Female reproductive fluid and post-mating sexual selection in two fish species with external fertilization

Post-mating sexual selection is the consequence of polyandry, a taxonomically widespread phenomenon that causes the ejaculates of different males to overlap and compete to fertilize the same set of eggs. Sperm competition has been traditionally considered the post-mating equivalent of male competition, and hence regarded as an intra-sexual process. However, the arena in which sperm competition occurs is largely shaped by the female reproductive environment, enabling females to interfere with, and potentially control, the outcome of sperm competition, a process known as cryptic female choice. The female reproductive fluid (FRF) has recently been identified as a mediator of cryptic female choice in both internal and external fertilizers, for its ability to differentially influence ejaculates of different males and bias fertilization towards specific partners. In this thesis, to gain a broad perspective of the role of FRF in post-mating sexual selection, I employ two externally fertilizing fish species with different reproductive systems: the zebrafish Danio rerio and the grass goby Zosterisessor ophiocephalus. In the zebrafish both pre- and post-mating competition between males (and ejaculates) are rather common, often resulting in multiple paternity in the clutches and limited female pre-mating choice. The grass goby is characterised by territorial-sneaker male reproductive tactics and a strong female pre-mating preference towards territorials, hampered by sneakers attempts to parasitize the spawning events, leading to intense sperm competition and multiple paternity in the clutches. In both species, cryptic female choice is therefore expected to play a key role in determining the reproductive outcome, enabling females to gain some control over the fertilization process in case of the zebrafish, and to reinforce their pre-mating preference for territorial males in case of the grass goby. The research conducted with the zebrafish uncovered novel proximate mechanisms of FRF role in post-mating sexual selection, acting on the gametes of both sexes. The results of paper I revealed for the first time the potential of FRF to increase multiple paternity in the offspring by prolonging the egg fertilization window, thus expanding the opportunities for sperm competition and for exerting cryptic female choice. The mechanism by which females of the zebrafish influence sperm competition through their FRF was investigated in paper II, where the variation in sperm velocity caused by FRF was found to directly predict the change in paternity share between rival males from water to FRF, providing the first evidence of the direct association in direction and magnitude of FRF phenotypic and fitness effects. With the grass goby, I explored the potential for a FRF-mediated directional cryptic choice towards the territorial phenotype, by investigating the previously unexplored effects of FRF concentration and interaction with male seminal fluid. The findings of paper III revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm performance and a permissive directional choice, favouring sperm depending on their proximity to the eggs during spawning. Such a mechanism was found to favour territorial males, but likewise advantage sperm of those sneakers able to get close to the eggs, allowing flexibility in the direction of female post-mating choice. Finally, in paper IV, our findings revealed a differential effect of FRF, mediated by its interaction with male seminal fluid, on the fertilization success of the different tactics, favouring territorial ejaculates, thus enabling females to reinforce their pre-mating choice. Ultimately, the combined results of this thesis, provide a key indication of the powerful selective role of FRF in the post-mating scenario, revealing new mechanisms by which females can tailor their influence over the ejaculate competition system.