Divergence of perivitelline fluid proteins in association with the change in egg deposition environment in apple snails: A transcriptomic and proteomic approach

Apple snails of the family Ampullariidae exhibit unusually diverse reproductive strategies: while some genera deposit eggs underwater, other genera deposit eggs above-the-water surface. Remarkably, the most derived taxa adopting aerial oviposition, especially Pomacea, display a wide array of egg shape and color among species. The shift from aquatic to aerial egg deposition must have been a difficult one, as evidenced by the dearth of such transitions in evolutionary history. Aerially deposited eggs of Pomacea are brightly colored and filled with nutritious polysaccharides and proteins. However, their eggs have very few predators and the embryos can develop under the strong heat and UV radiation of tropical areas. Our previous studies on the three most abundant lipoglycoprotein complexes from the egg perivitelline fluid (PVF) of the invasive snail Pomacea canaliculata have shown that they play critical roles in the embryonic development: provision of nutrients, protection from UV radiation, and unusual defenses against predation by a neurotoxin and a protease inhibitor. Using transcriptomics and proteomics, we have identified over 50 other proteins from the PVF of P. canaliculata, revealing many other potential roles for these reproductive proteins. We found that gene duplication was involved in the formation of these PVF proteins. Based on these, we propose a transcriptomic and proteomic approach to discover the PVF proteins from key basal and derived ampullariid genera/species exhibiting different traits such as egg colors and oviposition strategies, followed by a structure-function study of candidate proteins. To facilitate protein discovery, the transcriptome of each species will be sequenced and assembled, followed by proteomic analysis to identify and confirm the PVF proteins. Genes encoding PVF proteins will be cloned, and orthologs and paralogs will be analyzed to test alternative hypotheses regarding the mechanisms of their divergence. Functional characterization will then be conducted on selected PVF proteins to establish a link between their sequence divergence and acquisition of cellular function. Our specific aims are to discover the PVF proteins, identify the regions of genes and mechanisms that are responsible for the diversity of these proteins, and ultimately understand the relationship between PVF protein diversification and the contrasting egg-laying strategies of these apple snails. These results will contribute significantly to a better understanding of the evolution of proteins and protein families to facilitate the switch from underwater to aerial egg deposition, a transition that has occurred very rarely in the whole animal kingdom.