Consecutive matings reveal differential depletion of male ejaculate functions in Aedes aegypti (Diptera: Culicidae)

Repeated mating can progressively deplete sperm and seminal fluid proteins (SFPs), potentially altering male reproductive performance. In the dengue vector mosquito Aedes aegypti, males transfer decreasing quantities of sperm and SFPs with successive matings, yet the effects of ejaculate depletion on distinct components of the female post-mating response remain poorly understood. Here, we investigated how consecutive matings influence male ability to suppress female re-mating, sperm use patterns of re-mated females, and male recovery of sperm reserves. Males were mated to five consecutive females, and each female in the mating order was subsequently given the opportunity to re-mate with a competitor male. Additionally, we quantified sperm stored in the seminal vesicles of virgin males, males immediately after five consecutive matings, and males allowed to recover for two or four days following depletion. Female re-mating probability progressively increased from 26.1% after a male's first mating to 72.6% after his fifth mating, displaying a marked decline in male ability to induce post-mating refractoriness. In contrast, although first-male paternity (P1) declined with mating order, males continued to sire the majority of offspring even after the fifth mating. Repeated mating depleted sperm reserves by approximately 86%, but males partially replenished sperm within two days, with no additional recovery detected after four days. Together, our results demonstrate that distinct reproductive functions display different sensitivities to ejaculate depletion, providing an explanation for how relatively modest reductions in ejaculate quality can alter some female post-mating responses while fertility remains comparatively resilient.