A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital

Modifications of metabolic pathways are important in insecticide resistance evolution. Mutations leading to changes in expression levels or substrate specificities of cytochrome P450 (P450), glutathione-S-transferase (GST) and esterase genes have been linked to many cases of resistance with the responsible enzyme shown to utilize the insecticide as a substrate. Many studies show that the substrates of enzymes are capable of inducing the expression of those enzymes. We investigated if this was the case for insecticides and the enzymes responsible for their metabolism. The induction responses for P450s, GSTs and esterases to six different insecticides were investigated using a custom designed microarray in Drosophila melanogaster. Even though these gene families can all contribute to insecticide resistance, their induction responses when exposed to insecticides are minimal. The insecticides spinosad, diazinon, nitenpyram, lufenuron and dicyclanil did not induce any P450, GST or esterase gene expression after a short exposure to high lethal concentrations of insecticide. DDT elicited the low-level induction of one GST and one P450. These results are in contrast to induction responses we observed for the natural plant compound caffeine and the barbituate drug phenobarbital, both of which highly induced a number of P450 and GST genes under the same short exposure regime. Our results indicate that, under the insecticide exposure conditions we used, constitutive over-expression of metabolic genes play more of a role in insect survival than induction of members of these gene families.     

Ethiprole resistance in Nilaparvata lugens (Hemiptera: Delphacidae): possible mechanisms and cross-resistance

This article reports the current status of ethiprole resistance in Nilaparvata lugens Stål in the central region of Thailand, together with the associated resistance mechanisms. A resistance survey found that a field population had developed 308.5-fold resistance to ethiprole. Further selection with ethiprole for nine generations in the laboratory led to 453.1-fold ethiprole resistance. However, following this selection procedure, the resistance of N. lugens to other insecticides decreased to about one-third of its original resistance. This result implies that there is no cross-resistance between ethiprole and other kinds of insecticides in this pest. In an in vivo study of synergisms, triphenyl phosphate (TPP) exhibited a strong synergism (SR 4.2) with ethiprole in the resistant hoppers, piperonyl butoxide (PBO) also showed significant synergistic effects with ethiprole (1.6), but diethyl maleate (DEM) did not show any obvious synergism with ethiprole (1.2). An in vitro biochemical study indicated that esterase activity increased with ethiprole resistance in N. lugens, that P450 monooxygenase activity also increased significantly with high resistance, but that glutathione S-transferase activity did not. These results reveal that increases in esterase activity and P450 monooxygenase activity cause the ethiprole resistance observed in the field populations of N. lugens. Whether the mechanisms for ethiprole resistance involve target-site sensitivity is not yet known; further molecular analysis is required. However, an analysis of insecticide cross-resistance and the insecticide application history of the resistant populations indicated that target resistance was present and that rotation between insecticides with different modes of action will provide a key countermeasure to maintain the efficacy of ethiprole.     

Selection for fenpropathrin and fenpropathrin + acephate resistance in the silverleaf whitefly (Homoptera: Aleyrodidae)

A large, genetically diverse pool of Bemisia argentifolii Bellows & Perring was collected in 1994 from different crops, and a mixed colony was established in the laboratory. Subsets of this colony were reared on cotton plants held in large Plexiglas cages, and adult whiteflies were selected for resistance to fenpropathrin and to fenpropathrin + acephate (1:5). Selection was performed by exposing adults to treated glass vials at doses sufficient to give 60-80% mortality. Thirteen generations of adult selection with fenpropathrin + acephate yielded 856.3- and 1,289.3-fold tolerance (using lethal concentration ratio), respectively, to fenpropathrin and to fenpropathrin + acephate, indicating additive genetic variation for resistance in the source population. Selection with fenpropathrin alone yielded only a 10.9-fold increase in tolerance to fenpropathrin at the end of the selection period. There was no significant change in tolerance to fenpropathrin + acephate in this strain. Contrary to expectations, the early onset and the magnitude of resistance attained on selection with fenpropathrin + acephate compared with fenpropathrin alone indicates that this mixture evidently possesses a high degree of selectivity for development of resistance in B. argentifolii. Estimates of realized heritability of resistance to fenpropathrin and to fenpropathrin + acephate in B. argentifolii (in the fenpropathrin + acephate-selected strain) showed that they were significantly higher in the first half of selection (six generations), in both instances. Rearing of the fenpropathrin + acephate-resistant strain under conditions free of insecticides for six generations did not result in any significant decline in resistance, indicating that resistance is fairly stable.     

