胰岛素信号通路关键基因在杀虫剂诱导黑肩绿盲蝽生殖中的功能解析

Cyrtorhinus lividipennis is a predominant predator of the rice planthoppers. Our previous study indicated that insecticides triazophos, deltamethrin, imidacloprid and avermectin could induced the reproduction of Cyrtorhinus lividipennis. Furthermore, we found insecticide treatment increased the relative expression level of genes in insulin signaling pathway and RNAi of the gene caused the reduction of fecundity of C. lividipennis, which indicated that insulin signaling pathway had function in the insecticide-induced reproduction of C. lividipennis. In this project, we would firstly investigate the effect of insecticides (triazophos and avermectin) treatment on the levels of expression and phosphorylation of the key genes (InR, PI3K, Akt) related to insulin signaling pathway in the predator. Subsequently, using molecular, behavioral, and immunohistochemical technologies, we would examine the regulatory function of key genes related to insulin signaling pathway on the reproduction capability, population growth, reproductive behavior, vitellogenesis and oogenesis in insecticide-treated C. lividipennis. Our results will provide valuable information for improving our understanding of the mechanisms of insecticide-induced reproduction regulated by hormonal signaling molecular in C. lividipennis. This study will also help us to select appropriate insecticides (e.g. by stimulation of reproduction of natural enemies) having high compatibility with C. lividipennis to control rice pest insects.

黑肩绿盲蝽是稻飞虱的重要捕食性天敌。我们前期研究发现亚致死浓度三唑磷、溴氰菊酯、吡虫啉、阿维菌素等杀虫剂处理诱导黑肩绿盲蝽生殖，但相关的作用机制尚不清楚。初步研究结果表明胰岛素信号通路参与了杀虫剂诱导的黑肩绿盲蝽生殖调控。本项目以三唑磷、阿维菌素为供试药剂，以黑肩绿盲蝽为研究对象，首先研究亚致死浓度杀虫剂对黑肩绿盲蝽胰岛素信号通路关键基因（InR, PI3K, Akt）时空表达动态和磷酸化的影响；再采用分子生物学、昆虫行为学、免疫组织化学等方法，研究胰岛素信号通路关键基因对杀虫剂处理的黑肩绿盲蝽生殖力、种群增长、生殖行为、卵黄形成、卵子发生的调控作用，以揭示胰岛素信号通路对杀虫剂诱导黑肩绿盲蝽生殖的调控机制。本研究可为阐明农药对天敌昆虫生殖刺激的激素和分子调控机制提供理论依据；同时为选择合适的农药（有利于天敌生殖）与天敌协同防治害虫提供指导。

黑肩绿盲蝽是稻飞虱等水稻害虫的重要天敌。项目组前期发现吡虫啉、阿维菌素等杀虫剂可增强黑肩绿盲蝽的繁殖力，且胰岛素信号通路可能参与调控该现象。本项目研究胰岛素信号通路相关基因调控杀虫剂刺激黑肩绿盲蝽生殖的机制，取得以下结果：（1）克隆了胰岛素信号通路关键基因ClInR、ClPI3K和ClAKT的全长，发现3个基因在成虫期、成虫的中肠和卵巢组织高表达。吡虫啉处理后2 d，3个基因在雌虫体内的表达量分别上升了4.09、4.73和2.04倍，且吡虫啉诱导了ClAKT磷酸化水平升高。阿维菌素处理后2 d，3个基因的表达量分别上升了255.5%、52.5%和122.8%。（2）沉默ClInR、ClPI3K和ClAKT使ClVg的表达分别下降92.1%、72.1%和57.8%，抑制黑肩绿盲蝽雌虫卵黄发生和卵母细胞成熟，并使其产卵量分别下降53.9%、50.8%和48.5%，证明胰岛素信号通路正向调控黑肩绿盲蝽的生殖。（3）吡虫啉刺激ClVg表达量上调154. 8%，促进Vg蛋白的表达和卵巢发育，还可使dsAKT处理的雌虫产卵量回升20.8%，说明吡虫啉通过刺激胰岛素通路基因的表达诱导其生殖。（4）明确了黑肩绿盲蝽的交配行为主要有爬背、触角敲打和交配试探、插入、调转身体、雌雄虫分离等5个步骤，分为求偶、交配前期、交配和交配后期4个阶段。发现吡虫啉处理可诱导黑肩绿盲蝽交配时间延长53.9%，日产卵量增加19.2%，表明吡虫啉刺激黑肩绿盲蝽生殖机制与生殖行为调控相关。（5）进一步研究证明胰岛素信号通路可正向调控黑肩绿盲蝽体内保幼激素含量。本项目成功揭示了杀虫剂诱导黑肩绿盲蝽胰岛素信号通路关键基因的表达，促进雌虫卵黄发生和卵巢成熟，从而诱导黑肩绿盲蝽的生殖。项目成果为进一步明确天敌昆虫黑肩绿盲蝽的生殖调控机理提供了基础，对田间科学用药和保护利用天敌进行水稻害虫绿色防控具有指导意义。