Molecular regulation of diapause in Culex pipiens

描述（由申请人提供）：蚊子和大多数昆虫一样，每年只有几个月能够觅食和繁殖。剩下的几个月以一种被称为滞育的休眠形式度过。本提案的目的是了解控制西尼罗病毒载体北方家蚊（库蚊）成年发育停滞的分子机制，其长期目标是破坏生命周期中这一脆弱阶段。本提案的三个具体目标集中在编程滞育状态的分子途径上。胰岛素信号是产生滞育表型（如发育停止、脂肪积累、寿命延长和抗逆性增强等特征）的关键中介，本项目研究了胰岛素信号传导的上下游途径。我们的第一个目标是通过胰岛素和叉头转录因子（FOXO）的下游工作来确定导致滞育表型的途径。利用FOXO抗体和ChIP测序，FOXO下游基因将被鉴定，它们在滞育中的推测作用将通过包括RNA干扰（RNAi）在内的功能分析进行评估。第二个目标，胰岛素信号的上游，是确定时钟基因在蚊子进入滞育时使用的短日照信号转导中的作用。克隆了库蚊的主要生物钟基因，分析了长日（非滞育）和短日（滞育）下的表达模式，并利用RNAi技术分析了它们在滞育编程中的功能。第三个目标是将光周期反应与下游胰岛素/FOXO通路联系起来。在破坏生物钟机制后，我们将监测这些变化对胰岛素/FOXO通路中基因表达模式的影响。有了这些信息，就有可能开始构建从光周期感知到滞育表型执行的途径。任何滞育反应的改变（如滞育预防、滞育开始或终止的延迟、能量储存或利用的损害、胁迫耐受性的降低）都可以作为破坏蚊子生命周期这一关键阶段的工具。

DESCRIPTION (provided by applicant): Mosquitoes, like most insects, are able to feed and reproduce only a few months each year. The remaining months are spent in a form of dormancy known as diapause. It is the objective of this proposal to understand the molecular mechanisms that regulate this developmental arrest in adults of the northern house mosquito, Culex pipiens, the vector of West Nile virus, with the longterm goal of disrupting this vulnerable phase of the life cycle. The three specific goals of this proposal focus on the molecular pathway that programs the diapauses state. Insulin signaling is a key intermediary in generating the diapause phenotype (traits such as a halt in development, fat accumulation, extended lifespan and increased stress resistance), and this project investigates pathways both up- and downstream of insulin signaling. Our first goal is to identify the pathways leading to the diapause phenotype by working downstream of insulin and FOXO (forkhead transcription factor). Using a FOXO antibody and ChIP sequencing, genes downstream of FOXO will be identified and their putative roles in diapause will be evaluated with functional assays including RNA interference (RNAi). The second goal, upstream of insulin signaling, is to identify the role of clock genes in the transduction of the short day length signals that are used by the mosquito to program entry into diapause. The major clock genes from C. pipiens will be cloned, expression patterns will be evaluated under both long day lengths (nondiapause-inducing) and short day lengths (diapause-inducing), and their function in programming diapause will be evaluated using RNAi. The third aim is to link the photoperiodic response to the downstream insulin/FOXO pathway. After disrupting the clock mechanism we will monitor the impact of these changes on gene expression patterns in the insulin/FOXO pathway. With this information, it will be possible to begin construction of the pathway leading from perception of photoperiod to execution of the diapause phenotype. Any alterations in the diapause response (e.g. diapause prevention, delay in onset or termination, impairment of energy storage or utilization, reduced stress tolerance) could be used as tools for disrupting this critical phase of the mosquito life cycle.