Mechanisms of last male precedence in a moth: sperm displacement at ejaculation and storage sites

In many polyandrous species, the last male to mate sires most of the offspring. This phenomenon has been explained as the result of male manipulation, postcopulatory female choice, or both. However, in most previous studies, direct evidence for these explanations is lacking. Here, we report our work on a polyandrous moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). We show that the last male sperm precedence occurs by sperm displacement operating at both sperm ejaculation and storage sites. Thus, the second male physically displaces the first male's spermatophore with his own in the bursa copulatrix. However, females usually allow most sperm of the first males to move to the spermatheca before they accept a second copulation. The intermating duration, which is positively correlated with the number of the first male's sperm moving to the spermatheca, may be the product of mate choice and sexual conflict between the sexes. After the female accepts the second mating, she ejects ≈50% of the first male's sperm from the spermatheca to accommodate the second male's sperm. Spermathecal contractions appear to be the mechanism for sperm ejection. Before oviposition occurs, ≈75% of eupyrene sperm in the spermatheca of the twice-mated female are from the second male. The outcome of sperm displacement before and after sperm storage appears to be the result of male × female interactions. We demonstrate that ejaculate size in the bursa copulatrix by the second male, and sperm stratification and flushing in the spermatheca, do not play a role in last male sperm precedence.