CPR和P450基因在昆虫抗药性中的作用研究进展

P450酶系在昆虫代谢农药中有重要作用,NADPH-细胞色素P450还原酶（NADPH-cytochrome P450 reductase,CPR）和细胞色素P450（P450）在该酶系起核心作用。昆虫具有P450超基因家族,但只有一个单一的CPR基因,CPR是昆虫所有参与农药代谢的P450酶的唯一电子供体,其影响P450活性。P450基因的高水平表达在害虫抗药性中具有重要作用,P450基因介导的昆虫抗药性是最重要的代谢抗性类型。不同P450基因的高表达的调控机制不同,引起P450基因过量表达的原因可能有P450基因的编码区突变、顺式作用元件和反式作用因子变化、基因扩增等。细胞色素P450介导的抗药性存在一定程度的进化可塑性,即同种昆虫不同种群对相同的农药产生抗药性时,导致抗性产生的P450基因不同;同一昆虫品系在某种农药的抗性选择压力下,影响抗性的P450基因的种类和表达特性会随着持续的农药选择而发生变化。最近的研究显示,CPR的变异和昆虫抗药性相关,但是昆虫CPR基因介导抗药性的机制还缺乏深入研究。全面阐释P450酶系介导昆虫抗药性的机制、建立基于P450基因表达量变化与CPR突变的抗性分子标记,对于害虫抗药性治理具有重要意义。     

昆虫抗药性机理：行为和生理改变及解毒代谢增强（英文）

杀虫剂抗性是指“生物的一个品系发展了对该生物正常种群中大多数个体具有致死作用剂量的杀虫药剂的能力”。行为改变、生理学上的变化或代谢解毒等抗性机制能够降低毒物到达靶标的有效剂量。行为抗性是指减少昆虫与毒物接触或使昆虫能够存活于对大多数对正常个体致死（或有害）的环境中的任何行为。生理学改变的机制包括杀虫剂对表皮的穿透性降低、增加对药剂阻隔（sequestration）或储存和加速杀虫剂的排泄。细胞色素P450、水解酶和谷胱甘肽S-转移酶是杀虫药剂代谢解毒的主要3大酶系。细胞色素P450是一个超基因家族,是生物体内对外源性和内源性化合物解毒代谢或活化最重要的酶系。在许多害虫中发现P450介导的解毒代谢增加导致了对杀虫药剂抗性的增加。谷胱甘肽S-转移酶是可溶性的 二聚体蛋白,与代谢解毒、大量内源性和外源性化合物的排泄有关,许多昆虫中证明其抗药性与该酶活性增加有关。水解酶实际上是一组异源的酶类,其对抗药性的作用包括通过基因扩增增加酶量,作为结合蛋白隔离杀虫药剂或通过增加酶的活性加强对药剂的水解作用。     

P450s in plant-insect interactions

Cytochrome P450 monooxygenases (P450s) are integral in defining the relationships between plants and insects. Secondary metabolites produced in plants for protection against insects and other organisms are synthesized via pathways that include P450s in many different families and subfamilies. Survival of insects in the presence of toxic secondary metabolites depends on their metabolism by more limited groups of P450s. Examples of functionally characterized plant and insect P450s known to be involved in these interactions are discussed in terms of their diversities, reactivities and regulators. These and future examples, which will be uncovered as the fields of plant biology and entomology converge on this interesting area, provide much insight into the array of plant metabolites that are mainline defenses against insects, the range of insect monooxygenases that inactivate these compounds and the evolutionary processes occurring as these organisms wage daily battles with one another. Molecular perspectives on these interactions will provide the scientific community with information critical for genetic manipulation of these organisms aimed at enhancing plant resistance to insects and eliminating insect resistance to natural plant toxins and synthetic insecticides.     

Cytochrome P450 MA expression in insecticide-resistant German cockroaches (Dictyoptera: Blattellidae).

Cytochrome P450 monooxygenases are detoxification enzymes commonly involved in insecticide resistance by insects. Recently, an overexpressed form of this enzyme, P450 MA, was purified from an insecticide-resistant strain of German cockroach, Blattella germanica (L.), and polyclonal antisera (anti-P450 MA) was produced. To test hypotheses that the overexpressed condition of P450 MA has evolved in > 1 geographic location and that P450 MA might be involved in insecticide resistance to specific insecticides, investigations were conducted using 4 insecticide-resistant and 1 susceptible German cockroach strains. In western blots that used anti-P450 MA antiserum as a probe, substantial differences in expression of P450 MA were observed. Strains showing the highest P450 MA expression had both the highest tolerance to the organophosphate insecticide chlorpyrifos and cytochrome P450-mediated demethylation activity. Results support the hypothesis that cytochrome P450 MA is potentially overexpressed in insecticide-resistant populations on a global scale.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

