We used discovery, bottom up proteomics to provide the first in-depth accessory gland proteome in D. pseudoobscura. Computational bioinformatics identified Ͼ500 proteins in the secretory pathway of which 163 were annotated as extracellular., and therefore candidate, seminal fluid proteins. We further compared molecular rates of evolution between intra-and extracellular proteins, showing a hierarchy of rapid evolution with putative seminal fluid proteins evolving more rapidly than other secreted proteins, and those proteins evolving more rapidly than intra-cellular proteins. Graphical Abstract Highlights• First deep proteomic coverage of an accessory gland proteome in Drosophila.• Discovery proteomics identified Ͼ3000 proteins of the D. pseudoobscura accessory gland proteome.• Identified 132 putative novel seminal fluid proteins in this species.• Demonstrated the exoproteome as the most rapidly evolving subcellular component of the proteome.
Despite holding a central role in fertilization, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis‐signalling between the male ejaculate and the female during and after mating resulting in postmating prezygotic (PMPZ) isolation between populations. Crosses between Drosophila montana populations show PMPZ isolation in the form of reduced fertilization success in both noncompetitive and competitive contexts. Here we test whether male ejaculate proteins produced in the accessory glands or ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. We find more than 150 differentially abundant proteins between populations that may contribute to PMPZ isolation, including a number of proteases, peptidases and several orthologues of Drosophila melanogaster Sfps known to mediate fertilization success. Males from the population that elicit the stronger PMPZ isolation after mating with foreign females typically produced greater quantities of Sfps. The accessory glands and ejaculatory bulb show enrichment for different gene ontology (GO) terms and the ejaculatory bulb contributes more differentially abundant proteins. Proteins with a predicted secretory signal evolve faster than nonsecretory proteins. Finally, we take advantage of quantitative proteomics data for three Drosophila species to determine shared and unique GO enrichments of Sfps between taxa and which potentially mediate PMPZ isolation. Our study provides the first high‐throughput quantitative proteomic evidence showing divergence of reproductive proteins between populations that exhibit PMPZ isolation.
word count: 270 23 24 Total word count: 5085 25 2 ABSTRACT 26 Despite holding a central role for fertilisation success, reproductive traits often show elevated 27 rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within 28populations is predicted to cause mis-signalling between the male ejaculate and female 29 reproductive tract between populations resulting in postmating prezygotic (PMPZ) isolation. 30Crosses between populations of Drosophila montana show PMPZ isolation in the form of 31 reduced fertilisation success in both noncompetitive and competitive contexts. Here we test 32 whether male ejaculate proteins deriving from either the accessory glands or the ejaculatory 33 bulb differ between populations using liquid chromatography tandem mass spectrometry. We 34 find more than 150 differentially abundant proteins between populations which may contribute 35 to PMPZ isolation. These proteins include a number of proteases and peptidases, and several 36 orthologs of D. melanogaster Sfps, all known to mediate fertilisation success and which mimic 37 PMPZ isolation phenotypes. Males of one population typically produced greater quantities of 38 Sfps and the strongest PMPZ isolation occurs in this direction. The accessory glands and 39 ejaculatory bulb have different functions and the ejaculatory bulb contributes more to 40 population differences than the accessory glands. Proteins with a secretory signal, but not 41 Sfps, evolve faster than non-secretory proteins although the conservative criteria used to 42 define Sfps may have impaired the ability to identify rapidly evolving proteins. We take 43 advantage of quantitative proteomics data from three Drosophila species to determine shared 44 and unique functional enrichments of Sfps that could be subject to selection between taxa and 45 subsequently mediate PMPZ isolation. Our study provides the first high throughput quantitative 46 proteomic evidence showing divergence of reproductive proteins implicated in the emergence 47 IMPACT SUMMARY (word count: 300) 51Identifying traits that prevent successful interbreeding is key to understanding early stages of 52 the formation of new species, or speciation. Reproductive isolation arising prior to and during 53 fertilisation frequently involves differences in how the sexes interact. In internally fertilising taxa, 54 such interactions are mediated between the female reproductive tract where fertilisation occurs 55 and the receipt of the ejaculate necessary for fertilisation. Because ejaculate proteins are at 56 least partially responsible for these interactions, differences in male ejaculate protein 57 composition could negatively impact fertilisation success, generating reproductive isolation. 58While the biological classes of ejaculate proteins are shared across all animal taxa, proteins 59 that are secreted by males tend to show rapid evolution in gene expression and genetic 60 sequence. Thus, reproductive proteins are suggested as prime targets facilitating reproductive 61 iso...
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