Evolutionary quantitative proteomics of reproductive interactions in Drosophila

Abstract

Reproductive traits evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of male and female reproductive proteins and how their interaction mediates fertilisation success. Species in the Drosophila virilis clade exhibit interspecific postmating prezygotic reproductive isolation, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Here we present a novel method to simultaneously identify and quantify transferred male ejaculate proteins and the female reproductive proteome using multiplexed Tandem-Mass-Tag (TMT) isobaric labelling of the lower female reproductive tract before and immediately after mating in three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species. We also identified over 2000 proteins that provide the first description of the female reproductive tract proteome outside of the D. melanogaster group. The female reproductive tract proteome shows significant divergence between species, especially female-specific serine-type endopeptidases, which showed differential abundance between species and elevated rates of molecular evolution that is similar to that of male seminal fluid proteins. We also found that only ~1/3 of male ejaculate proteins are likely produced in the accessory glands and code for a signal peptide sequence. Finally, we assessed the utility of species-specific compared to single species query databases for protein identification and quantification from multiple species samples. Our results show that using species-specific query databases dramatically improves protein identification and quantification.