Exploring the correlation between deltamethrin stress and Keap1-Nrf2-ARE pathway from Drosophila melanogaster RNASeq data

Deltamethrin (DM) is widely used in a variety of pest control, resulting in serious drug resistance. Keap1-Nrf2-ARE is the antioxidant stress pathway. We identified 268 genes differentially expressed (DEGs) in Drosophila Kc cells treated with DM, including up-regulated 180 genes and down-regulated 88 genes compared with the control group (fold-change≥2, qValue≤0.001) by RNA-seq, they are mainly linked to metabolic process, stimulation response, immune system process. When the cells are treated with DM in the case of overexpression of the Keap1 gene, the cytochrome P450 family genes were significantly down-regulated, and some diseases-related genes and non-coding genes also changed. Our data shown that Keap1-Nrf2-ARE pathway may play an important role in DM stress, which will provide a new direction for studying the mechanism of insect resistance.     

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

Background

Permethrin 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 Findings

To 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.

Conclusions

These 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.     

飞蝗解毒酶系活力测定方法

飞蝗Locusta migratoria是重要的农业害虫,代谢抗性是飞蝗主要的农药抗性机制之一。与代谢抗性相关的解毒酶系主要有:非专一性酯酶系(Non-specficesterases,ESTs)、谷胱甘肽S-转移酶系(Glutathione S-transferases,GSTs)和细胞色素P450单加氧酶系(Cytochrome P450 monooxygenases,P450s),解毒酶系活力的测定是研究飞蝗农药代谢机制的重要途径。本文详细介绍了飞蝗解毒酶系的测定方法,为蝗虫及其他昆虫解毒酶系的测定提供参考。     

Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus

Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCq(G0) and MAmCq(G0)) and their permethrin selected offspring (HAmCq(G8) and MAmCq(G6)). While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCq(G8) and MAmCq(G6) were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCq(G8) and MAmCq(G6) mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCq(G8) and MAmCq(G6); of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment.     

甜菜夜蛾田间种群对甲氨基阿维菌素苯甲酸盐和高效氯氰菊酯的代谢抗性机制

甜菜夜蛾Spodoptera exigua(Hübner)云南晋宁、上海奉贤和江苏六合种群对甲氨基阿维菌素苯甲酸盐抗性为45～437倍,对高效氯氰菊酯抗性为211～555倍,对其它药剂抗性不明显。这3个田间种群3龄幼虫多功能氧化酶、谷胱甘肽S-转移酶和酯酶的活力分别为室内敏感种群的2.7～8.4倍、1.9～8.6倍和1.6～5.7倍。多功能氧化酶抑制剂PBO和酯酶抑制剂DEF对甲氨基阿维菌素苯甲酸盐的增效比为1.2～4.3和1.3～7.7;PBO和DEF对高效氯氰菊酯的增效比为1.8～58和3.6～245;谷胱甘肽S-转移酶抑制剂DEM对这2种药剂均无增效作用。上述结果表明,解毒代谢增强可能是甜菜夜蛾田间种群对甲氨基阿维菌素苯甲酸盐和高效氯氰菊酯的重要抗性机理,与酯酶和多功能氧化酶活性升高有关,与谷胱甘肽S-转移酶活性升高无关。本文的研究结果还表明,对于代谢抗性机理复杂的多抗性田间种群,根据不同解毒酶抑制剂对药剂的增效作用判断不同解毒代谢酶在抗性形成中的作用更加可靠。     

