Improving Anopheline fitness and resistance through fat body insulin signaling

AbstractGenetically modified mosquitoes resistant to Plasmodium development have been proposed as an alternativestrategy to reduce malaria transmission. Several proof-of-principle studies have validated the idea thatgenetically modified mosquitoes can be refractory to the malaria parasite development, without incurringsignificant fitness loads. Recently, signaling cascades have been exploited to manipulate both parasiteresistance and fitness. For example, manipulation of the insulin/insulin growth factor 1 signaling (IIS) pathwayhas been shown to confer Plasmodium resistance when up or down regulated via unique mechanisms, andextended lifespan when downregulated. However, the impact of IIS in other mosquito tissues has not beenexplored as extensively. The fat body of mosquitoes and other model invertebrates performs a number offunctions, including storage of nutrients, production of yolk proteins and the synthesis of antimicrobial peptides.IIS has been implicated in these fat body processes leading to control of immunity, lifespan, metabolism, andreproduction. We previously generated a transgenic Anopheles stephensi line with increased insulin signalingin the peripheral fat body. Surprisingly, these transgenic mosquitoes survived significantly longer than theirnon-transgenic siblings, while in nearly every other organism and tissue increased IIS leads to a decrease inlifespan. To define how fat body IIS controls lifespan and to determine the impact fat body IIS has onreproduction, nutrient metabolism and Plasmodium resistance we will complete the following studies. Work inDrosophila suggest that fat body insulin signaling suppresses the expression of neuronal insulin-like peptides(ILPs) leading to increased lifespan. Thus, we will first examine transcript and peptide expression patterns ofkey AsILPs and knockdown putative AsILP targets via RNAi or Crispr knockout to validate the link betweenILPs and lifespan extension. Our transgenic mosquito line also synthesized significantly more yolk protein thannon-transgenic controls, although this did not translate into increased egg production during the first twogonotrophic cycles. Therefore, we will next conduct lifetime fecundity assays and development assays on theprogeny to determine if an increase in lifetime reproductive fitness occurs. Third, due to the critical and wellestablished role of IIS and the fat body on nutrient metabolism we will also quantify lipid, glycogen, glucoseand trehalose levels at various physiological stages. Finally, the fat body is a key immune tissue regulating theproduction of anti-microbial peptides. As such we will assess the expression patterns of key immune genesand challenge transgenic mosquitoes with the most important human malaria parasite, Plasmodium falciparum.By the end of this project we will have a solid understanding of how fat body insulin signaling affects a range ofphysiologies impacting mosquito fitness and parasite resistance and will have developed new tools to generatehighly fit Anopheles stephensi mosquitoes resistant to Plasmodium falciparum parasites.