Molecular mode of action of Juvenile hormone analogs

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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.