Diet-derived vicilins detected in eggs laid by a double-mated female Callosobruchus maculatus originate from nuptial gifts donated by both male partners

•Callosobruchus maculatus females are polyandrous.•Both females and males sequester vicilins from the cowpea host.•Males donate vicilin to females as a seminal nuptial gift.•Double mated females deposit vicilins from different males in the eggs.

The eggshell and the cement that attaches the eggs of the seed-beetle Callosobruchus maculatus to the seeds of cowpea (Vigna unguiculata) contain vicilins, the main storage protein found in seeds. These proteins are derived from both males and females, which originally sequester them during the larval phase from their seed hosts. In this test, females received vicilin from different males during mating as a seminal nuptial gift, and transmitted this sequestered blend of proteins to the eggs. The presence of vicilin in the cement and eggshell was demonstrated by light microscopy and Western blotting. Quantification using ELISA showed that the concentration of vicilin from seminal vesicles of a virgin male is approximately double that measured following the first copula to a virgin female, suggesting that the investment of vicilin by the males is very important. This is the first demonstration showing that at least two different males can make a direct contribution of a sequestered protein which is used in oogenesis.