Female remating decisions and a shorter inter-mating interval diminish last-male sperm precedence

Highly variable within and across species, patterns of sperm use not only are often driven by post-copulatory sexual selection but can also be impacted by experimental design. In investigations of paternity bias using competitive double matings, the inter-mating interval is a temporal factor that can affect sperm use patterns if the first male's sperm is used or lost at an appreciable rate between matings or if its viability or relative competitiveness is influenced by the time since ejaculation. Rapid loss of first-male sperm within the female after mating has been established in the seed beetle (Callosobruchus maculatus), a model system in sperm competition studies. However, our understanding of sperm precedence in this species, which disproportionately favors the last (second) male to mate, is based on long inter-mating intervals. Here, I determine the effect of a shortened inter-mating interval on second-male paternity (P-2) and, importantly, the extent to which females in this species are willing to remate immediately. I find that P2 is significantly reduced when females remate immediately than when they remate 24 or 48 h after the first mating and that immediate remating is common, indicating that there is a substantial potential for female remating decisions to influence the intensity of sperm competition within species. To understand the variation in inter-mating intervals from a female perspective, I further identify key differences between females that did and did not remate at three inter-mating intervals (0, 24, and 48 h after the initial mating) and discuss potential mechanisms for the observed variation in female refractoriness. Significance statement Females often mate with multiple males, and in insects it is common for the last male to mate to obtain the greatest share of paternity. One possible explanation for this bias towards last-male sperm use is the passive loss of sperm from the first male with time since mating. Here, I examine the effect of different intervals between matings in a seed beetle species to determine if first-male paternity is increased when females remate sooner. My results show that first males do achieve higher paternity when females remate sooner, which is more common than expected in this species. I also show the differences between females that do and do not remate, which can help explain potential causes for variation in female remating behavior. Together, these results show the significant impact that female remating decisions can have on male competition and, ultimately, their reproductive success.