Summary Post-copulatory sexual selection can select for sperm allocation strategies in males [1, 2] but males should also strategically allocate non-sperm components of the ejaculate [3, 4] such as seminal fluid proteins (Sfps). Sfps can influence the extent of post-copulatory sexual selection [5–7] but little is known of the causes or consequences of quantitative variation in Sfp production and transfer. Using Drosophila melanogaster, we demonstrate that Sfps are strategically allocated to females in response to the potential level of sperm competition. We also show that males who can produce and transfer larger quantities of specific Sfps have a significant competitive advantage. When males were exposed to a competitor male, matings were longer and more of two key Sfps, sex peptide [8] and ovulin [9], were transferred, indicating strategic allocation of Sfps. Males selected for large accessory glands (AGs, a major site of Sfp synthesis) produced and transferred significantly more sex peptide, but not more ovulin. Large AG males also had significantly increased competitive reproductive success. Our results show that quantitative variation in specific Sfps is likely to play an important role in post-copulatory sexual selection and that investment in Sfp production is essential for male fitness in a competitive environment.

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We assessed the extent to which traits related to ejaculate investment have evolved in lines of Drosophila melanogaster that had an evolutionary history of maintenance at biased sex ratios. Measures of ejaculate investment were made in males that had been maintained at male-biased (MB) and female-biased (FB) adult sex ratios, in which levels of sperm competition were high and low, respectively. Theory predicts that when the risk of sperm competition is high and mating opportunities are rare (as they are for males in the MB populations), males should increase investment in their few matings. We therefore predicted that males from the MB lines would (1) exhibit increased investment in their first mating opportunities and (2) deplete their ejaculates at a faster rate when mating multiply, in comparison to FB males. To investigate these predictions we measured the single mating productivity of males from three replicates each of MB and FB lines mated to five wild-type virgin females in succession. In contrast to the first prediction, there was no evidence for differences in productivity between MB and FB line males in their first matings. The second prediction was upheld: mates of MB and FB males suffered increasingly reduced productivity with successive matings, but the decline was significantly more pronounced for MB than for FB males. There was a significant reduction in the size of the accessory glands and testes of males from the MB and FB regimes after five successive matings. However, the accessory glands, but not testes, of MB males became depleted at a significantly faster rate than those of FB males. The results show that male reproductive traits evolved in response to the level of sperm competition and suggest that the ability to maintain fertility over successive matings is associated with the rate of ejaculate, and particularly accessory gland, depletion.

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.

Theory predicts that males adapt to sperm competition by increasing their investment in testis mass to transfer larger ejaculates.Experimental and comparative data support this prediction. Nevertheless, the relative importance of sperm competition in testis size evolution remains elusive, because experiments vary only sperm competition whereas comparative approaches confound it with other variables, in particular male mating rate. We addressed the relative importance of sperm competition and male mating rate by taking an experimental evolution approach. We subjected populations of Drosophila melanogaster to sex ratios of 1:1, 4:1, and 10:1 (female:male). Female bias decreased sperm competition but increased male mating rate and sperm depletion. After 28 generations of evolution, males from the 10:1 treatment had larger testes than males from other treatments. Thus, testis size evolved in response to mating rate and sperm depletion, not sperm competition. Furthermore, our experiment demonstrated that drift associated with sex ratio distortion limits adaptation; testis size only evolved in populations in which the effect of sex ratio bias on the effective population size had been compensated by increasing the numerical size. We discuss these results with respect to reproductive evolution, genetic drift in natural and experimental populations, and consequences of natural sex ratio distortion.