Wolbachia-mediated regulation of EscrGST1 modulates pesticide resistance in Eucryptorrhynchus scrobiculatus.

BackgroundPermethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.Methodology/Principal FindingsTo determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.ConclusionsThese findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

The involvement of cytochrome b5 in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b5. Anti-b5 antiserum inhibited the reduction of cytochrome b5 by NADH-cytochrome b5 reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b5 is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b5. The results suggest that cytochrome b5 involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved.

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.

Recently, there are many studies suggesting antibacterial, antifungal, and anthelmintic agents as alternative chemicals to insecticides. In this study, the oxidative and genotoxic effect of Piperazine, a clinically important hexahydropyrazine anthelmintic, on Galleria mellonella L. hemolymph tissue by adding artificial diet were investigated. Galleria mellonella larvae were reared until 7th larval stage in artificial diet containing 0.001, 0.01, 0.1, and 1 g piperazine per 100 g of diet. Using hemolymph collected from 7th-instar larvae, the amount of lipid peroxidation final product malondialdehyde (MDA), protein oxidation product protein carbonyl (PCO), and detoxification enzymes glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cyt P450) activity, comet assay were measured. According to the results obtained, when the piperazine high concentrations tested with the control group were compared, statistically significant differences were found in MDA, PCO content, cyt P450, GST activity, and comet assay in the hemolymph of the insect. While MDA content was 0.01 ± 0.0021 nmol/mg protein in the control group, this amount increased approximately 2-fold at the highest concentration (0.0231 ± 0.0050 nmol/mg protein). On the other hand, when the control group and the highest piperazine concentration were compared in the GST and cyt P450 activity, it was determined that there was a statistically significant increase. We obtained similar results in comet assay and micronucleus formation data. This study showed that the tested piperazine concentrations caused significant changes in the detoxification capacity, oxidative stress, and genotoxic markers in the insect's hemolymph tissue.

The diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae), is one of the most important pests of cruciferous crops in Iran and is controlled mostly by fenvalerate. The susceptibility to fenvalerate was investigated for four populations of P. xylostella. Bioassay results indicated significant differences among the populations tested. The highest level of resistance to fenvalerate was obtained for the Khuzestan (Khz) population (resistance ratio = 9.5). Survival was suppressed by diethyl maleate, piperonylbutoxide, and triphenyl phosphate, which confirmed that resistance to fenvalerate is caused by glutathione S-transferases (GSTs), mixed function oxidases, and esterases, respectively. Up to 8.6-, 2-, 2.7-, and 1.75-fold increases in GSTs, esterase (α-naphthylacerate and β-naphthyl acetate as substrate), and cytochrome P450 monooxygenases activities in resistant strains when compared with the susceptible one, were observed, respectively. The expression of six GST genes of P. xylostella including GSTs1, GSTe2, GSTe4, GSTo4, GSTd4, and GSTd5 were analyzed. The quantitative PCR analysis showed that three of the PxGSTs had the highest expression levels in the Khz population. Two of the GSTs (GSTd4 and PxGSTe2) exhibited highest expression level in both Khz and Alborz (Alb) populations. Therefore, PxGST genes were involved in fenvalerate resistance in P. xylostella. Overall, the mechanisms of insecticide resistance in diamondback moth populations in four regions of Iran were related to GST, esterase, and cytochrome P450 monooxygenase activities.

Susceptibility to two insecticides (profenophos and cypermethrin) as influenced by host plants viz., cauliflower, soybean and castor in comparison to artificial diet and changes in detoxification enzymes in tobacco caterpillar, Spodoptera litura were studied under laboratory. In comparison to larvae reared on artificial diet, those reared on cauliflower were tolerant to profenophos and cypermethrin by, 1.41 and 6.6 respectively, while the larvae reared on soybean showed 1.32 and 2.00-fold more tolerance to these insecticides. However, the larvae reared on castor were found to be more susceptible to profenophos than those reared on artificial diet. The host plants had a significant influence on levels of detoxification enzymes in S. litura. Elevated levels of carboxylesterase (20.72-fold) and cytochrome P450 mono-oxygenase (3.10-fold) were observed in larvae reared on cauliflower as compared to diet fed larvae. Activity of glutathione S-transferase (1.56-fold) was higher in larvae reared on soybean. Enhanced activity of detoxification enzymes in larvae of S. litura reared on different host plants could be correlated with insecticide susceptibility. Positive and significant (p < 0.05) correlation was observed between the detoxification enzyme (CarE and GST) activity in different host fed larvae and LD50 of profenophos (r = 0.77; r = 0.93). Activity of cytochrome P450 mono-oxygenase also had positive correlation with LD50 of cypermethin (r = 0.98). The present studies have shown that differential susceptibility of S. litura population could be correlated with the diet/host plants and levels of detoxifications enzymes like carboxylesterases, glutathione S-transferase and cytochrome P450 mono-oxygenase.

