Female novelty and male status dynamically modulate ejaculate expenditure and seminal fluid proteome over successive matings in red junglefowl

Abstract

Theory predicts that males will strategically invest in ejaculates according to the value of mating opportunities. While strategic sperm allocation has been studied extensively, little is known about concomitant changes in seminal fluid (SF) and its molecular composition, despite increasing evidence that SF proteins (SFPs) are fundamental in fertility and sperm competition. Here, we show that in male red junglefowl, Gallus gallus, along with changes in sperm numbers and SF investment, SF composition changed dynamically over successive matings with a first female, immediately followed by mating with a second, sexually novel female. The SF proteome exhibited a pattern of both protein depletion and enrichment over successive matings, including progressive increases in immunity and plasma proteins. Ejaculates allocated to the second female had distinct proteomic profiles, where depletion of many SFPs was compensated by increased investment in others. This response was partly modulated by male social status: when mating with the second, novel female, subdominants (but not dominants) preferentially invested in SFPs associated with sperm composition, which may reflect status-specific differences in mating rates, sperm maturation and sperm competition. Global proteomic SF analysis thus reveals that successive matings trigger rapid, dynamic SFP changes driven by a combination of depletion and strategic allocation.

Figure 1

Patterns of ejaculate investment over a mating sequence. (a) Average number of sperm (Log-transformed, standardised against maximum number of sperm for each male) over the mating sequence comprising the first five matings with the first female (empty data points), and the 6^th mating with a novel, second female (filled data point). (b) The average volume of seminal fluid (SF) allocated by males across the six mating opportunities. (c) The average proportion of SF protein present in the ejaculates produced over the mating sequence. Vertical bars represent SEM. Note that (a) and (b) include mating opportunities that resulted in no ejaculation, thus capturing both male mating propensity and ejaculate investment when mating occurred (see Figs S1–S3 for additional information).

Figure 2

Seminal fluid proteome dynamics across successive matings. (A) Hierarchical clustering analysis. SF samples across four successive matings. Proteins were organized by dendrogram order, with the distance matrix computed using the euclidean method. AU (Approximately Unbiased) (red) p-value and BP (Bootstrap Probability) (green) values were computed by multiscale bootstrap resampling. (B) Fuzzy clustering analysis was conducted to identify global patterns of protein abundance differences that are associated with the mating sequence. Protein abundance patterns relative to the cluster average are depicted for each protein (purple: high identity; green: lower identity). Note: For simplicity, only one of two clusters exhibiting a pattern of protein depletion is displayed.

Figure 3

Seminal fluid proteome dynamics in dominant and subdominant males. (A) Hierarchical clustering analysis. SF samples across four successive and proteins were organized by dendrogram order, with the distance matrix computed using the euclidean method. Approximately Unbiased p-value (red) and Bootstrap probability (green) value were computed by multiscale bootstrap resampling. (B) Principal component analysis of SF samples from dominant and subdominant males across four successive matings. Principal components 2 and 3 are displayed. (C) Piano analysis displays enriched functional groups of proteins that differ in abundance between the SF sample of dominant and subdominant males when mated to the second, novel female. Node size is proportional to the total number of proteins in each functional category (total number of proteins in the comparison = 746).