Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms

Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests.     

Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus

A cytochrome P450 gene, Cyp9m10, is more than 200-fold overexpressed in a pyrethroid resistant strain of Culex quinquefasciatus, JPal-per. The haplotype of this strain contains two copies of Cyp9m10 resulted from recent tandem duplication. In this study, we discovered and isolated a Cyp9m10 haplotype closely related to this duplicated Cyp9m10 haplotype from JHB, a strain used for the recent genome project for this mosquito species. The isolated haplotype (JHB-NIID-B haplotype) shared the same insertion of a transposable element upstream of the coding region with JPal-per strain but not duplicated. The JHB-NIID-B haplotype was considered to have diverged from the JPal-per lineage just before the duplication event. Cyp9m10 was moderately overexpressed in larvae with the JHB-NIID-B haplotype. The overexpressions in JHB-NIID-B and JPal-per haplotypes were developmentally regulated in similar pattern indicating both haplotypes share a common cis-acting mutation responsible for the overexpressions. The isolated moderately overexpressed haplotype conferred resistance, however, its efficacy was relatively small. We hypothesized that the first cis-acting mutation modified the consequence of the subsequent duplication in JPal-per lineage to confer stronger phenotypic effect than that if it occurred before the first cis-acting mutation.     

泉州地区小菜蛾对三氟氯氰菊酯和阿维菌素的抗性监测

用浸叶法对泉州地区小菜蛾对三氟氯氰菊酯和阿维菌素的抗性进行了监测。结果表明,小菜蛾对三氟氯氰菊酯和阿维菌素的抗性呈逐年上升趋势,2003年的分别为2000年的7．4和4．3倍。     

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.     

表皮穿透和GABAA受体不敏感性在小菜蛾对阿维菌素抗性中的作用

用氚标记阿维菌素点滴处理阿维菌素敏感（ABM-S）和抗性（ABM-R）种群小菜蛾幼虫,结果显示,在5～360 min内的7个不同处理时间,ABM-R种群的平均表皮穿透量比ABM-S种群少1.5倍,处理24 h后,ABM-R种群仍有45.9%的3H-阿维菌素滞留于体表,而ABM-S种群却有98.4%的药剂穿透表皮。放射配体结合分析表明,GABAA受体结合性质的改变是小菜蛾对阿维菌素的另一抗性机制,ABM-S种群（K_d=10.9368±0.4374 nmol/L）和ABM-R种群（K_d=9.8328±0.3933　nmol/L）的受体亲和力无显著差异,但抗性种群的最大结合量（B_max=71.2842±4.9910　fmol/mg 蛋白）比敏感种群（B_max=112.0255±7.8418　fmol/mg 蛋白）降低63.6%,即抗性是受体数目的减少而非结构上的改变。     

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。     

Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations

Cytochrome P450 enzymes catalyze a number of oxidations in nature including the difficult hydroxylations of unactivated positions in an alkyl group. The consensus view of the hydroxylation reaction 10 years ago was that a high valent iron-oxo species abstracts a hydrogen atom from the alkyl group to give a radical that subsequently displaces the hydroxy group from iron in a homolytic substitution reaction (hydrogen abstraction-oxygen rebound). More recent mechanistic studies, as summarized in this review, indicated that the cytochrome P450-catalyzed "hydroxylation reaction" is complex, involving multiple mechanisms and multiple oxidants. In addition to the iron-oxo species, another electrophilic oxidant apparently exists, either the hydroperoxo-iron intermediate that precedes iron-oxo or iron-complexed hydrogen peroxide formed by protonation of the hydroperoxo-iron species on the proximal oxygen. The other electrophilic oxidant appears to react by insertion of OH(+) into a C-H bond to give a protonated alcohol. Computational work has suggested that iron-oxo can react through multiple spin states, a low-spin ensemble that reacts by insertion of oxygen, and a high-spin ensemble that reacts by hydrogen atom abstraction to give a radical.     

