米氏沙雷菌与白纹伊蚊抗药性的相关性研究

Correlation research between Serratia oryzae and insecticide-resistance of Aedes albopictus

目的探究肠道共生菌米氏沙雷菌(Serratia oryzae)与白纹伊蚊(Aedes albopictus)溴氰菊酯抗性的关系。方法从白纹伊蚊肠道细菌分离培养米氏沙雷菌,通过抗生素处理及回饲法构建米氏沙雷菌富集白纹伊蚊,CDC Blottle检测富集前后抗药性。转录组分析富集前后白纹伊蚊代谢解毒酶基因的变化,ELASA法进行3种代谢解毒酶活性测定,RACE全长克隆差异基因及序列分析。结果抗生素处理及回饲富集米氏沙雷菌后,采用细菌菌落直接计数法及16S rRNA基因绝对定量法验证,与空白对照组差异均有统计学意义,即米氏沙雷菌富集白纹伊蚊构建成功。CDC Blottle实验结果显示,米氏沙雷菌富集白纹伊蚊在恢复24 h后死亡率显著低于对照组。在抗性品系和敏感品系白纹伊蚊米氏沙雷菌富集组和对照组转录组测序显示,米氏沙雷菌富集会导致代谢解毒酶基因上调表达,CYP9f2和CCEAE4分别上调表达5.07倍和11.35倍;酶活性检测显示,富集组3种酶活性均显著提高。结论米氏沙雷菌与白纹伊蚊的抗药性相关,通过上调蚊虫的代谢解毒酶的表达和酶活性增强,参与白纹伊蚊的抗药性发生。

Objective To investigate the relationship between intestinal commensal bacterium Serratia oryzae and deltamethrin resistance in Aedes albopictus.Methods Serratia oryzae was isolated and cultured from the intestinal bacteria of Aedes albopictus,which enriched by Serratia oryzae was constructed by antibiotic treatment and back-feeding method.Drug resistance was detected by CDC Bottle before and after enrichment.The changes in metabolic detoxification enzyme genes in Aedes albopictus were analyzed by transcriptome analysis before and after enrichment.The activities of three metabolic detoxification enzymes were determined by the ELISA method.Full-length differential genes were cloned by RACE and sequences were analyzed.Results The results of the direct bacterial colony counting method and the 16S rRNA gene absolute quantification method,after enrichment of Serratia oryzae by antibiotic treatment and back-feeding method,showed significant differences from those of the blank control group,indicating that the enrichment of Serratia oryzae in Aedes albopictus was successfully constructed.Subsequent CDC Bottle experiments showed that the mortality of Aedes albopictus enriched by Serratia oryzae was significantly lower than that of the control group after 24 h of recovery.Transcriptome sequencing in the Serratia oryzae enriched and control groups of resistant and sensitive strains of Aedes albopictus showed that the expression of metabolic detoxification enzyme genes was up-regulated by the enrichment of Serratia oryzae,among which CYP9f2 and CCEAE4 were up-regulated by 5.07 and 11.35 times,respectively.Meanwhile,the enzyme activity assay displayed that the activities of three enzymes in the enriched group were significantly increased.Conclusion Serratia oryzae is associated with resistance in Aedes albopictus and involvement in the development of drug resistance in Aedes albopictus by up-regulating the expression and activity of metabolic detoxification enzymes in mosquitoes.