Manipulation of insulin signaling in the fat body and ovaries of mosquitoes to regulate reproduction

Mosquito reproduction is regulated by a complex series of hormonal cues. In the ovaries and fat body the insulin/IGF1 signaling (IIS) cascade regulates steroidogenesis and vitellogenesis respectively. Phosphotase and tensin homolog (PTEN) is a direct antagonist of IIS. Using RNAi, knockdown of AaegPTEN or its splice variant AaegPTEN6 resulted in a 15-63% increase in egg production with no effect on egg viability during the first reproductive cycle. Knockdown of aother splice variant, AaegPTEN3, did not effect reproduction. However, RNAi has limitations. The dsRNA injections resulted in knockdown of AaegPTEN in both fat body and ovaries making it impossible to elucidate whether the ovaries, fat body, or both tissues are responsible for the increased egg production. Also, the transient nature of RNAi knockdown prevents us from determining whether increased reproduction occurs throughout the mosquito lifespan or whether there is a life history change to earlier reproduction. To address these questions we are generating transgenic mosquitoes with either increased or decreased IIS in the fat body and ovary. We have successfully engineered transgenic lines using the vitellogenin promoter linked to an insulin signaling inhibitor, PTEN, or to its activator, Akt. We have characterized how manipulating IIS specifically in fat body impacts mosquito lifespan and egg production during multiple reproductive cycles. We have also identified a putative ovary specific promoter expressed in the ovarian follicle cells where the IIS cascade regulates steroidogenesis. We have linked this promoter to an IIS cascade activator and are generating transgenic lines with modified IIS in the ovaries.