Monandry and its underlying mechanisms in the rhinoceros beetle Trypoxylus dichotomus

Although females of many insects suppress remating after an initial mating, the duration and mechanisms of this post-copulatory refractory period vary widely across species. We investigated the refractory period, remating rate, and underlying mechanisms in the Japanese rhinoceros beetle Trypoxylus dichotomus under laboratory conditions. Over 90% of females rejected mating attempts for at least 28 days after initial copulation, a duration exceeding the lifespan of wild females. When females were initially paired with recently-mated males, whose spermatophore size was reduced by 60%, the remating rate increased slightly to 15% but remained low. Therefore, females of this species typically mate only once in their lifetime, representing a rare example of monandry among insects, particularly within Scarabaeidae. To explore the mechanisms underlying refractoriness, we injected aqueous extracts of spermatophores into the female hemocoel. This treatment did not suppress subsequent mating. Additionally, females experimentally separated from males during copulation readily accepted remating, indicating that genital insertion and associated mechanical stimulation alone are insufficient to induce refractoriness. Collectively, our results indicate that inducing refractoriness relies on male-derived substances functioning locally within the reproductive tract, likely interacting with mechanical cues from spermatophore filling. Finally, females mated with recently-mated males did not differ from those mated with unmated males in fecundity or egg hatch rate, indicating that a single copulation provides sufficient sperm for lifetime fertility even when spermatophore size is reduced. We suggest that the large spermatophores produced by unmated males have evolved through post-copulatory sexual selection to prevent females from remating.