We investigated the effects of host species and resource (carbon dioxide, nitrate) availability on activity of detoxication enzymes in the gypsy moth,Lymantria dispar. Larvae were fed foliage from quaking aspen or sugar maple grown under ambient or elevated atmospheric CO2, with low or high soil NO 3 (-) availability. Enzyme solutions were prepared from larval midguts and assayed for activity of cytochrome P-450 monooxygenase, esterase, glutathione transferase, and carbonyl reductase enzymes. Activity of each enzyme system was influenced by larval host species, CO2 or NO 3 (-) availability, or an interaction of factors. Activity of all but glutathione transferases was highest in larvae reared on aspen. Elevated atmospheric CO2 promoted all but transferase activity in larvae reared on aspen, but had little if any impact on enzyme activities of larvae reared on maple. High NO 3 (-) availability enhanced activity of most enzyme systems in gypsy moths fed high CO2 foliage, but the effect was less consistent for insects fed ambient CO2 foliage. This research shows that gypsy moths respond biochemically not only to interspecific differences in host chemistry, but also to resource-mediated, intraspecific changes in host chemistry. Such responses are likely to be important for the dynamics of plantinsect interactions as they occur now and as they will be altered by global atmospheric changes in the future.

Juvenile hormone (JH) analog insecticides are relatively nontoxic to vertebrates and provide efficient control of key arthropod pests. One JH analog, pyriproxyfen, has provided over a decade of exceptional management of whiteflies in cotton of the southwestern United States. Thwarting resistance to pyriproxyfen in Bemisia tabaci (Gannadius) (a.k.a. Bemisia argentifolii Bellows and Perring) has been the focus of an integrated resistance management program because this insecticide was first registered for use in Arizona cotton in 1996. Resistance levels have increased slowly in field populations in recent years but have not demonstrably affected field performance of pyriproxyfen. Resistant strains have been isolated and studied in the laboratory to determine the mechanism of resistance and identify optimal strategies for controlling resistant whiteflies. Synergism bioassays showed that resistance in a laboratory-selected strain QC02-R, was partially suppressible with piperonyl butoxid (PBO) and diethyl maleate (DEM) but not with S, S, S-tributyl phosphorotrithioate (DEF). Consistent with the synergism bioassay results, enzymatic assays revealed that the enzyme activities of cytochrome P450 monooxygenases (P450) and glutathione S-transferases (GST) but not esterases were significantly higher in the pyriproxyfen-resistant QC02-R strain than in the susceptible strain. These results indicate that both P450 and GST are involved in whitefly resistance to pyriproxyfen.

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.

High glutathione S-transferase (GST) activity may contribute to colorectal cancer prevention. Functional polymorphisms are known in the GSTM1, GSTT1, GSTA1 and GSTP1 genes. The influence of these GST polymorphisms and recent fruit and vegetable consumption on GST levels and activity has not been investigated simultaneously in a human population. Also, it is not clear if blood GST activity reflects rectal GST activity. Therefore, we determined GST polymorphisms in 94 patients scheduled for sigmoidoscopy. Rectal GST isoenzyme levels (GSTM1, GSTM2, GSTT1, GSTA and GSTP1) were measured by quantitative western blotting, and rectal and white blood cell total GST activities were measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Vegetable and fruit consumption was assessed by dietary record. As expected, the GSTM1 and GSTT1 deletion polymorphisms, and the GSTA1 g.-69C-->T polymorphism significantly affected the respective isoenzyme levels. Also, rectal GST isoenzyme levels differed between those with and without recent consumption of Alliaceae, Cucurbitaceae, Apiaceae and citrus fruit. Rectal GST activity, however, was not clearly influenced by fruit and vegetable consumption. It was most significantly determined by the GSTP1 c.313A-->G polymorphism; compared with the 313AA genotypes, the 313AG and 313GG genotypes showed 36 and 67 nmol/min/mg protein (P < 0.001) lower GST activity, respectively. The correlation between rectal and white blood cell GST activities was low (r = 0.40, P < 0.001), and the relevance of the various genetic and dietary factors appeared to differ between the two tissues. In conclusion, this study indicates that the GST enzyme system is influenced by both GST polymorphisms and consumption of fruits and vegetables. The latter appeared more important for individual rectal GST isoenzyme levels than for total GST activity, which could affect detoxification of isoenzyme-specific substrates. The study results do no support the use of white blood cell GST activity as a surrogate measure for rectal GST activity.

