A cuticular protein essential for sperm dimorphism and male fertility in Bombyx mori

In Lepidoptera, sperm develop as two distinct types: fertile eupyrene and non-fertile apyrene sperm. Although apyrene sperm do not fertilize eggs, they support fertilization through auxiliary functions. However, the molecular mechanisms underlying this dimorphism remain poorly understood. Here, we identify the Cuticular Protein Hypothetical 19 precursor (BmorCPH19) gene as essential for sperm development in Bombyx mori. Using 70 μm cell strainers, we isolated transcriptionally active apyrene spermatocytes and an eupyrene-enriched fraction containing mature sperm bundles from 3-day-old pupal testes, when morphs are distinct and separable. Transcriptomic profiling revealed 737 differentially expressed genes, including BmorCPH19, unexpectedly enriched in the eupyrene fraction -suggesting a novel role beyond cuticle-related activity. qRT-PCR analysis showed that BmorCPH19 expression in the testes peaks during eupyrene differentiation in the 6-day-old fourth-instar larva and the 1-day-old prepupa. Isolated sperm morphs confirmed consistent BmorCPH19 enrichment in eupyrene sperm, with maximal accumulation in late pupae. Functional assays demonstrated its role in sperm fate: RNAi knockdown increased the apyrene-to-eupyrene ratio, while overexpression favored eupyrene sperm but impaired fertility, yielding more non-viable eggs. These findings identify BmorCPH19 as the first cuticular protein essential for lepidopteran sperm dimorphism and reveal its dosage-sensitive role in balancing sperm differentiation and reproductive success, offering a potential molecular target for pest control.