小菜蛾对苦皮藤素抗性选育及交互抗性测定

敏感小菜蛾Plutella xylostella (L.)经苦皮藤素20代的抗性选育,其抗性增长21.57倍。选育的小菜蛾抗性品系对杀虫双、杀螟丹和叶蝉散分别有4.63、4.11和3.71倍的交互抗性;对溴氰菊酯、氯菊酯、氯氰菊酯分别有0.22、0.01和0.26倍的负交互抗性。高抗杀螟丹、杀虫双小菜蛾品系对苦皮藤素无明显交互抗性,而高抗溴氰菊酯小菜蛾品系对苦皮藤素有3.61倍的交互抗性。     

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (k_cat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (k_cat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.     

Mutations in Saccharomyces cerevisiae sterol C5-desaturase conferring resistance to the CYP51 inhibitor fluconazole

Understanding fluconazole resistance is important as it emerged as a serious clinical problem for this CYP51, sterol 14alpha-demethylase, inhibitor. One mechanism, observed first in Saccharomyces cerevisiae, was through defective sterol C5-desaturase (Erg3p) required to form the fungistatic sterol end-product resulting from CYP51 inhibition, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol. Here, we report molecular changes resulting in both blocked mutants and also leaky mutants in which reduced ergosterol levels were detected. Blocked mutants exhibited nonsense and frameshift mutations, while leaky mutants contained missense mutations that were generally in conserved positions based on the alignment of sterol C5-desaturases and located mainly between residues 250 and 282.     

Contributions of three-site mutations in acetylcholinesterase and cytochrome P450 to genetic variation in susceptibility to organophosphate insecticides within a natural population of Drosophila melanogaster

In this study, we attempted to elucidate the two resistance factors conferring resistance to organophosphates within the Katsunuma population of Drosophila melanogaster (Meigen), one of which has been mapped on the second chromosome and the other on the third chromosome. With regard to the second chromosome factor, we tested susceptibility to malathion of 54 recombinant inbred lines with recombination between ltd and vg. Analyses of variance (ANOVAs) showed highly significant variation in susceptibility to malathion between recombinant lines. In addition, susceptibility of the second-chromosome resistant line to malathion was increased with additional application of piperonyl butoxide, suggesting a member of the Cyp gene family located between ltd and vg. With regard to the third-chromosome factor, we conducted inhibition assays of acetylcholinesterase (AChE) with respect to fenitroxon and carbaryl, to evaluate the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. I ₅₀ values of resistant lines, isolated from this population, were about 15 times higher for fenitroxon, and about two times higher for carbaryl, than those of susceptible lines, suggesting the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. We further investigated the genetic variation in the acetylcholinesterase (Ace) gene within the newly collected Katsunuma population, by using the allele-specific polymerase chain reaction (PCR) approach, and revealed that within this population there were high frequencies of resistant-type mutations at three sites in the Ace gene, which play critical roles in altering sensitivity of AChE to organophosphate and carbamate insecticides.     

昆虫抗药性相关细胞色素P450基因的表达调控机制

杀虫剂的频繁持续使用,必然导致昆虫产生抗药性.大量研究事例表明参与杀虫剂解毒的细胞色素P450(简称P450)过量表达是昆虫对不同类型杀虫剂产生抗性的重要原因,但目前人们对P450基因过量表达机制的认识还非常有限.近十年来,随着生命科学与相关研究技术的发展,有关昆虫P450基因表达调控机制的研究取得了实质性的进展.本文...     

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.     

Metabolism of Methoxychlor by the P450‐Monooxygenase CYP6G1 Involved in Insecticide Resistance of Drosophila melanogaster after Expression in Cell Cultures of Nicotiana tabacum

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1‐trichloro‐2,2‐bis(4‐methoxyphenyl)ethane, 1 ) against the background of endogenous enzymes of the corresponding non‐transgenic culture. The Cyp6g1‐transgenic cell culture metabolized 96% of applied methoxychlor (45.8 μg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 μg), whereas inhibition in the non‐transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid ( 6 ; 20 μg, 24 h) in the Cyp6g1‐transgenic culture by 82% in the presence of piperonyl butoxide (200 μg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor ( 1 ). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.     

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。