Haihua Wu

Chronic accumulation of cadmium and its effects on antioxidant enzymes and malondialdehyde in Oxya chinensis (Orthoptera: Acridoidea)

The accumulation of cadmium (Cd) and its effects on antioxidant enzyme activities and malondialdehyde (MDA) levels of Chinese rice grasshopper (Oxya chinensis) were evaluated under the laboratory conditions. Our results showed that Cd accumulation in O. chinensis exhibited a concentration-dependent increase in both males and females under Cd pollution. Environmental Cd can lead to the absorption of large quantities of Cd, which induces oxidative damage in insects by altering antioxidant defense enzyme systems. Our results demonstrated that Cd stress caused a significant decrease in glutathione peroxidase (GPx) levels and a significant increase in superoxide (SOD) dismutase and catalase (CAT) activities. In the grasshoppers, the MDA content was also enhanced, with an increase in Cd concentrations and a positive correlation between them; for females from second instar nymphs to the adult stage, R(2) was 0.6467, 0.9136, 0.6516, 0.942 and 0.7182, whereas for males, it was 0.6467, 0.8239, 0.9302, 0.7861, 0.8632, respectively. We also observed differences in the effects of Cd between grasshoppers of different developmental stages and genders, which suggested that the insects developmental stage and sex should be considered when studying enzyme activity.

Effects of malathion and chlorpyrifos on acetylcholinesterase and antioxidant defense system in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of malathion and chlorpyrifos on acetylcholinesterase (AChE), esterase (EST) activity and antioxidant system after topical application with different concentration to Oxya chinensis. The results showed that malathion and chlorpyrifos inhibited EST, AChE activity and increased malondialdehyde (MDA) contents. A change in superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) activity combined with reduced glutathione (GSH) and total glutathione (tGSH) contents was found in O. chinensis after malathion and chlorpyrifos treatments. Malathion and chlorpyrifos increased SOD and CAT activity compared with the control. With the concentrations increasing, SOD and CAT activity showed the similar tendency, namely, SOD and CAT activity increased at the lower concentrations and decreased at the higher concentrations. The results showed that malathion and chlorpyrifos decreased significantly GR activity. GST and GPx activity at the studied concentrations of chlorpyrifos was lower than that of the control. However, no significance was observed. GPx and GST activity in malathion treated grasshoppers showed a biphasic response with an initial increase followed by a decline in its activity. Malathion and chlorpyrifos decreased GSH contents and the ratio of GSH/GSSG. The present findings indicated that the toxicity of malathion and chlorpyrifos might be associated with oxidative stress.

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.

Acute toxicity and sublethal effects of fipronil on detoxification enzymes in juvenile zebrafish (Danio rerio)

The acute toxicity of fipronil and its sublethal effects on detoxification enzymes (carboxylesterases (CarEs), glutathione S-transferases (GSTs), and 7-ethoxycoumarin O-deethylase (ECOD)) in zebrafish (Danio rerio) were investigated. The results indicated that the 24-h LC50 of fipronil for zebrafish was 220.4 μg/L (95% CI: 173.7-272.4 μg/L). Sublethal concentrations of fipronil did not cause significant changes in CarEs activities. In the liver and muscle tissues, GST activities at the tested concentrations did not significantly differ from those in the control. In the brain and gill tissues, GST activities at a concentration of 4 μg/L were significantly lower than those at a concentration of 2 μg/L. The results suggest that CarEs and GSTs were not suitable biomarkers for fipronil effects in D. rerio. A significant induction in the ECOD activities in the brain, gill, liver, and muscle tissues was observed compared with the control. Moreover, the dose-dependent responses of the ECOD activity were observed after treatment with sublethal concentrations of fipronil in the range of 2-20 μg/L. The results suggested that ECOD could be a suitable biomarker of fipronil effects in D. rerio.

