Data Sheet 1_Proteomic analysis of dimorphic sperm in the cabbage white butterfly, Pieris rapae.xlsx

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.

精子二态性（sperm dimorphism）指单个雄性产生两种截然不同的精子形态，是一种广泛存在却仍具谜题性的生殖现象。在鳞翅目（Lepidoptera）昆虫中，具有受精能力的有核精子（eupyrene sperm）与无核精子（apyrene sperm）共存；后者虽无法使卵子受精，却是成功繁殖所必需的。尽管精子二态性分布广泛且被推测具有适应价值，但目前学界对该性状的分子机制认知仍较为有限。
本研究以菜粉蝶（Pieris rapae）——一种新兴的性选择与交配后互作研究模型——为对象，对其二态性精子的蛋白质组（proteome）进行了表征。本研究采用高分辨率无标记质谱（label-free mass spectrometry）技术，共鉴定出超过1600种蛋白质，这一数量几乎是此前其他鳞翅目昆虫报道的蛋白质数量的两倍。差异丰度分析结果显示，有核精子中富集了与离子转运、液泡酸化相关的蛋白质，而无核精子则富集了与线粒体功能及呼吸作用相关的蛋白质。
令人意外的是，通过与另外两种鳞翅目昆虫——君主斑蝶（Danaus plexippus）和烟草天蛾（Manduca sexta）——进行比较同源性分析，本研究发现菜粉蝶与烟草天蛾的精子同源蛋白质数量，多于其与亲缘关系更近的君主斑蝶的同源蛋白质数量，这凸显了精子蛋白质组复杂的进化动态。
综上，本研究的发现深化了学界对鳞翅目昆虫精子功能与多样性的认知，阐明了有核精子与无核精子各自的独特功能，并为未来开展精子进化、性选择以及生殖蛋白质功能相关研究奠定了基础。