Last male sperm precedence is modulated by female remating rate in Drosophila melanogaster

Following multiple matings, sperm from different males compete for fertilization within the female reproductive tract. In many species, this competition results in an unequal sharing of paternity that favors the most recent mate, termed last male sperm precedence (LMSP). Much of our understanding of LMSP comes from studies in Drosophila melanogaster that focus on twice‐mated females with standardized latencies between successive matings. Despite accumulating evidence indicating that females often mate with more than two males and exhibit variation in the latency between matings, the consequences of mating rate on LMSP are poorly understood. Here, we developed a paradigm utilizing D. melanogaster in which females remated at various time intervals with either two or three transgenic males that produce fluorescent sperm (green, red, or blue). This genetic manipulation enables paternity assessment of offspring and male‐specific sperm fate examination in female reproductive tracts. We found that remating latency had no relationship with LMSP in females that mated with two males. However, LMSP was significantly reduced in thrice‐mated females with short remating intervals; coinciding with reduced last‐male sperm storage. Thus, female remating rate influences the relative share of paternity, the overall clutch paternity diversity, and ultimately the acquisition of indirect genetic benefits to potentially maximize female reproductive success.