肠道微生物对小菜蛾耐受溴氰虫酰胺胁迫的作用与机制

Diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a primary pest of cruciferous crops. Long period chemical control results in frequently insecticide resistance for DBM. The novel pesticide cyantraniliprole is a succedaneous chemical that could used to reduce insecticide resistance. However, it still has the risk for resistance. Early researches showed that symbioses in DBM affected the host sensitivity to insecticide and also insecticide resistance. Up until now, whether the chemical sensitivity of DBM to cyantraniliprole which targets at ryania will be influenced by any symbioses is still unclear. In this study, a sensitive population of DBM will be selected here, a resistant population of DBM will be obtained by continuous feeding the sensitive DBM with cyantraniliprole. Afterwards, gut samples of the two populations were sampled. For the two populations, detoxifying enzyme activity of the intestinal microorganism will be compared; the metabolic function of the larval gut microorganism will be analyzed and compared by Biolog-ECO technology; the microbial community and gene function of the gut microorganism of DBM will be analyzed by metagenomics; the differential microorganism will be screened, and cyantraniliprole sensitivity test of the microorganism will be verified in the sensitivity population of DBM. On the basis of above research, the gut microflora which affect the sensitivity of DBM to cyantraniliprole and their function will be verified. Result of this study will show the insecticide mechanism of DBM to the chemical cyantraniliprole from intestinal microorganism, and will provide scientific evidence for the resistance management of cyantraniliprole .

小菜蛾是危害十字花科作物的主要害虫，长期化学防治导致小菜蛾产生了广泛的抗药性。新型杀虫剂溴氰虫酰胺是减缓抗性的替代药剂，但仍存在抗药性风险。前期研究发现，小菜蛾肠道共生微生物会影响宿主对药剂的敏感性和抗性。作用于鱼尼丁受体的溴氰虫酰胺对小菜蛾的敏感性是否也会受到共生微生物的影响，目前还缺乏了解。本项目以小菜蛾敏感品系为对象，通过药剂驯服获得其溴氰虫酰胺耐/抗性品系，再以2个品系肠道微生物为对象，分析肠道微生物在解毒酶活性上的差异；采用Biolog-ECO技术分析其在微生物代谢功能上的差异；采用宏基因组学分析其在微生物群落结构与基因功能上的差异；分离筛选差异可培养微生物，并以小菜蛾为对象进行溴氰虫酰胺敏感性验证。在此基础上，明确影响小菜蛾对溴氰虫酰胺敏感性变化的肠道微生物类群及其功能特征。研究结果将从肠道微生物的角度阐明小菜蛾对溴氰虫酰胺产生抗性的机理，对溴氰虫酰胺抗性治理提供理论依据。

小菜蛾是危害十字花科作物的主要害虫，长期化学防治导致小菜蛾产生了广泛的的抗药性。新型杀虫剂溴氰虫酰胺是减缓抗性的替代药剂，但仍存在抗药性的风险。前期研究发现，小菜蛾肠道共生微生物会影响宿主对药剂的敏感性和抗性。溴氰虫酰胺对小菜蛾的敏感性是否也会受到共生微生物的影响，目前还缺乏了解。本项目测定了小菜蛾对溴氰虫酰胺的LC50为0.143 mg/L。分离培养法发现小菜蛾肠道可培养优势细菌为蒙氏肠球菌Enterococcus mundtii和血杆菌Sanguibacter sp.，宏基因组学分析发现优势细菌属均小孢子虫属（Nosema）和肠球菌属（Enterococcus）。溴氰虫酰胺处理显著降低厚壁菌门、肠球菌属相对丰度，增加肠道细菌群落多样性、均匀度和丰富度；降低担子菌门、镰刀菌属和散尾鬼笔属等的相对丰度，增加平脐蠕孢属相对丰度,增加了微生物的代谢功能。溴氰虫酰胺处理增强了肠道CarE和GST活性，降低了MFO活性。Biolog-ECO碳源代谢表型分析发现，溴氰虫酰胺0.1和 1 mg/L浸叶饲喂和直接作用对小菜蛾肠道菌群代谢均无显著性影响，而10和100 mg/L溴氰虫酰胺直接作用于肠道可降低肠道微生物的碳源代谢能力。小菜蛾肠道优势细菌蒙氏肠球菌益生特性分析发现其对藤黄微球菌，苏云金芽孢杆菌及大肠杆菌均表现出抑菌活性，具较强的自凝聚习性和疏水习性，pH 5.0-10.0的环境下都能存活，具有较强的脱氨酶活性、无脱羧酶活性。转录组学分析结果显示，溴氰虫酰胺处理组和对照组小菜蛾样本中有33个解毒酶基因差异表达，包括7种CYPs、2种CCEs和1种GSTs上调，13种CYPs、7种CCEs和3种GSTs下调；共有16个免疫相关基因差异表达，包括3个肽聚糖识别蛋白（PGRP）基因上调、2个C型凝集素基因上调，3个抗菌肽基因（cecropin和2个gloverins）下调, qRT-PCR验证试验结果显示溴氰虫酰胺处理后，一个PGRP基因和CYP6J1基因表达上调，而cecropin基因、两个gloverin基因和一个溶菌酶基因表达下调。结合本研究结果及相关文献报道蒙氏肠球菌可分泌多种抗菌肽，推测小菜蛾肠道优势细菌蒙氏肠球菌可能通过影响昆虫免疫系统间接参与小菜蛾耐受溴氰虫酰胺的胁迫。