Sperm attraction by female reproductive fluid in a fish with an unconventional fertilisation strategy

Active sperm attraction is a widespread function of female reproductive fluids (FRF), identified in both internal and external fertilisers. However, it remains unknown whether FRF retains this function in species where gamete encounters are mediated by physical processes. Such systems, where fertilisation occurs within confined or flow-driven microhabitats, may relax selection on chemotactic sperm guidance. Hence, we hypothesised that the sperm attractant properties of FRF may be lost in species with unconventional fertilisation modes. We tested this hypothesis using the bitterling, a small fish that parasitise freshwater mussels. Bitterling deposit sperm and eggs inside the mussel gills and gamete interactions are facilitated by the water current generated by mussel respiration. Using a recently developed sperm selection chamber and the European bitterling, we find that more sperm accumulate in the FRF channel compared to the water control. Moreover, European bitterling sperm showed no preference for conspecific over a distantly related heterospecific FRF. Our results suggest that bitterling FRF resembles that of species with conventional fertilisation modes, implying that it could mediate sperm selection and cryptic female choice. We discuss alternative evolutionary scenarios underlying the persistence of sperm attractant properties of bitterling FRF despite the shift to a physically mediated fertilisation environment.