Proteomic analysis of dimorphic sperm in the cabbage white butterfly, Pieris rapae.

Sperm dimorphism, the production of two distinct sperm morphs by a single male, is a widespread but enigmatic reproductive phenomenon. In Lepidoptera, fertilizing eupyrene sperm coexist with anucleate apyrene sperm, which cannot fertilize eggs but are nevertheless required for successful reproduction. Despite the prevalence and presumed adaptive significance of sperm dimorphism, the molecular basis of this trait remains limited. Here, we characterize the proteome of dimorphic sperm in the Cabbage White butterfly, Pieris rapae, an emerging model for sexual selection and postcopulatory interactions. Using high-resolution, label-free mass spectrometry, we identified more than 1,600 proteins, nearly doubling the number of proteins previously reported for other lepidopteran species. Differential abundance analyses revealed eupyrene sperm were enriched for proteins linked to ion transport and vacuolar acidification, while apyrene sperm were enriched for mitochondrial and respiratory functions. Unexpectedly, comparative homology analyses with two other Lepidoptera, Danaus plexippus and Manduca sexta, showed P. rapae shared more homologous sperm proteins with M. sexta than with the more closely related D. plexippus, highlighting complex evolutionary dynamics of sperm proteomes. Together, these findings expand our understanding of sperm function and diversity in Lepidoptera, highlight the distinct roles of eupyrene and apyrene sperm, and provide a foundation for future studies of sperm evolution, sexual selection, and reproductive protein function.