Male accessory gland-derived carboxypeptidase B regulates sperm activation in the spermatophores of Grapholita molesta

Grapholita molesta is a globally significant fruit-boring pest. Females maximize reproductive efficiency through optimal sperm utilization following a single mating event. Post-mating sperm activation is critical for reproductive success. Although sperm activation in G. molesta relies on the spermatophore microenvironment, the molecular regulators and associated metabolic pathways remain poorly understood. Here, we report that a male accessory gland-derived carboxypeptidase B (GmCPB) is essential for this process. GmCPB exhibited peak expression in the accessory gland during the pre-maturation stage; RNAi-mediated knockdown of GmCPB in males substantially reduced fertility, while mating success and longevity were unaffected. Mating with GmCPB-knockdown males had no effect on spermatophore formation or sperm transfer, whereas sperm activation was markedly impaired. Untargeted metabolomic analysis further revealed that multiple metabolic pathways exhibited significant dysregulation in the spermatophores of females after mating with GmCPB- knockdown males. Our findings propose a functional framework for CPB characterization and establish a molecular foundation for population suppression strategies targeting male accessory gland proteins in G. molesta.