Evolutionary proteomics reveals distinct patterns of complexity and divergence between lepidopteran sperm morphs

Abstract

Spermatozoa are one of the most strikingly diverse animal cell types. One poorly understood example of this diversity is sperm heteromorphism, where males produce multiple distinct morphs of sperm in a single ejaculate. Typically, only one morph is capable of fertilization and the function of the non-fertilizing morph, called parasperm, remains to be elucidated. Sperm heteromorphism has multiple independent origins, including Lepidoptera (moths and butterflies), where males produce a fertilizing eupyrene sperm and an apyrene parasperm, which lacks a nucleus and nuclear DNA. Here we report a comparative proteomic analysis of eupyrene and apyrene sperm between two distantly related lepidopteran species, the monarch butterfly (Danausplexippus) and Carolina sphinx month (Manduca sexta). In both species, we identified approximatey 700 sperm proteins, with half present in both morphs and the majority of the remainder specific to eupyrene sperm. Apyrene sperm thus have a distinctly less complex proteome. Gene Ontology (GO) analysis revealed proteins shared between morphs tend to be associated with canonical sperm cell structures (e.g. flagellum) and metabolism (e.g. ATP production). GO terms for morph-specific proteins broadly reflect known structural differences, but also suggest a role for apyrene sperm in modulating female neurobiology. Comparative analysis indicates that proteins shared between morphs are most conserved between species as components of sperm, while morph-specific proteins turn over more quickly, especially in apyrene sperm. The rapid divergence of apyrene sperm content is consistent with a relaxation of selective constraints associated with fertilization and karyogamy. On the other hand, parasperm exhibit greater evolutionary lability, which may reflect adaptive response to shifting regimes of sexual selection. Additionally, we provide the first (to our knowledge) scanning electron micrographs of lepidopteran sperm.