Proteomics Reveals Novel Drosophila Seminal Fluid Proteins Transferred at Mating

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Abstract
Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction. Across many species, males transfer both sperm and seminal proteins to their mates. These proteins increase male reproductive success by improving sperm competitive ability and modifying female behavior. In Drosophila, seminal proteins increase female rates of egg-laying and sperm storage and reduce a female's willingness to mate with subsequent suitors. Several male seminal proteins have been extensively characterized, and others have been predicted based on gene expression patterns, yet the full set of proteins that is transferred to females has not been defined. Here we introduce a new proteomic method that identifies transferred seminal proteins in recently mated females and quantifies their relative abundance. We confirm many of the predicted seminal proteins and discover a number of novel seminal fluid components. Some of these proteins show elevated rates of evolution, consistent with their involvement in sexual selection or sexual conflict, and many have arisen by tandem gene duplication. By using this method in three species of Drosophila, we identified lineage-specific components of seminal fluid. Additionally, we developed and validated a method to identify completely new genes in the D. melanogaster genome. These transferred proteins are now targets for follow-up genetic, biochemical, and evolutionary analysis.