Cytochrome P450 monooxygenases and insecticide resistance in insects

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies. Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain. The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli. When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance. Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

细胞色素P450介导抗性的进化可塑性

细胞色素P450是超基因家族,由其介导的杀虫剂代谢解毒的增强是昆虫产生抗药性的普遍而主要的机制。近年的研究表明,细胞色素P450介导的代谢抗性表现出一定程度的进化可塑性:即使是同种昆虫的不同种群在相同种类杀虫剂的胁迫下,进化选择出的抗性相关的细胞色素P450也有所不同,抗性的产生也可以是几种不同细胞色素P450协同作用或控制P450表达的调控因子的不同。     

Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellus

Cytochrome P450 monooxygenases (CYP s) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYP s which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYP s viz. CYP 417A2v2, CYP 425A1v2, and CYP 4DJ 1 from CYP 4 family of Laodelphax striatellus were randomly selected for experiments. CYP 417A2v2 and CYP 425A1v2 were found expressed successfully in Sf9 cell line while CYP 4DJ 1 was not expressed successfully and out of two expressed CYP s, only CYP 417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, p‐nitroanisole and ethoxycoumarin were preferentially metabolized by CYP 417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min^?1 mg protein^?1, respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP 417A2v2. Incubation of imidacloprid with CYP 417A2v2 of L. striatellus and subsequent HPLC , LC ‐MS , and MS /MS analysis revealed the formation of imidacloprid metabolites, that is, 4′ or 5′hydroxy‐imidacloprid by hydroxylation. This result implies the exemption of CYP s character that it is not always, all the CYP s degrading insecticides being selected and overexpressed in resistant strains and the degrading CYP s without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYP s in insecticide degradation can provide insight for better understand of insecticide resistance development.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

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.     

Comparison of Drosophila cytochrome P450 metabolism of natural and model substrates

Metabolism of some insecticides and toxic natural plant compounds is known to involve cytochrome P450 enzymes. Correlations between insecticide resistance and deethylation of the model substrate, 7-ethoxycoumarin, have prompted its use in screens for potentially resistant insect populations. The applicability of this model substrate as an indicator of the enzyme activities and inductive responsiveness of cytochrome P450 isoforms involved in the metabolism of carnegine was investigated. This toxic isoquinoline alkaloid is found in the host-plants of some species of cactophilic Drosophila. The results show that the ethoxycoumarin (ECOD) assay does not accurately predict carnegine metabolism either quantitatively or with respect to the overall pattern of activity. Therefore, the ECOD assay may be as isozyme-specific in insects as has already been demonstrated in mammals and its use as an indicator of general P450 activity is questionable.     

Toxicity, synergism, and neurological effects of novel volatile insecticides in insecticide-susceptible and -resistant Drosophila strains

Naturally derived volatile insecticides from the heterobicyclic and formate ester classes were investigated using a combination of volatility and synergist bioassays. In these studies, Drosophila melanogaster (Meigen) was used as a model for other medically important dipterans. In addition to a susceptible strain (Canton-S), three mutant strains were tested that included a strain resistant by P450-based metabolism (Hikone-R) and two resistant neurological mutant strains; one voltage-gated sodium channel mutant (para(ts-1)) and one GABA-gated chloride channel mutant (Rdl). In general, the 11 tested insecticides displayed a diversity of toxicity, metabolism, and resistance characteristics that correlate with their structural diversity. Several important trends were revealed by these studies, including hydrolase- and cytochrome P450 (P450)-based activation, P450-based resistance, distinct patterns of neurological activity, and negative cross-resistance with established insecticides. These findings provide important insight into the metabolism and modes of action for the volatile insecticides. These findings also suggest potential approaches for insecticide deployment in integrated vector management and resistance management programs.     

细胞色素P450介导的昆虫抗药性的分子机制

细胞色素P450(简称P450) 对杀虫剂的代谢作用直接影响到昆虫对杀虫剂的耐受性和杀虫剂对昆虫的选择性,由P450介导的杀虫剂代谢解毒作用的增强是昆虫产生抗药性的常见而重要的机制。P450介导的杀虫剂代谢抗性具有普遍性、交互抗性与进化可塑性的特点,涉及P450基因重复与基因扩增、基因转录上调以及结构基因的变异等多样化的分子机制,并且多重机制的共同作用可以导致高水平抗药性。这些研究发现说明,无论是昆虫抗药性机制的研究,还是抗药性监测与治理都要有动态的、因地制宜的理念。     

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.