There is extensive evidence from a wide range of taxa that supports sperm competition theory, including studies that show males can adapt to sperm competition by producing more competitive ejaculates. Several traits, including sperm concentration and velocity, influence ejaculate quality by altering male reproductive success when sperm compete. Emerging evidence shows that males can strategically alter ejaculate quality in response to cues, such as the presence of a male competitor, that signal changed sperm competition risk. However, when rapid adjustments to ejaculates occur it is unclear whether changes to sperm performance are due to changes in seminal fluid composition and/or the production of new sperm. Furthermore, if changing sperm performance is mediated by seminal fluid, the mechanisms underlying the way sperm and seminal fluid interact in most taxa are poorly understood. Here, I used a series of experiments on an externally fertilising fish, chinook salmon (Oncorhynchus tshawytscha), a species with a dynamic social environment in which males adopt alternative reproductive tactics that exposes them to different sperm competition risk. Overall, my aim was to determine whether male chinook salmon can make rapid strategic adjustments to ejaculate quality, and to further understand the proximate mechanisms behind such adjustments, focusing on sperm velocity - a key trait that impacts reproductive success in salmonids.

In Chapter two, I report results from a comprehensive series of experiments on fully grown “hooknose” males. Using a two-stage social status manipulation, I tested whether ejaculate quality traits respond rapidly to changes in sperm competition risk. I then used in-vitro ejaculate manipulations to determine if changes to sperm velocity are mediated by seminal fluid. Finally, using in-vitro fertilisation trials in which sperm compete under simulated natural spawning conditions, I assessed the paternity share of males using both manipulated and unmanipulated ejaculates. I found that both sperm number and velocity were linked to social position; subdominant males that have greater sperm competition risk produced higher quality ejaculates compared to dominant males. Furthermore, males changing from dominant to subdominant status responded to this increase in sperm competition risk by producing ejaculates with faster swimming sperm within 48 hours. By manipulating ejaculates, I found that rapid changes in sperm velocity were mediated by seminal fluid and found that the effect of seminal fluid on sperm velocity directly impacted paternity share and therefore reproductive success.

Chapter two provides compelling support for seminal fluid having “quality” driven effects that influence sperm from all males similarly rather than a “targeted” negative effect on rival male sperm. However, two studies have reported such targeted effects in externally fertilising fish, but both compared averages across treatment groups. In Chapter three, I test the hypothesis that support for quality driven effects is only apparent when relative sperm velocity between males in each pair is considered. I tested this hypothesis using an experiment that manipulated ejaculates from male chinook salmon with different life-histories, “hooknose” males and early maturing “precocious” males. Although comparison of averages across treatment groups suggested targeted effects on rival sperm, the alternate approach found a significant correlation between relative sperm velocity and changes in sperm velocity caused by rival seminal fluid. These results further support a quality driven rather than targeted effect and provide further information about the possible proximate mechanism of sperm and seminal fluid interaction.

To gain further insight on the potential mechanism of sperm and seminal fluid interaction identified in chapters two and three, I used proteomic methods to characterise the seminal fluid proteome in chinook salmon. Growing evidence for invertebrate species shows that seminal fluid proteins (SFPs) have evolved key functional roles in sperm competition. However, relatively little is known about SFPs in vertebrate species. Chapters four and five explore the chinook salmon seminal fluid proteome, using seminal fluid collected during the social status manipulation experiment in chapter two and a combination of pre-fractionation techniques followed with analysis by mass spectrometry. In Chapter four, the chinook salmon seminal fluid proteome is described and compared to the three previous proteomic studies in teleost fish. To identify candidate SFPs that may be linked to the underlying mechanism, in Chapter five I assess the correlation between SFP abundance, male social status and ejaculate quality traits. I show that SFP composition is influenced by social status, and identify several SPFs correlated with sperm velocity and sperm concentration that are part of energy metabolism, defence and signalling pathways previously shown to influence sperm function. In summary, using a series of behavioural manipulation, ejaculate manipulation, in vitro sperm competition experiments and proteomic analyses on chinook salmon males with alternative reproductive tactics, I provide unequivocal evidence that sperm competition risk drives patterns of investment in ejaculate quality. Furthermore, I show that adjustment of sperm velocity, a key trait determining ejaculate competitiveness and impacting male reproductive success in salmonids, occurs via investment in seminal fluid. My results provide support that males invest in high quality seminal fluid that affects the velocity of sperm from all males similarly, rather than targeting and reducing the velocity of sperm from rival males. I also provide a detailed analysis of the chinook salmon seminal fluid proteome and identify a candidate list of SFPs associated with ejaculate quality, that will be critical for the identification of proximate mechanisms underlying sperm and seminal fluid interactions that influence male reproductive success. These combined results represent a significant advance in our understanding of post-copulatory sexual selection and the evolution of adaptations to sperm competition risk.