Sperm counts in the reproductive tract of doubly-mated femalesAssays were performed to evaluate how sperm dynamics in the reproductive tract of doubly-mated females differs when the second-to-mate experimental male is either possessing (Sdic+ ) or lacking (Sdic- ) the entire D. melanogaster-specific Sdic multigene family. Virgin females were first mated to a reference Sdic+ male and then to one of the two possible experimental males (Sdic+ or Sdic- ) two days later. Females were randomly assigned to one of three time points after the end of the second mating period (30 min, 1 h and 45 min, and 2 days) and subsequently flash frozen. Female reproductive tracts were dissected and the sperm from the different males were counted using a fluorescent microscope. Reference and experimental sperm fluoresce red and green, respectively.Sperm counting was performed for the bursa and both sperm storage organs, i.e. the paired spermatheca and the seminal receptacle.Sperm counts in doubly-mated females.xlsx

Sperm competition is a post-copulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to D. melanogaster, Sdic, is important for the outcome of sperm competition in doubly-mated females, although the mechanistic nature of this phenotype remained unresolved. Here we compared doubly-mated females, second mated to either Sdic knockout or non-knockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first-to-mate male’s sperm within the female’s sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.