Proteomic consequences of male mate preference learning and female phenotype in the butterfly Bicyclus anynana

Mate preference learning, where individuals use past social experience to choose mates, is prevalent in many species. Yet its consequences on reproductive investment, and whether individuals differentially invest in reproduction based on their learned perception of the mates phenotype, are unknown. We addressed these questions using the butterfly Bicyclus anynana, where males acquire individual preferences for artificially painted 0-UV dorsal hindwing spotted (DHSN) females, who then lay more eggs. We measured the spermatophore proteins transferred by naive and experienced males to females manipulated to have preferred (0-DHSN) and unpreferred (2-DHSN) wing patterns. Using data independent acquisition (DIA), we identified 2144 proteins in the B. anynana spermatophore transferred to the female during mating. Experienced males that mated with preferred and unpreferred females had more differentially abundant (DA) proteins in their spermatophores that have functions in circadian rhythms, oogenesis, and neural signalling than naive males. Proteins associated with oogenesis were DA in spermatophores transferred to preferred females, which may contribute to why preferred females lay more eggs. Overall, our study provides evidence for the role of experience-induced behavioural plasticity in tailoring male ejaculates in butterflies and identifies proteins that influence female physiology and behaviours which directly affect the pairs overall reproductive fitness.