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

Lepidopteran insects are unique in producing two different types of sperm, apyrene sperm (without nuclei, non-fertile) and eupyrene sperm (with nuclei, fertile) (Shepherd and Bonk, 2021). When stored in the seminal vesicle and ductus ejaculatorius duplex of unmated males, both types of sperm are immotile and eupyrene sperm are incapable of fertilization (Nagaoka et al., 2017a). During mating, two kinds of sperm and other secretions (mainly accessory gland proteins; Acps) are transferred by males, and the ejaculate is fully mixed in the spermatophore that is formed wholly within the bursa copulatrix (BC) of mated females (Osanai et al., 1989, Friedländer et al., 2005). Within the spermatophore, the transferred sperm are activated as follows: first, the apyrene sperm gain sufficient motility; second, the eupyrene sperm bundles dissociate; and finally, dissociated eupyrene sperm acquire fertility (Friedländer et al., 2005). Full Sperm activation in vivo is a key step for reproductive success. Hence, sperm-activating factors are biorational molecular targets for population control (Seth et al., 2023).

The activation of both apyrene and eupyrene sperm results from the exposure of the transferred sperm to numerous male-derived activating factors (Yadav et al., 2024). A solid body of evidence indicates that multiple serine proteases are required for sperm activation in the spermatophore of several lepidopteran species including Bombyx mori (serine protease 2, also known as Initiatorin) (Nagaoka et al., 2012), Spodoptera litura (serine protease 2 and trypsin-like serine protease) (Bi et al., 2022, Yadav et al., 2024), and S. frugiperda (serine protease snake-like 1, SPSL1) (Qian et al., 2023). Moreover, another peptidase, angiotensin converting enzyme (ACE), also plays a major role in sperm activation. In Lacanobia oleracea, male accessory gland (AG)-derived ACE was transferred to the spermatheca and BC of females and may contribute to energy provision for sperm motility (Ekbote et al., 2003). In B. mori, captopril (specific ACE inhibitor) could block the motility activation of sperm. Specifically, seminal plasma-derived ACE protein is encoded by the BmAcre2 gene (Nagaoka et al., 2017b). These proteolysis regulators trigger cascade reaction of sperm activation inside the spermatophore (Nagaoka et al., 2012, Yadav et al., 2024).

The oriental fruit moth (OFM), Grapholita molesta (Lepidoptera: Tortricidae), is among the most dominant Rosaceae fruit-boring pests worldwide. Female moths lay a large number of eggs on the surface of fruits or twigs (Ahn et al., 2012). After hatching, the first instar larvae bore into the fruit, causing direct damage to the fruit tissue. In OFM, single mating is mostly a female trait, and gravid OFM females can lay eggs continuously for at least 11 days (de Morais et al., 2012). During the oviposition period, females must maintain the vitality of stored sperm. Sperm activation in the spermatophore is the primary step for sperm migration into the spermathecae for long-term storage and ultimately egg fertilization (Cheng et al., 2023). However, the functional characterization of sperm-activating factors in OFM is underexplored, limiting our understanding of its reproductive success. Our previous transcriptomic studies have shown that “carboxypeptidase activity” is critical for maintaining sperm activation, but the specific genes have not been identified and characterized (Cheng et al., 2022). Further on, comparative proteomic studies of AGs (before and after mating) and spermatophore (after mating) revealed the carboxypeptidase B (CPB) may be a key activating factor. Until now, only one study in B. mori has reported that seminal CPB activated by Initiatorin is crucial in triggering sperm maturation (Sakakura et al., 2022). However, global metabolic signatures in the spermatophore remain poorly understood. Hence, our aims were (a) to determine the role of GmCPB in the OFM male fertility, especially in sperm activation; (b) to examine whether GmCPB is a potential biorational molecular target for OFM population control; (c) to explore the metabolite profiles within the spermatophore after GmCPB knockdown.

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