Sperm counts in the reproductive tract of doubly-mated females

Assays were performed to evaluate how sperm dynamics in the reproductive tract of doubly-mated females differs when the second male to mate lacks or carries the species-specific multigene family Sdic of Drosophila melanogaster in the genome. Virgin females were first mated to a reference male and two days later to one of the two possible experimental males (with Sdic, Sdic+, or without Sdic, Sdic-). 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 counted using a fluorescent microscope; the sperm from the reference and experimental males glow in red and green respectively. Sperm counting was performed for both the bursa and the sperm storage organs, i.e. the paired spermatheca and the seminal receptacle.