Molecular mode of action of Juvenile hormone analogs

项目摘要 成年蚊子传播的病原体会导致人类患上致命的疾病。因此，防止成人 出现已被用作阻止病原体传播的一种策略。保幼激素(JH)预防 变态和成虫羽化；保幼激素类似物(JHA)，如甲基戊二烯、氢戊烯和 吡虫草醚已被商业化，用于控制蚊子和其他病媒。然而， JHAS在杀死蚊子和其他昆虫方面的分子作用模式仍然知之甚少。JHAS是 被认为是通过防止幼虫-幼虫蜕变而起作用的。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，在双翅目昆虫中，如黄热病蚊子、埃及伊蚊、Jha 施药不能阻止幼虫-蛹的变态；处理的幼虫会经历幼虫蜕皮并死亡 在蛹阶段。初步研究表明，蛹是由甲氧基丁二烯处理过的幼虫发育而来的 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 击倒艾。埃及伊蚊编码蜕皮酮诱导蛋白93F(E93)基因的同源基因，已知 调节幼虫组织程序性细胞死亡(PCD)。Ae.埃及伊蚊e93基因敲除动物 与在JHA处理的幼虫中观察到的一样，表明JHA抑制e93的表达，从而防止 幼虫组织死亡，导致同时含有幼虫和蛹组织的蛹死亡。这 假设将通过在两个特定目标下进行实验来检验。在第一个特定目标中，幼虫 将对接受PCD的组织进行检查，以确定JHA治疗和JHA治疗的异同 E93淘汰赛。经JHA处理和E93基因敲除的幼虫和蛹组织中PCD的状态 将通过细胞凋亡和自噬标记染料染色进行研究。在第二个具体目标中，RNA和 将使用ATAC测序、RNAi和报告分析来识别与幼虫组织有关的基因 JHA作用于e93的重塑及其表达调控机制。的功能 将研究E93作为先驱和激活转录因子的作用。建议的结果 研究将通过为媒介控制方法的发展提供信息来影响医学。这个 拟议研究的结果将回答长期以来关于JHA在蚊子和 增加我们对JH ACTION的理解，这有助于开发新的、高度活跃的和安全的方法来 控制蚊子和其他导致人类疾病的病原体媒介。

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.