Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance

BACKGROUND: Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450–substrate interactions. RESULTS: Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X‐ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also ‘V’‐shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT–CYP6G1 complex and a non‐resistant CYP6A2 homology model implies that tight‐fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. CONCLUSION: The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities. Copyright © 2010 Society of Chemical Industry     

Metabolism of imidacloprid in Apis mellifera

Biotransformation of imidacloprid and the appearance of olefin and 5-hydroxyimidacloprid metabolites in the honeybee were studied by HPLC-MS/MS analysis. Honeybees were treated orally with imidacloprid at 20 and 50 microg kg(-1) bee. Imidacloprid was metabolised relatively quickly and thoroughly. Twenty minutes after the beginning of imidacloprid ingestion, the sum of the residues from the three compounds amounted to only 70% of the actual given dose. Imidacloprid, 5-hydroxyimidacloprid and olefin represented, respectively, 50%, 9% and 8% of the actual ingested dose. Six and 24 h, respectively, after ingestion of imidacloprid at 20 and 50 microg kg(-1) bee, imidacloprid could no longer be detected in the honeybee. Imidacloprid had a half-life ranging between 4.5 and 5 h and was rapidly metabolised into 5-hydroxyimidacloprid and olefin. Except 5-hydroxyimidacloprid in the 20 microg kg(-1) treatment, these two metabolites presented a peak value 4 h after ingestion of the 20 and 50 microg kg(-1) doses. This time fully coincided with the appearance of mortality induced by imidacloprid after acute oral intoxication. These results suggested that the immediate neurotoxicity symptoms are due to the action of imidacloprid, whereas 5-hydroxyimidacloprid and/or olefin are involved in honeybee mortality. In addition, it was likely that the 30% of residues undetected 20 min after intoxication were imidacloprid metabolites, although not 5-hydroxyimidacloprid or olefin. Thus, 5-hydroxyimidacloprid and olefin could not be the major metabolites in the worker bees.     

烟粉虱细胞色素CYP6EM1基因的克隆及对吡虫啉抗性的作用

烟粉虱是一种世界性农业害虫,其防治手段以化学防治为主,新烟碱类杀虫剂吡虫啉常年用于防治烟粉虱,田间烟粉虱已经形成严重的抗药性。本研究通过分析烟粉虱吡虫啉抗性和敏感种群,发现细胞色素CYP6EM1基因在吡虫啉抗性品系中上调了4.7倍,进而克隆了其全长基因,进行了荧光定量PCR分析,发现该基因在吡虫啉抗性烟粉虱3龄若虫期和雄虫成虫期过量表达,并且在抗性成虫胸部和腹部过量表达。最后通过RNA干扰的方法使成虫的CYP6EM1基因表达量下降了54.8%,之后发现当烟粉虱暴露于吡虫啉时死亡率显著升高了39.65%,这表明CYP6EM1与烟粉虱对吡虫啉抗性的形成相关。研究结果对于揭示烟粉虱对吡虫啉产生抗性的机制有帮助,也为烟粉虱抗性水平田间监测及烟粉虱综合治理提供理论依据。     

Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster

Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, cypermethrin, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No obvious abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.     

Spinosad resistance in female Musca domestica L. from a field-derived population

BACKGROUND: Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. RESULTS: In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC₅₀, whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. CONCLUSION: This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry     

Resistance evolution in Drosophila: the case of CYP6G1

The massive use of DDT as an insecticide between 1940 and 1970 has resulted in the emergence of a resistant population of insects. One of the main metabolic mechanisms developed by resistant insects involves detoxification enzymes such as cytochrome P450s. These enzymes can metabolise the insecticide to render it less toxic and facilitate its elimination from the organism. The P450 Cyp6g1 was identified as the major factor responsible for DDT resistance in Drosophila melanogaster field populations. In this article, we review the data available for this gene since it was associated with resistance in 2002. The knowledge gained on Cyp6g1 allows a better understanding of the evolution of insecticide resistance mechanisms and highlights the major role of transposable elements in evolutionary processes. © 2016 Society of Chemical Industry     

Identification and expression profiles of nine glutathione S-transferase genes from the important rice phloem sap-sucker and virus vector Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae)

BACKGROUND: Glutathione S‐transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised. RESULTS: The cloned GSTs of L. striatellus belonged to six cytosolic classes and a microsomal subgroup. Exposure to sublethal concentrations of each of the six insecticides, DDT, chlorpyrifos, fipronil, imidacloprid, buprofezin and beta‐cypermethrin, quickly induced (6 h) up‐expression of LsGSTe1. The expression of LsGSTs2 was increased by chlorpyrifos, fipronil and beta‐cypermethrin. Furthermore, exposure of L. striatellus to fipronil, imidacloprid, buprofezin and beta‐cypermethrin increased the expression of the LsGSTm gene after 24 or 48 h. CONCLUSION: This work is the first identification of GST genes from different GST groups in Auchenorrhyncha species and their induction characteristics with insecticide types and time. The elevated expression of GST genes induced by insecticides might be related to the enhanced tolerance of this insect to insecticides and xenobiotics. Copyright © 2012 Society of Chemical Industry     

