羧酸酯酶介导的小菜蛾对氟虫腈的抗性

Fipronil-resistance mediated by carboxylesterases in the diamondback moth,Plutella xylostella( Lepidoptera: Plutellidae)

【目的】羧酸酯酶(carboxylesterases,Car Es)是昆虫重要的解毒代谢酶之一,可以介导靶标昆虫对多种杀虫剂的代谢抗性。本研究检测了羧酸酯酶对小菜蛾Plutella xylostella抗药性的介导功能,旨在阐明羧酸酯酶在小菜蛾代谢解毒中的生理生化和分子机理。【方法】采用点滴法测定氟虫腈对小菜蛾敏感种群和抗氟虫腈种群的毒力,以及羧酸酯酶抑制剂磷酸三苯酯(triphenyl phosphate,TPP)对氟虫腈的增效作用;以LC30和LC50浓度的氟虫腈处理抗性小菜蛾,测定药剂处理后Car Es酶活性的变化;利用qRT-PCR技术分析Pxae22和Pxae31两个基因在小菜蛾不同发育阶段、组织和种群的表达模式;利用dsRNA干扰Pxae22和Pxae31后观察基因的表达变化和小菜蛾3龄幼虫对药剂敏感性的变化。【结果】TPP可以削弱小菜蛾3龄幼虫对氟虫腈的抗性,增效倍数约为6倍;使用较低剂量(LC30和LC50)氟虫腈处理小菜蛾3龄幼虫后,处理组Car Es比活力明显高于对照,提示氟虫腈对小菜蛾Car Es活性具有诱导作用。对羧酸酯酶基因Pxae22和Pxae31在小菜蛾不同发育阶段、4龄幼虫不同组织和不同种群3龄幼虫中的表达模式分析发现,这两个基因在小菜蛾4龄幼虫中的表达量最高;在4龄幼虫中以中肠组织中的表达量较高,头、表皮、脂肪体中的表达量很低;抗性种群中的表达量显著高于敏感种群。通过干扰Pxae22和Pxae31后的qRT-PCR验证,两个基因的表达量均显著降低,进一步的氟虫腈毒力测定发现,干扰Pxae22和Pxae31后的小菜蛾3龄幼虫对氟虫腈的敏感性分别增加了1.63倍和1.73倍。【结论】羧酸酯酶在小菜蛾对氟虫腈解毒代谢中具有重要作用;Pxae22和Pxae31是小菜蛾的两个抗性相关基因,其表达水平的变化直接影响小菜蛾对氟虫腈的敏感性。

【Aim】Carboxylesterases（ Car Es） are one of the most important detoxification enzymes in insects,and are implicated in the metabolic resistance of many different classes of insecticides. The present study aims to investigate the mediated functions of carboxylesterases in fipronil resistance of the diamondback moth,Plutella xylostella（ L.）,and thus to reveal the physiological,biochemical,and molecular mechanisms of carboxylesterases in metabolic detoxification of insects. 【Methods 】 Topical application was used to test the toxicity of fipronil to the susceptible population（ SP） and the fipronilresistant population（ FRP） and the synergistic effect of triphenyl phosphate（ TPP）,a carboxylesterase inhibitor,on fipronil. The LC30 and LC50concentrations of fipronil were used to treat FRP,and the effect on the carboxylesterase activity after treatment was determined. Stage- and tissue-specific expression patterns of Pxae22 and Pxae31 were profiled by qRT-PCR. Expression levels of Pxae22 and Pxae31 andthe insecticide susceptibility of the 3rd instar larvae were also examined after RNA interference（ RNAi）.【Results】The results showed that TPP decreased the resistance of the 3rd instar larvae of P. xylostella to fipronil by about six times. After treatment with the lower doses（ LC30 and LC50） of fipronil,the carboxylesterase activity in the treated 3rd instar larvae of P. xylostella was significantly higher than that of the control,suggesting that the carboxylesterase activity in P. xylostella could be induced by fipronil.Developmental expression profiling of genes exhibited that Pxae22 and Pxae31 had the highest expression levels in the 4th instar larvae. Tissue expression profiling of genes showed that Pxae22 and Pxae31 were highly expressed in the midgut of the 4th instar larvae,while lowly expressed in the head,cuticle and fat body. The expression levels of the two genes in the fipronil-resistant population were significantly higher than those in the susceptible population,respectively. The qRT-PCR analysis showed that after RNAi of Pxae22 and Pxae31,the expression of the two genes was significantly suppressed,and the sensitivity of the 3rd instar larvae of P. xylostella to fipronil increased by 1. 63 times and 1. 73 times,respectively.【Conclusion】 Carboxylesterases play important roles in metabolic detoxification of fipronil in P.xylostella. Two Car E genes,Pxae22 and Pxae31,are involved in insecticide resistance,and the variation of their expression levels can directly influence the susceptibility of P. xylostella to fipronil.