Xueyao Zhang

Characterization and functional analysis of four glutathione S-transferases from the migratory locust, Locusta migratoria.

Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni2+-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu2+ and Cd2+). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos.

LmCYP4G102: An oenocyte-specific cytochrome P450 gene required for cuticular waterproofing in the migratory locust, Locusta migratoria

Cytochrome P450 superfamily proteins play important roles in detoxification of xenobiotics and during physiological and developmental processes. To contribute to our understanding of this large gene family in insects, we have investigated the function of the cytochrome P450 gene LmCYP4G102 in the migratory locust Locusta migratoria. Suppression of LmCYP4G102 expression by RNA interference (RNAi) does not interfere with moulting but causes rapid loss of body weight - probably due to massive loss of water, and death soon after moulting. Accordingly, maintaining these animals at 90% relative humidity prevented lethality. Consistently, RNAi against LmCYP4G102 provoked a decrease in the content of cuticular alkanes, which as an important fraction of cuticular hydrocarbons have been shown to confer desiccation resistance. In addition, the cuticle of LmCYP4G102-knockdown locusts was fragile and easier deformable than in control animals. Presumably, this phenotype is due to decreased amounts of cuticular water that is reported to modulate cuticle mechanics. Interestingly, LmCYP4G102 was not expressed in the epidermis that produces the cuticle but in the sub-epdiermal hepatocyte-like oenocytes. Together, our results suggest that the oenocyte-specific LmCYP4G102 plays a critical role in the synthesis of cuticular hydrocarbons, which are important for cuticle waterproofing and mechanical stability in L. migratoria

RNA interference to reveal roles of β-N-acetylglucosaminidase gene during molting process in Locusta migratoria.

Abstract  β‐N‐acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a β‐N‐acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full‐length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233‐ and 589‐nucleotide non‐coding regions at the 5′‐ and 3′‐ends, respectively. Phylogenetic analysis grouped the cDNA‐deduced LmNAG1 protein with the enzymatically characterized β‐N‐acetylglucosaminidases in group I. Analyses of stage‐ and tissue‐dependent expression patterns of LmNAG1 were carried out by real‐time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double‐stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1‐injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.

Heterologous expression and characterization of a sigma glutathione S-transferase involved in carbaryl detoxification from oriental migratory locust, Locusta migratoria manilensis (Meyen)

Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and resistance to insecticides in insects. In the present study, a sigma-class GST gene (LmGSTs3) was identified from the locust, Locusta migratoria manilensis. Its full-length cDNA sequence is 828 bp containing an open reading frame (ORF) of 612 bp that encodes 204 amino acid residues. The predicted protein molecular mass and pI are 23.4 kDa and 7.62, respectively. Recombinant LmGSTs3 was heterologously expressed in Escherichia coli as a soluble fusion protein. Its optimal activity was observed at pH 8.0. Incubation for 30 min at temperatures below 40 °C scarcely affected activity. The LmGSTs3 at pH values between 4.0 and 11.0 retained more than 80% of its original activity. Ethacrynic acid and cibacron blue were very effective inhibitors of LmGSTs3 with I50-values 1.7 and 3.7 μM, respectively. In response to heavy metal (CuSO4, CdCl2) exposure there was a concentration-dependent and time-dependent decrease in activity. The nymph mortalities after carbaryl treatment increased 38.7% after LmGSTs3 were silenced. These results suggest that LmGSTs3 may be involved in carbaryl detoxification in L. migratoria manilensis.

Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria

A 1578-bp cDNA of a cytochrome P450 gene (CYP9AQ2) was sequenced from the migratory locust, Locusta migratoria. It contains an open reading frame (ORF) of 1557 bp that encodes 519 amino acid residues. As compared with other known insect cytochrome P450 enzymes, the overall structure of its deduced protein is highly conserved. The expression of CYP9AQ2 was relatively higher in nymphal stages than in egg and adult stages, and the highest expression was found in fourth-instar nymphs, which was 8.7-fold higher than that of eggs. High expression of CYP9AQ2 was observed in foregut, followed by hindgut, Malpighian tubules, brain and fat bodies, which were 75~142-fold higher than that in hemolymph. Low expression was found in midgut, gastric cecum and hemolymph. The expression of CYP9AQ2 was up-regulated by deltamethrin at the concentrations of 0.04, 0.08, and 0.12 µg/mL and the maximal up-regulation was 2.6-fold at LD10 (0.04 µg/mL). RNA interference-mediated silencing of CYP9AQ2 led to an increased mortality of 25.3% when the nymphs were exposed to deltamethrin, suggesting that CYP9AQ2 plays an important role in deltamethrin detoxification in L. migratoria. Computational docking studies suggested that hydroxylation of the phenoxybenzyl moiety might be one of the deltamethrin metabolic pathways by CYP9AQ2.

