protecting against the oxidative stress induced by insecticides.

Identification and expression profiles of fifteen delta-class glutathione S-transferase genes from a stored-product pest, Liposcelis entomophila (Enderlein) (Psocoptera: Liposcelididae).

Glutathione S-transferases (GSTs) comprise a diverse family of enzymes found ubiquitously in aerobic organisms and they play important roles in insecticide resistance. In this study, we tested the sensitivities of Liposcelis entomophila, collected from four different field populations, to three insecticides. The results showed that the insects from Tongliang population had a relatively higher tolerance to malathion and propuxor than insects from other field populations. The insecticide sensitivities of different populations detected in psocids may be due to the different control practices. Through sequence mining and phylogenetic analyses, we identified 15 delta class GST genes that contained the conserved motifs of the GSTs. Quantitative real-time PCR (Q-PCR) analysis indicated that the 15 GST genes were expressed at all tested developmental stages, and 12 GST genes had significantly higher expression levels in adulthood than in egg stage. The expression levels of 15 GST genes in different field populations showed that 9 GST genes were significantly higher in Tongliang population compared to other populations. Furthermore, Q-PCR confirmed that the expression of several delta class GSTs was upregulated at different times after malathion, propuxor and deltamethrine exposure with the LC50 concentration of insecticide. Taken together, these findings showed that delta class GST genes have various expression levels in different developmental stages and different field populations, and they were up-regulated in response to insecticide exposure, which suggested that these GSTs may be associated with insecticide metabolism in psocids.

Comparative RNA-sequencing profiling reveals novel Delta-class glutathione S-transferases relative genes expression patterns in Tribolium castaneum

Glutathione S-transferases (GSTs) are a large group of enzymes having both detoxification roles conferring insecticide resistance and specialist metabolic functions. Tribolium castaneum GST Delta 1 (TcGSTd1) has been found playing crucial role in insecticide resistance and biological processes in insect species. However, the regulatory system of TcGSTd1 has still rarely been known. Comparing the transcriptome profile of RNAi treated larvae (ds-TcGSTd1) and control larvae of T. canstaneum by using RNA-sequencing, we obtained 14,284,085 sequence reads aligned with 13,275 genes. And 512 differentially expressed genes (DEGs) were identified from ds-TcGSTd1 treated group. Est/CCE, CYP, MRPs were significantly down-regulated in ds-TcGSTd1 group when compared with control group, which illustrated that they cooperated with TcGSTd1 to reduce the activity of cellular metabolism system. While, SNO was up-regulated in ds-TcGSTd1 insects suggested it may also involve in detoxifying alkaloid of insect metabolism system. These results established that TcGSTd1 not only acts as a vital gene for phase II cellular detoxification but also participates in phase 0, I, and III cellular detoxification by cooperating with CSPs, OBPs, CYP9, ESTB1, CCE6, MRPs and other detoxification genes. Knockdown of TcGSTd1 also suppressed several genes encoding antioxidant enzymes, e.g. CuZnSOD, Duox, Prx, HPX, CPO, and MCORP. Suggested that they may modulate the function of TcGSTd1 on lifespan, immune, development and reproduction. All these results shed the new insights into the regulatory mechanism of TcGSTd1 involved in insect physiology and could further facilitate the research of suitable and sustainable managements for the pest control.