Gene expression alterations in doxorubicin resistant MCF7 breast cancer cell line

Many molecular mechanisms contribute to the development of doxorubicin resistance and different cancers can express wide and diverse arrays of drug-resistance genes. The aim of this study was to identify the changes in gene expression associated with the development of doxorubicin resistance in MCF7 breast cancer cell line. The doxorubicin resistant MCF7 cell line was developed by stepwise selection of MCF7 cells and was tested using the MTT assay. The alterations in gene expression were examined using the real-time based PCR array. The findings showed an up-regulation of many phase I/II metabolizing genes, specifically, the CYP1A1 and the CYP1A2 that were up-regulated by 206- and 96-fold respectively. Drug efflux pump genes were also up-regulated profoundly. TOP2A was strongly down-regulated by 202-fold. Many other changes were observed in genes crucial for cell cycle, apoptosis and DNA repair. The findings of this project imply that the development of doxorubicin resistance is a multi-factorial process.     

Susceptibility and detoxifying enzyme activity in two spider mite species (Acari: Tetranychidae) after selection with three insecticides

Changes in the susceptibility and detoxifying enzyme activity were measured in laboratory strains of Banks grass mite, Oligonychus pratensis (Banks), and twospotted spider mite, Tetranychus urticae Koch, that were repeatedly exposed to three insecticides. Three strains of each mite species were exposed to one of two pyrethroids, bifenthrin, and lambda-cyhalothrin, or an organophosphate, dimethoate, for 10 selection cycles at the LC60 for each insecticide. A reference or nonselected strain of each mite species was not exposed to insecticides. After 10 cycles of exposure, susceptibility to the corresponding insecticides, bifenthrin, lambda-cyhalothrin, and dimethoate, decreased 4.5-, 5.9-, and 289.2-fold, respectively, relative to the reference strain in the respective O. pratensis strains, and 14.8-, 5.7-, and 104.7-fold, respectively, relative to the reference strain in the respective T. urticae strains. In the bifenthrin-exposed O. pratensis strain, there was a 88.9-fold cross-resistance to dimethoate. In the dimethoate-exposed T. urticae strain, there was a 15.9-fold cross-resistance to bifenthrin. These results suggest that there may be cross-resistance between dimethoate and bifenthrin. The reduced susceptibility to dimethoate remained stable for three months in the absence of selection pressure in both mites. The decrease in susceptibility in the O. pratensis strains exposed to bifenthrin, lambda-cyhalothrin, and dimethoate was associated with a 4.7-, 3.0-, and 3.6-fold increase in general esterase activity, respectively. The decrease in susceptibility in the T. urticae strains exposed to bifenthrin and lambda-cyhalothrin was associated with a 1.3- and 1.1-fold increase in general esterase activity, respectively. The mean general esterase activity was significantly higher in the pyrethroid-exposed O. pratensis and T. urticae strains than in the nonselected strain. There was no significant increase in esterase activity in the dimethoate-exposed T. urticae strain. The decrease in susceptibility to insecticides was also associated with reduced glutathione S-transferase 1-chloro-2, 4-dinitrobenzene conjugation activity, but this did not appear to be related to changes in insecticide susceptibility. These results suggest that in these mites, the general esterases may play a role in conferring resistance to pyrethroids. However, some other untested mechanism, such as target site insensitivity, must be involved in conferring dimethoate resistance.     

The classification of esterases: an important gene family involved in insecticide resistance--a review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.     