Two metallothionein genes in Oxya chinensis: molecular characteristics, expression patterns and roles in heavy metal stress.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification in living organisms. In the present study, we cloned two MT genes (OcMT1 and OcMT2) from Oxya chinensis, analyzed the expression patterns of the OcMT transcripts in different tissues and at varying developmental stages using real-time quantitative PCR (RT-qPCR), evaluated the functions of these two MTs using RNAi and recombinant proteins in an E. coli expression system. The full-length cDNAs of OcMT1 and OcMT2 encoded 40 and 64 amino acid residues, respectively. We found Cys-Cys, Cys-X-Cys and Cys-X-Y-Z-Cys motifs in OcMT1 and OcMT2. These motifs might serve as primary chelating sites, as in other organisms. These characteristics suggest that OcMT1 and OcMT2 may be involved in heavy metal detoxification by capturing the metals. Two OcMT were expressed at all developmental stages, and the highest levels were found in the eggs. Both transcripts were expressed in all eleven tissues examined, with the highest levels observed in the brain and optic lobes, followed by the fat body. The expression of OcMT2 was also relatively high in the ovaries. The functions of OcMT1 and OcMT2 were explored using RNA interference (RNAi) and different concentrations and treatment times for the three heavy metals. Our results indicated that mortality increased significantly from 8.5% to 16.7%, and this increase was both time- and dose-dependent. To evaluate the abilities of these two MT proteins to confer heavy metal tolerance to E. coli, the bacterial cells were transformed with pET-28a plasmids containing the OcMT genes. The optical densities of both the MT-expressing and control cells decreased with increasing concentrations of CdCl2. Nevertheless, the survival rates of the MT-overexpressing cells were higher than those of the controls. Our results suggest that these two genes play important roles in heavy metal detoxification in O. chinensis.

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.

Effect of Dietary Cadmium on the Activity of Glutathione S-Transferase and Carboxylesterase in Different Developmental Stages of the Oxya chinensis (Orthoptera: Acridoidea)

ABSTRACT Glutathione S-transferases (GSTs) and carboxylesterases (CarEs) play important roles in the detoxification of endogenous and exogenous compounds. In this study, the biochemical effects of dietary cadmium (Cd) on the activities of GST and CarE in different developmental stages of the rice grasshopper Oxya chinensis Thunberg were studied. The results showed that the effects of the Cd concentration and developmental stage on GST activity were statistically significant. GST activity in O. chinensis increased at the highest Cd concentration in most nymphs, suggesting that GST is typically inducible by Cd. However, GST activity was inhibited in adults under Cd stress owing to life-stagespecific physiological characteristics. The results showed that the substrates, developmental stage, and Cd concentration had statistically significant effects on CarE activity. In most studies of CarE activity, the interaction between any two studied factors was statistically significant, although the interaction effects of the substrates, developmental stages, and Cd concentrations were not significant, which implied that the insect physiological condition and the external environmental may affect CarE activity. The results suggest that the insects life stage and enzyme substrates should be considered when enzyme activity under Cd stress is studied.

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.

Antioxidant enzymes and their role in phoxim and carbaryl stress in Caenorhabditis elegans

Pesticide exposure can induce oxidative stress and cause changes to antioxidant enzymes in living organisms. In the present study, the effects of phoxim (an organophosphorus insecticide) and carbaryl (a carbamate insecticide) on antioxidant enzyme activity and gene expression were investigated in the model organism Caenorhabditis elegans. The results show that phoxim exposure can induce superoxide dismutase (SOD) and catalase (CAT) activities and decrease glutathione peroxidase (GPx) activity at lower concentrations. The expression levels of sod-3, sod-5, ctl-1, gpx-6, and gpx-8 were up-regulated after treatment with phoxim. The mRNA expression levels of sod-5, ctl-1 and gpx-6 were increased approximately 70-, 170- and 130-fold, respectively, in the 0.25mM treatment group compared to the control group. Carbaryl exposure decreased SOD activity and induced CAT and GPx activities. The addition of carbaryl up-regulated the expression of sod-5, ctl-1, ctl-3 and gpx-8. Specifically, ctl-1 expression increased approximately 10-fold, and gpx-8 expression increased <30-fold in the 0.5mM treatment group relative to the control group. The transcript level of sod-5 increased >20-fold, and ctl-3 increased approximately 10-fold in the 1mM treatment group. The functions of the antioxidant enzymes during oxidative stress caused by the two insecticides were investigated using deletion mutants. The LC50 values phoxim for the of sod-3 (tm760), sod-5 (tm1146), ctl-1 (ok1242), ctl-3 (ok2042) and gpx-8 (tm2108) mutant strains were lower than those observed for the N2 strain. The LC50 values of carbaryl for the ctl-1 (ok1242), ctl-3 (ok2042) and gpx-6 (tm2535) deletion mutant strains decreased in comparison to the N2 strain. The results suggest that these two insecticides caused oxidative stress and changed altered the antioxidant enzyme activities and their gene expressions in C. elegans. The sod-3, sod-5, ctl-1, ctl-3, gpx-6, and gpx-8 encoding enzymes may play roles in defending cells from oxidative stress caused by these two insecticides.