北京、山东和湖南地区烟粉虱抗药性及CYP4v2和CYP6CX1 mRNA水平表达量分析

于2011年采集北京、山东和湖南三地的烟粉虱,进行B、Q隐种鉴定,并测定4种杀虫剂的抗药性,同时通过荧光定量PCR分析CYP4v2和CYP6CX1两个基因的mRNA水平的表达量。结果表明,北京、湖南和长沙烟粉虱均为Q隐种。抗药性监测表明,北京和湖南种群对阿维菌素敏感,山东种群抗性水平较低,而对烟碱类药剂噻虫嗪出现不同程度的抗药性,其中湖南地区烟粉虱对噻虫嗪的抗药性达到49.08倍的高抗水平,北京和山东地区也达到中抗水平。另外,这3个地区的种群对毒死蜱和联苯菊酯抗性水平都较低。通过qRT-PCR分析三地的CYP4v2和CYP6CX1基因表达量,发现相对于敏感种群CYP4v2基因在北京、山东和湖南3个地理种群中分别过量表达3.85倍、19.57倍和10.78倍,而CYP6CX1基因在北京种群中过量表达20.55倍。结果提示田间烟粉虱的细胞色素P450基因CYP4v2和CYP6CX1过量表达可能会是烟粉虱抗药性的形成机制之一。     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

Differential metabolism of sulfoximine and neonicotinoid insecticides by Drosophila melanogaster monooxygenase CYP6G1

Sulfoxaflor [N-[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl]ethyl]-λ⁴-sulfanylidene] cyanamide] is in development as the first product from the new sulfoximine class of insect control agents. Highly effective against a variety of sap-feeding pest insects, available data indicate no cross-resistance to sulfoxaflor in pest insect strains that exhibit high levels of resistance to neonicotinoids and other insecticides. In vitro studies of the cytochrome P450 monooxygenase CYP6G1 from Drosophila melanogaster, expressed in a Drosophila cell line, show very high levels of metabolism for a variety of neonicotinoids, but not for sulfoxaflor and its chloropyridine-analog. A sulfoxaflor analog with nitrogen in place of the carbon in the bridge between the pyridine and sulfoximine moiety shows a modest degree of metabolism. In silico homology modeling of the CYP6G1 with the sulfoximines and neonicotinoids suggests that steric effects may limit interactions of the sulfoximines with the reactive heme-oxo complex. A distinct relationship was identified for the summed Hückel charges and the degree of metabolism observed. These observations help explain the lack of sulfoxaflor metabolism by CYP6G1, and in turn provide a basis for the lack of cross-resistance to sulfoxaflor in insecticide resistant strains of pest insects.     

新疆MEAM1烟粉虱隐种对吡虫啉的抗性遗传力分析及交互抗性测定

采用Tabashnik的域性状指标分析了新疆MEAM1(Middle-East-Asia-Minor l)烟粉虱隐种对吡虫啉的抗性现实遗传力(h2)和不同致死率下的抗性发展速率,同时测定了抗性种群对不同类型杀虫剂的交互抗性。结果表明,在30%~50%较低的选择压力下,新疆MEAM1烟粉虱隐种连续汰选8代后,对吡虫啉的抗性上升28.01倍,抗性现实遗传力h2为0.429 7。假设田间种群现实遗传力为实验室筛选估算值的1/2,即h2=0.214 9,对新疆MEAM1烟粉虱隐种对吡虫啉的抗性发展速率估算结果表明:在药剂选择压力为50%~60%下,若使其对吡虫啉的抗性增长10倍,则需要生长10~8代;而在药剂选择压力为70%~90%下,若使其抗性增长10倍,则仅需要生长6~4代。表明新疆MEAM1烟粉虱隐种对吡虫啉产生抗性的风险很大。交互抗性测定结果显示:抗性种群对同类型的杀虫剂吡虫清和噻虫嗪分别产生了10.78倍和4.75倍的中等至低水平交互抗性;对多杀菌素、毒死蜱、吡丙醚和高效氯氰菊酯的敏感性有所降低;对阿维菌素、氟啶虫胺腈和乙基多杀菌素等杀虫剂则无交互抗性。     