Global metabolic regulation analysis for<Emphasis Type="Italic"> Escherichia coli</Emphasis> K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement

Regulation of the main metabolic pathways of Escherichia coli K12 was investigated based on 2-dimensional electrophoresis (2DE) and the measurement of enzyme activities. The cells were grown aerobically in different carbon sources, such as glucose, acetate, gluconate or glycerol. Microaerobic cultivation was also conducted with glucose as a carbon source. Fifty-two proteins could be identified based on 2DE, and 26 enzyme activities from the main metabolic pathways-including glycolysis, pentose phosphate pathway, TCA cycle, Entner-Doudoroff pathway and fermentative pathway-were assayed. These enzyme activities, together with global and quantitative protein expression, gave us a clear picture of metabolic regulation. The results show that, compared with the control experiment with glucose as a carbon source under aerobic conditions, glycolytic enzymes were slightly up-regulated (<2-fold), TCA cycle enzymes were significantly down-regulated (2- to 10-fold), and fermentative enzymes such as pfl and adhE were highly up-regulated (>10-fold) under microaerobic conditions in glucose medium. When acetate was used as a carbon source, pfkA, pykF, ppc and zwf were down-regulated, while fbp, pckA, ppsA and mez were significantly up-regulated. Glyoxylate enzymes such as aceA and aceB were strongly up-regulated (>10-fold) and TCA-cycle-related enzymes were also up-regulated to some extent. With gluconate as a carbon source, edd, eda, fbp and TCA cycle enzymes were up-regulated. With glycerol as a carbon source, fbp and TCA cycle enzymes were up-regulated, while ackA was significantly down-regulated. Protein abundance obtained by 2DE correlated well with enzyme activity, with a few exceptions (e.g., isocitrate dehydrogenase), during aerobic growth on acetate.     

橘全爪螨对双甲脒的抗性选育及其P450基因的表达差异分析

为了对双甲脒进行抗性风险评估, 弄清P450基因在橘全爪螨Panonychus citri抗药性中的作用, 在室内用双甲脒对橘全爪螨进行了抗性选育和交互抗性研究, 同时分析了橘全爪螨双甲脒抗性和敏感品系P450基因表达差异。经过12代抗性选育, 获得了橘全爪螨双甲脒抗性品系, 与敏感品系比较, 橘全爪螨对双甲脒的抗性倍数达到26.32倍。抗性风险评估表明, 橘全爪螨对双甲脒抗性遗传力h²为0.148。螺螨酯、 丁醚脲、 炔螨特和三唑锡对抗性品系的LC₅₀分别为敏感品系的16.85, 4.98, 2.13和2.05倍, 表明双甲脒抗性品系对螺螨酯、 丁醚脲、 炔螨特和三唑锡具有明显的交互抗性。阿维菌素、 苯丁锡、 哒螨灵、 矿物油对抗性品系LC₅₀分别为敏感品系的1.10, 1.21, 0.67和0.99倍, 表明双甲脒抗性品系对上述4种药剂没有显著的交互抗性。基因差异性分析发现, 抗性品系中有16条P450基因发生了上调, 27条P450基因发生了下调, 其中CYP389A6上调倍数最高［log₂ratio (RS/SS)=11.526］, CYP389A2下调倍数最高［log2ratio(RS/SS) =-12.683］, 由此推断, CYP389A6上调和CYP389A2下调可能是橘全爪螨对双甲脒产生抗性的重要原因。     

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.     

Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non–alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.     

Role of cytochrome P450s in insecticide resistance: impact on the control of mosquito-borne diseases and use of insecticides on Earth

The fight against diseases spread by mosquitoes and other insects has enormous environmental, economic and social consequences. Chemical insecticides remain the first line of defence but the control of diseases, especially malaria and dengue fever, is being increasingly undermined by insecticide resistance. Mosquitoes have a large repertoire of P450s (over 100 genes). By pinpointing the key enzymes associated with insecticide resistance we can begin to develop new tools to aid the implementation of control interventions and reduce their environmental impact on Earth. Recent technological advances are helping us to build a functional profile of the P450 determinants of insecticide metabolic resistance in mosquitoes. Alongside, the cross-responses of mosquito P450s to insecticides and pollutants are also being investigated. Such research will provide the means to produce diagnostic tools for early detection of P450s linked to resistance. It will also enable the design of new insecticides with optimized efficacy in different environments.