Evolutionary rate covariation identifies new members of a protein network required for Drosophila melanogaster female post-mating responses

Abstract

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.

Figure 1. Proteins in the SP network show a significantly elevated signature of ERC.

This pairwise matrix shows ERC values (above diagonal) and their corresponding empirical p-values (below diagonal) between the seven known members of the SP network. Red shading indicates correlations with empirical p<0.05; more intense shading indicates a stronger correlation.

Figure 2. Fertility assays for new candidate SP network proteins identified by ERC.

Each graph depicts the mean (± SE) number of eggs laid on each day of a 10-day fertility assay (knockdown: KD, dashed line; control: cont, solid line). For each male-expressed gene, knockdown or control males were mated to wild-type females. For each female-expressed gene, wild-type males were mated to knockdown or control females. Knockdown of each gene shown had a highly significant effect (corrected p<10⁻⁶ in all cases) on overall fertility; results of statistical testing for fertility on each day of the assay are shown on each graph. Control data points are offset horizontally from knockdown data points to facilitate comparison, but all flies in each experiment were transferred from one vial to the next at the same time each day. Samples sizes for each treatment range from 11 to 28. One representative biological replicate (out of 2–3 for each gene) is shown.

Figure 3. SP retention in mated females, 4 days after mating.

Western blots probed with antibodies to SP or alpha-tubulin (loading control). Proteins were isolated from lower female reproductive tracts 4 days after mating. Gene names to the left of each pair of blots indicate which gene was (KD) or was not (cont) knocked down in the mating pair. Across all experiments, the number of female reproductive tract (RT) equivalents used for each condition ranged from 13 to 20; however, for any given gene, the number of RT equivalents compared between KD and control was within 2 RTs.

Figure 4. Production, transfer and processing of SP network proteins in males knocked down for aquarius, antares or intrepid.

Western blots were probed with either an antibody to an SP network protein or a loading control. Alpha-tubulin was used as the loading control for blots of CG9997, CG17575 and SP. Since CG1652 and CG1656 sometimes co-migrated with tubuiln, loading controls for these proteins were either a consistently observed cross-reactive band or tubulin. Proteins were isolated from male reproductive tracts (“male” columns) or lower female reproductive tracts dissected 1 hour after the start of mating (“female” columns). “KD” indicates males knocked down for aqrs, antr or intr or females mated to a knockdown male, while “cont” indicates control males or females mated to a control male. Arrows next to the blots for CG9997 indicate the ∼45 (top) and ∼36-kDa (bottom) forms of the protein . Within each blot, the amount of RT equivalents loaded for each sex was equal. Across blots, male lanes contain 0.5–1 RT equivalents; female lanes contain 2–4 RT equivalents.

Figure 5. Average number of sperm stored in the seminal receptacles (SR) of wild-type females mated to knockdown or control males for new SP network proteins.

Average number of sperm in female SRs at 2(A) or 10 days (B) after mating to aqrs, antr or intr knockdown (KD, gray) or control (cont, black) males. Each bar indicates the mean; error bars indicate 1 standard error. *, p<0.01; **, p<0.002; n.s. = not significant. Samples sizes for each treatment range from 11 to 18.

Figure 6. An expanded network of proteins is required for SP to bind sperm and to be utilized in mated females.

(A) The SP network. Colors of protein names indicate predicted protein functional classes: red = protease or protease homolog; green = cysteine-rich secretory protein (CRISP); dark blue = C-type lectin; light blue oval = SP; purple = unknown function. Boxes indicate proteins discovered by ERC; other proteins were described previously , . Intrepid acts upstream of SP-SPR signaling, but at present we cannot position it further. (B) New members of the SP network function at steps consistent with their signals of ERC. New network proteins are shown in rows; known network proteins are shown in columns. Each cell indicates the empirical p-value associated with the protein's pair ERC value. P-values less than 0.05 are shaded in red; more intense shading indicates a stronger correlation.