Pyrethroid insecticide and milkweed cardenolide interactions on detoxification enzyme activity and expression in monarch caterpillars

Cycloxaprid is a novel cis-configuration neonicotinoid insecticide that is effective against a wide range of insect pests, including those that are resistant to conventional neonicotinoids. In this study, life table parameters were applied to estimate the cycloxaprid-induced sublethal effects on Aphis gossypii. The results indicated that the LC20 (0.81 mg a.i. L−1) of cycloxaprid significantly decreased the pre-oviposition period in first-progeny adults. Additionally, the life expectancy of F1 generation adults was reduced. However, no significant differences were observed for the intrinsic rate of increase (ri), finite rate of increase (λ), net reproductive rate (R0), or mean generation time (T) of F1 individuals. Therefore, resurgence in the A. gossypii population induced by a low concentration of cycloxaprid might not occur. Additionally, the response of the detoxification enzymes showed that cycloxaprid at the LC20 inhibited cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) activities at 6 h after exposure. Such inhibition of P450 and GST activities could lead to a decrease in the metabolism of cycloxaprid, which would increase the efficacy of cycloxaprid. Therefore, our results contribute to the assessment of the overall effects of cycloxaprid on A. gossypii.

BackgroundThe yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood.ResultsThe present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR.ConclusionThese results suggest that the high level of insecticide resistance found in Ae. aegypti mosquitoes from Martinique island is the consequence of both target-site and metabolic based resistance mechanisms. Insecticide resistance levels and associated mechanisms are discussed in relation with the environmental context of Martinique Island. These finding have important implications for dengue vector control in Martinique and emphasizes the need to develop new tools and strategies for maintaining an effective control of Aedes mosquito populations worldwide.

BackgroundThe yellow fever mosquito Aedes aegypti is the major vector of dengue, yellow fever, Zika, and Chikungunya viruses. Worldwide vector control is largely based on insecticide treatments but, unfortunately, vector control programs are facing operational challenges due to mosquitoes becoming resistant to commonly used insecticides. In Southeast Asia, resistance of Ae. aegypti to chemical insecticides has been documented in several countries but no data regarding insecticide resistance has been reported in Laos. To fill this gap, we assessed the insecticide resistance of 11 Ae. aegypti populations to larvicides and adulticides used in public health operations in the country. We also investigated the underlying molecular mechanisms associated with resistance, including target site mutations and detoxification enzymes putatively involved in metabolic resistance.Methods and resultsBioassays on adults and larvae collected in five provinces revealed various levels of resistance to organophosphates (malathion and temephos), organochlorine (DDT) and pyrethroids (permethrin and deltamethrin). Synergist bioassays showed a significant increased susceptibility of mosquitoes to insecticides after exposure to detoxification enzyme inhibitors. Biochemical assays confirmed these results by showing significant elevated activities of cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and carboxylesterases (CCE) in adults. Two kdr mutations, V1016G and F1534C, were detected by qPCR at low and high frequency, respectively, in all populations tested. A significant negative association between the two kdr mutations was detected. No significant association between kdr mutations frequency (for both 1534C and 1016G) and survival rate to DDT or permethrin (P > 0.05) was detected. Gene Copy Number Variations (CNV) were detected for particular detoxification enzymes. At the population level, the presence of CNV affecting the carboxylesterase CCEAE3A and the two cytochrome P450 CYP6BB2 and CYP6P12 were significantly correlated to insecticide resistance.ConclusionsThese results suggest that both kdr mutations and metabolic resistance mechanisms are present in Laos but their impact on phenotypic resistance may differ in proportion at the population or individual level. Molecular analyses suggest that CNV affecting CCEAE3A previously associated with temephos resistance is also associated with malathion resistance while CNV affecting CYP6BB2 and CYP6P12 are associated with pyrethroid and possibly DDT resistance. The presence of high levels of insecticide resistance in the main arbovirus vector in Laos is worrying and may have important implications for dengue vector control in the country.

Functional analysis of CYP6AE68, a cytochrome P450 gene associated with indoxacarb resistance in Spodoptera litura (Lepidoptera: Noctuidae)