Longer matings increase male competitive fertilization success in Drosophila melanogaster

Copulation duration is a variable behaviour that is often extended by males beyond the time needed for sperm transfer. In many insect species, these prolonged matings have been linked to higher male reproductive success with virgin females. However, most matings in multiply mating species involve non-virgin females, whose role in shaping the evolution of this behaviour remains underexplored. Using hemiclonal analysis in Drosophila melanogaster, we tested whether males benefit from extended matings with non-virgin females by comparing competitive fertilization success (P2) between males from three long-mating and three short-mating hemiclone lines. We confirmed that differences among hemiclone lines in male mating duration with virgin females persisted with non-virgin females and that these longer matings were associated with higher P2, demonstrating a clear fitness benefit to males. These findings build on past work showing that longer matings increase male defensive reproductive success, highlighting the adaptive value of prolonged matings for males in multiply mating species and underscoring the importance of including non-virgin females in studies of sexual selection and reproductive behaviour.