RNA interference of cytochrome P450 CYP6F subfamily genes affects susceptibility to different insecticides in Locusta migratoria

BACKGROUND Many insect cytochrome P450s (CYPs) play critical roles in detoxification of insecticides. The CYP6 family is unique to the class Insecta, and its biochemical function has essentially been associated with the metabolism of xenobiotics. In this study, we sequenced and characterised the full-length cDNAs of five CYP genes from Locusta migratoria, a highly destructive agricultural pest worldwide. RESULTS The five genes were predominantly expressed in brain, guts, fat bodies or Malpighian tubules. CYP6FE1, CYP6FF1 and CYP6FG1 were expressed at higher levels in fourth-instar nymphs than in other developmental stages. CYPFD2 is specifically expressed in adults, whereas CYP6FD1, CYP6FD2 and CYP6FE1 showed significantly lower expression in eggs than in other developmental stages. Deltamethrin suppressed CYP6FD1 expression in third-instar nymphs and upregulated the expression level of CYP6FD2, CYP6FF1 and CYP6FG1 at the dose of LD10 . Efficient RNA interference-mediated gene silencing was established for four of the five CYP genes. Silencing of CYP6FF1 increased the nymphal mortality from 23 to 50% in response to deltamethrin. Silencing of CYP6FD2 and CYP6FE1 increased the nymphal mortality from 32 to 72 and 66%, respectively, to carbaryl. CONCLUSION Three of the four CYP6F subfamily genes in L. migratoria were associated with the detoxification of deltamethrin or carbaryl. The role of CYPs in insecticide detoxification appears to be both gene and insecticide specific.

Molecular Basis for Antioxidant Enzymes in Mediating Copper Detoxification in the Nematode Caenorhabditis elegans

Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC50) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC50 values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC50 values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes.

Effects of chlorpyrifos on glutathione S-transferase in migratory locust, Locusta migratoria.

Chlorpyrifos is a typical organophosphate pesticide and is among the most widely used worldwide. The objective of the present investigation was to assess the effect of chlorpyrifos exposure on glutathione S-transferase in Locusta migratoria. In the present study, chlorpyrifos (0.1, 0.2, and 0.4mgg(-1) body weight) was topically applied in the abdomen of locusts. The GST activity, mRNA levels of ten L. migratoria GSTs and protein levels of four representative GSTs were detected. The results showed that chlorpyrifos treatment caused significant decrease of 1,2-dichloro-4-nitrobenzene (DCNB) and p-nitro-benzyl chloride (p-NBC) activities, whereas 1-chloro-2,4-dinitrobenzene (CDNB) activity was not altered in locusts. The mRNA levels of seven L. migratoria GSTs, including LmGSTs2, LmGSTs3, LmGSTs4, LmGSTs5, LmGSTs6, LmGSTt1, and LmGSTu1, were decreased after chlorpyrifos exposure. The protein levels of LmGSTs5, LmGSTt1 and LmGSTu1 were significantly decreased at higher doses of chlorpyrifos. However, chlorpyrifos elevated the mRNA and protein expression of LmGSTd1. It indicated that LmGSTd1 might contribute to the resistance of locust to organophosphate pesticides such as chlorpyrifos, whereas the decrease in other GSTs might be an economic compensation by the insect to differentially regulate the expression of enzymes involved in the detoxification of insecticides on the expense of those that are not.

Identification and characterization of two CYP9A genes associated with pyrethroid detoxification in Locusta migratoria

Cytochrome P450s (CYPs) constitute one of the largest gene super families and distribute widely in all living organisms. In this study, the full-length cDNA sequences of two LmCYP9A genes (LmCYP9AQ1 and LmCYP9A3) were cloned from Locusta migratoria. We analyzed the expression patterns of two LmCYP9A genes in various tissues and different developmental stages using real-time quantitative PCR. Then we evaluated the detoxification functions of the two LmCYP9A genes by testing mortalities with four kinds of pyrethroid treatment after RNA interference (RNAi), respectively. Combining with docking structure of two LmCYP9A genes, their detoxification properties were extensively analyzed. The full-length cDNAs of LmCYP9AQ1 and LmCYP9A3 putatively encoded 525 and 524 amino acid residues, respectively. Both LmCYP9A genes were expressed throughout the developmental stages. The expression of LmCYP9AQ1 in the brain was higher than that in other examined tissues, whereas the LmCYP9A3 was mainly expressed in the fat body. The mortalities of nymphs exposed to deltamethrin and permethrin increased from 27.7% to 77.7% and 27.7% to 58.3%, respectively, after dsLmCYP9A3 injection. While the mortalities of nymphs exposed to fluvalinate increased from 29.8% to 53.0% after LmCYP9AQ1 was silenced using RNA interference. Our results suggested that the two LmCYP9A genes may be involved in different pyrethroid insecticide detoxification in L. migratoria.

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.