Susceptibility to thiamethoxam of Musca domestica from Danish livestock farms

BACKGROUND: Neonicotinoid baits are currently replacing anticholinesterase baits for control of adult houseflies (Musca domestica L.). Introduction of new insecticides includes evaluation of their cross-resistance potential, which was assessed for thiamethoxam in field populations from Denmark. RESULTS: In feeding bioassay with a susceptible strain, thiamethoxam LC(50) at 72 h was 1.7 microg thiamethoxam g(-1) sugar, making it 19-fold, 11-fold and threefold more toxic to houseflies than azamethiphos, methomyl and spinosad respectively. The field populations were 6-76-fold resistant to thiamethoxam. There was no correlation between the toxicities of thiamethoxam and spinosad, dimethoate, methomyl, bioresmethrin or azamethiphos. The toxicity in feeding bioassay at 72 h of imidacloprid in a susceptible strain was 32 microg imidacloprid g(-1) sugar at LC(50), making it 19-fold less toxic to houseflies than thiamethoxam. There was a strong significant correlation between the toxicities of thiamethoxam and imidacloprid in field populations.CONCLUSION: Neonicotinoid-resistant houseflies were present at a detectable and noticeable level before thiamethoxam and imidacloprid were introduced for housefly control in Denmark. The toxicity of thiamethoxam is explained by other parameters than the toxicities of spinosad, dimethoate, methomyl, bioresmethrin or azamethiphos. The cross-resistance between thiamethoxam and imidacloprid indicates a coincidence of mechanism of the toxicity and resistance in the field populations.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Neural actions of imidacloprid and their involvement in resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say)

BACKGROUND: The development of resistance to imidacloprid in eastern US populations of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), threatens this critical use for neonicotinoid insecticides. Previous pharmacokinetic studies with resistant adult CPBs provided no explanation for the high resistance level (over 200-fold) to topically applied imidacloprid. The authors assessed the neural activity of imidacloprid by recording spontaneous activity from a motor nerve leaving the isolated central nervous system to compare the sensitivity of the latter to imidacloprid between susceptible and resistant CPBs. RESULTS: On the isolated central nervous system, imidacloprid was initially neuroexcitatory, and neuroinhibitory at higher concentrations. The neuroexcitatory action of imidacloprid was blocked by coapplication of a specific nAChR antagonist, methyllycaconitine, indicating that it is a result of action on nAChRs. The sensitivity to the neuroexcitatory and inhibitory activities of imidacloprid varied independently among individuals in each population. The sensitivity of the central nervous system of resistant CPBs to excitation by imidacloprid did not differ from that of susceptible insects, but the sensitivity to inhibition by imidacloprid was reduced 52- to 58-fold, indicating a possible change in the sensitivity of at least one subgroup of nAChRs. CONCLUSION: This study provides evidence that reduced nerve sensitivity to the blocking action of imidacloprid is associated with imidacloprid resistance in the CPB.     

小菜蛾CYP6BG1基因体外真核表达及其对氯虫苯甲酰胺的代谢

小菜蛾Plutella xylostella是为害十字花科作物的世界性重要害虫,对氯虫苯甲酰胺产生了严重抗性。已经明确小菜蛾CYP6BG1的过量表达与其对氯虫苯甲酰胺的抗性相关。本文进一步通过昆虫杆状病毒表达系统在Sf9细胞中表达了小菜蛾CYP6BG1和NADPH-细胞色素P450还原酶(CPR)蛋白,并检测Sf9细胞表达CYP6BG1蛋白后对氯虫苯甲酰胺的敏感变化及氯虫苯甲酰胺经CYP6BG1蛋白代谢后对小菜蛾幼虫毒力的变化。结果显示:Sf9细胞中与CPR共表达的CYP6BG1具有7-乙氧基香豆素O-脱乙基酶活性,且细胞对氯虫苯甲酰胺的敏感性显著下降。将表达的蛋白与不同浓度的氯虫苯甲酰胺孵育24 h后,对3龄小菜蛾幼虫致死率显著低于对照组。本研究结果间接表明CYP6BG1能够降解氯虫苯甲酰胺,从而增强小菜蛾对氯虫苯甲酰胺的抗性。     

Toxicities and sublethal effects of seven neonicotinoid insecticides on survival, growth and reproduction of imidacloprid-resistant cotton aphid, Aphis gossypii

BACKGROUND: Imidacloprid has been a major neonicotinoid insecticide for controlling Aphis gossypii (Glover) (Homoptera: Aphididae) and other piercing–sucking pests. However, the resistance to imidacloprid has been recorded in many target insects. At the same time, cross‐resistance of imidacloprid and other insecticides, especially neonicotinoid insecticides, has been detected. RESULTS: Results showed that the level of cross‐resistance was different between imidacloprid and tested neonicotinoid insecticides (no cross‐resistance: dinotefuran, thiamethoxam and clothianidin; a 3.68–5.79‐fold cross‐resistance: acetamiprid, nitenpyram and thiacloprid). In the study of sublethal effects, imidacloprid at LC₂₀ doses could suppress weight gain and honeydew excretion, but showed no significant effects on longevity and fecundity of the imidacloprid‐resistant cotton aphid, A. gossypii. However, other neonicotinoid insecticides showed significant adverse effects on biological characteristics (body weight, honeydew excretion, longevity and fecundity) in the order of dinotefuran > thiamethoxam and clothianidin > nitenpyram > thiacloprid and acetamiprid. CONCLUSION: The results indicated that dinotefuran is the most effective insecticide for use against imidacloprid‐resistant A. gossypii. To avoid further resistance development, the use of nitenpyram, acetamiprid and thiacloprid should be avoided on imidacloprid‐resistant populations of A. gossypii. Copyright © 2011 Society of Chemical Industry