Characterization of essential eggshell proteins from Aedes aegypti mosquitoes

Background Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti , coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. Results In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. Conclusions While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies.