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

Active sperm guidance towards the eggs is necessary for gametes to meet within the fertilisation window and is usually achieved through the action of female reproductive fluids (FRF hereafter) on sperm flagellar movement. However, it remains unknown whether FRF retains this function in species with unusual fertilisation strategies. Such examples include systems where gametes are released within confined microhabitats characterised by flow-driven physical processes that could facilitate sperm-egg encounters, hence selection on active sperm guidance may be relaxed. Alternatively, sperm attraction could be maintained by pleiotropic effects of genes involved in other reproductive functions of FRF. Here we tested whether FRF of species with unusual modes of fertilisation retain sperm attractant properties using the bitterling, a small cyprinid fish that parasitises 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 FRF over that of a distantly related Asian bitterling 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.