Up to 40% of the world population now live in areas where dengue mosquito vectors coexist with humans. Aedes aegypti are vectors for zoonotic diseases that affect hundreds of millions of individuals per year globally. We recently identified the eggshell organizing factor 1 (EOF1) protein using systematic RNA interference (RNAi) screening of mosquito lineage-specific genes. It was shown that eggs deposited by RNAi-EOF1 A. aegypti and A. albopictus mosquitoes were non-melanized, fragile, and contained nonviable embryos. Motivated by this discovery, we performed RNAi screening of eggshell proteins to determine putative downstream target proteins of intracellular EOF1. We identified several eggshell proteins as essential for eggshell formation in A. aegypti and characterized their phenotypes in detail by 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 mRNA expression is highly upregulated in response to blood feeding. Furthermore, we identified four secreted eggshell enzymes as important factors for controlling the processes of mosquito eggshell formation and melanization. These enzymes included three dopachrome converting enzymes and one cysteine protease. All eight characterized eggshell proteins were required for intact eggshell formation. However, their surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1. Still, it remains unclear how EOF1 influences eggshell formation and melanization. The use of proteomic analysis of eggshell proteins from RNAi-EOF1 assisted in the identification of additional proteins that could be regulated in EOF1 deficient eggshells.

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