Molecular mode of action of Juvenile hormone analogs

项目摘要 成年蚊子传播病原体，导致人类致命的疾病。因此，防止成人 羽化被用作阻断病原体传播的策略。保幼激素（JH）预防 变态和成虫羽化;保幼激素类似物（JHA），如甲氧普烯、烯虫乙酯和 吡丙醚已经商业化用于控制蚊子和其它疾病媒介。但 JHA在杀死蚊子和其他昆虫中的分子作用模式仍然知之甚少。JHA是 被认为是通过阻止幼虫蛹的变态而起作用的。这正是这些化合物在大多数情况下的作用方式。 鳞翅目昆虫。然而，在双翅目昆虫如黄热病蚊子、埃及伊蚊、JHA中， 应用不能防止幼虫蛹变态;处理过的幼虫经历蛹蜕皮并死亡 在蛹的阶段。初步研究表明，脓疱是由甲氧普烯处理的幼虫发展而来的， 由于幼虫组织的持续存在而死亡。采用转基因CRISPR/Cas9基因组编辑方法， 击倒爱编码蜕皮激素诱导蛋白93 F（E93）的基因的埃及伊蚊直系同源物，已知该蛋白93 F 调节幼虫组织的程序性细胞死亡（PCD）。Ae.埃及伊蚊E93基因敲除动物 与JHA处理的幼虫中观察到的相似，表明JHA抑制E93表达， 幼虫组织死亡，导致蛹中含有幼虫和蛹组织，导致其死亡。这 假设将通过在两个特定目标下进行实验来验证。在第一个特定的目标，幼虫 将检查接受PCD的组织，以确定JHA治疗与 E93击倒。JHA处理和E93基因敲除的幼虫和蛹的组织中PCD的状态 将通过用凋亡和自噬标记染料染色来研究。在第二个具体目标中，RNA和 将采用ATAC测序、RNAi和报告基因分析来鉴定与幼虫组织有关的基因 重塑和JHA通过E93发挥作用调节其表达的机制。的功能 E93作为一个先锋和激活转录因子将被研究。拟议的结果 研究将通过为制定病媒控制方法提供信息来影响医学。的 拟议研究的结果将回答长期存在的关于JHA对蚊子作用的问题， 增加我们对JH作用的理解，这可能有助于开发新的，高活性和安全的方法， 控制蚊子和其他导致人类疾病的病原体载体。

Project Summary Adult mosquitoes transmit pathogens that cause deadly diseases in humans. Therefore, preventing adult emergence has been used as a strategy to block transmission of pathogens. Juvenile hormones (JH) prevent metamorphosis and adult emergence; juvenile hormone analogs (JHA) such as methoprene, hydroprene, and pyriproxyfen have been commercialized for controlling mosquitoes and other disease vectors. However, the molecular mode of action of JHAs in killing mosquitoes and other insects remains poorly understood. JHAs are thought to act by preventing larval-pupal metamorphosis. This is precisely how these compounds work in most lepidopteran insects. However, in dipteran insects such as the yellow fever mosquito, Aedes aegypti, JHA application does not prevent larval-pupal metamorphosis; the treated larvae undergo pupal ecdysis and die during the pupal stage. Preliminary studies showed that the pupae developed from methoprene treated larvae die due to the persistence of larval tissues. A transgenic CRISPR/Cas9 genome editing method was employed to knockout Ae. aegypti ortholog of the gene coding for ecdysone induced protein 93F (E93) which is known to regulate programmed cell death (PCD) of larval tissues. The phenotypes of Ae. aegypti E93 knockout animals are like that observed in JHA treated larvae, suggesting that JHA suppresses E93 expression in preventing the death of larval tissues, resulting in pupae containing both larval and pupal tissues leading to their death. This hypothesis will be tested by conducting experiments under two specific aims. In the first specific aim, larval tissues undergoing PCD will be examined to identify similarities and differences between JHA treatment and E93 knockout. The status of PCD in tissues dissected from JHA treated and E93 knockout larvae and pupae will be studied by staining with apoptosis and autophagy marker dyes. In the second specific aim, RNA and ATAC sequencing, RNAi and reporter assays will be employed to identify genes involved in larval tissue remodeling and mechanisms of regulation of their expression by JHA working through E93. The function of E93 acting as a pioneer and an activating transcription factor will be studied. Results from the proposed research will impact medicine by providing information for the development of vector control methods. The results from the proposed studies will answer long-standing questions on JHA action in mosquitoes and increase our understanding of JH action, which could help to develop novel, highly active, and safe methods to control mosquitoes and other vectors of pathogens that cause diseases in humans.