Seminal fluid proteins (SFPs) are crucial mediators of sexual selection and sexual conflict. Recent studies have chiefly focused on environmentally induced plasticity as one source of variation in SFP expression, particularly in response to differing sperm competition levels. However, understanding the evolution of a trait in heterogenous environments requires estimates of both environmental and genetic sources of variation, as well as their interaction. Therefore, we investigated how environment (specifically mating group size, a good predictor of sperm competition intensity), genotype and genotype-byenvironment interactions affect seminal fluid expression. To do so, we reared 12 inbred lines of a simultaneously hermaphroditic flatworm Macrostomum lignano in groups of either two or eight worms and measured the expression levels of 58 putative SFP transcripts. We then examined the source of variation in the expression of each transcript individually and for multivariate axes extracted from a principal component analysis. We found that mating group size did not affect expression levels according to the single transcript analyses, nor did it affect the first principal component (presumably representing overall investment in seminal fluid production). However, mating group size did affect the relative expression of different transcripts captured by the second principal component (presumably reflecting variation in seminal fluid composition). Most transcripts were genetically variable in their expression level and several exhibited genotype-byenvironment interactions; relative composition also showed high genetic variation. Collectively, our results reveal the tightly integrated nature of the seminal fluid transcriptome and provide new insights into the quantitative genetic basis of seminal fluid investment and composition.

Highlights d Suckless-1 and suckless-2 are putative seminal fluid transcripts d Their expression is negatively genetically correlated with partner suck propensity d Knockdown of either transcript increases the likelihood of sucking after mating d They may therefore mediate sexual conflict over ejaculate fate

Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra‐ and interspecific sequence information, along with genomics and functional data, we examine the molecular evolution of approximately 300 SFPs in Drosophila. We found that 50–57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7–12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35–37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. Our results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution.

Sperm competition commonly occurs whenever females mate multiply, leading to variation in male paternity success. This can be due to variation in the various traits that might affect sperm competitive ability, which itself depends on both genetic and environmental factors, as well as on genotype‐by‐environment interactions (GEI). Seminal fluid is a major component of the male ejaculate that is often expected to mediate sperm competition, where different genotypes can differ in their seminal fluid expression as a response to different levels of sperm competition (i.e. exhibit GEI). We therefore here focussed on testing for GEI in expression of two recently identified seminal fluid transcripts, suckless‐1 and suckless‐2, which potentially modulate sperm competitive ability in the simultaneously hermaphroditic flatworm Macrostomum lignano via their effects on manipulating post‐mating partner behaviour and ultimately the fate of transferred ejaculates. In addition, we sought to test for GEI in sperm competitive ability in a standardized sperm competition (P1 and P2) assay, to investigate the relationship between natural variation in the expression of these seminal fluid transcripts generated through GEI and relative paternity success. We found GEI for the expression level of suckless‐1 and suckless‐2, as well as for sperm competitive ability. Moreover, we found a positive relation between the expression of suckless‐1 and relative paternity success (P1). This suggests that natural variation in the expression of this seminal fluid transcript indeed can influence sperm competition outcomes in M. lignano.

Background The rapid evolution of seminal fluid proteins (SFPs) has been suggested to be driven by adaptations to postcopulatory sexual selection (e.g. sperm competition). However, we have recently shown that most SFPs evolve rapidly under relaxed selective pressures. Given the role of SFPs in competition for fertilization phenotypes, like the ability to transfer and store sperm and the modulation of female receptivity and ovulation, the prevalence of selectively relaxed SFPs appears as a conundrum. One possible explanation is that selection on SFPs might be relaxed in terms of protein amino acid content, but adjustments of expression are essential for post-mating function. Interestingly, there is a general lack of systematic implementation of gene expression perturbation assays to monitor their effect on phenotypes related to sperm competition. Results We successfully manipulated the expression of 16 SFP encoding genes using tissue-specific knockdowns (KDs) and determined the effect of these genes’ perturbation on three important post-mating phenotypes: female refractoriness to remating, defensive (P1), and offensive (P2) sperm competitive abilities in Drosophila melanogaster. Our analyses show that KDs of tested SFP genes do not affect female refractoriness to remating and P2, however, most gene KDs significantly decreased P1. Moreover, KDs of SFP genes that are selectively constrained in terms of protein-coding sequence evolution have lower P1 than KDs of genes evolving under relaxed selection. Conclusions Our results suggest a more predominant role, than previously acknowledged, of variation in gene expression than coding sequence changes on sperm competitive ability in D. melanogaster.

The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post‐mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partner's (sperm recipient's) post‐copulatory behaviour to prevent or delay re‐mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioural data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs—Mlig‐pro46 and Mlig‐pro63—linked to both mating frequency and ‘suck’ frequency, a distinctive behaviour, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference‐induced knockdown to ask how the loss of Mlig‐pro46 and Mlig‐pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown of Mlig‐pro63 resulted in a significantly decreased suck propensity of mating partners. This suggests that Mlig‐pro63 may normally act as a cue in the ejaculate to trigger recipient suck behaviour and—given that other proteins in the ejaculate have the opposite effect—could be one component of an ongoing arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig‐pro46 and Mlig‐pro63 from a donor perspective remains enigmatic.

It has long been acknowledged that changes in the regulation of gene expression may account for major organismal differences. However, we still do not fully understand how changes in gene expression evolve and how do such changes influence organisms’ differences. We are even less aware of the impact such changes might have in restricting gene flow between species. Here, we focus on studies of gene expression and speciation in the <i>Drosophila</i> model. We review studies that have identified gene interactions in post-mating reproductive isolation and speciation, particularly those that modulate male-gene expression. We also address studies that have experimentally manipulated changes in gene expression to test their effect in post-mating reproductive isolation. We highlight the need for a more in-depth analysis of the role of selection causing disrupted gene expression of such candidate genes in sterile/inviable hybrids. Moreover, we discuss the relevance to incorporate more routinely assays that simultaneously evaluate the potential effects of environmental factors and genetic background in modulating plastic responses in male genes and their potential role in speciation.

Sperm competition commonly occurs whenever females mate multiply, leading to variation in male paternity success. This can be due to variation in the various traits that might affect sperm competitive ability, which itself depends on both genetic and environmental factors, as well as on genotype‐by‐environment interactions (GEI). Seminal fluid is a major component of the male ejaculate that is often expected to mediate sperm competition, where different genotypes can differ in their seminal fluid expression as a response to different levels of sperm competition (i.e. exhibit GEI). We therefore here focussed on testing for GEI in expression of two recently identified seminal fluid transcripts, suckless‐1 and suckless‐2, which potentially modulate sperm competitive ability in the simultaneously hermaphroditic flatworm Macrostomum lignano via their effects on manipulating post‐mating partner behaviour and ultimately the fate of transferred ejaculates. In addition, we sought to test for GEI in sperm competitive ability in a standardized sperm competition (P1 and P2) assay, to investigate the relationship between natural variation in the expression of these seminal fluid transcripts generated through GEI and relative paternity success. We found GEI for the expression level of suckless‐1 and suckless‐2, as well as for sperm competitive ability. Moreover, we found a positive relation between the expression of suckless‐1 and relative paternity success (P1). This suggests that natural variation in the expression of this seminal fluid transcript indeed can influence sperm competition outcomes in M. lignano.