The Prmt5-Vasa module is essential for dichotomous spermatogenesis in Bombyx mori

Spermatogenesis is a key process for the sexual reproduction species. In lepidopteran insects, dichotomous spermatogenesis is a notable feature, which produces eupyrene (nucleate) and apyrene (anucleate) spermatozoa. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilizes the egg, while apyrene sperm is necessary for eupyrene sperm migration. In Drosophila, Prmt5 acts as a type II arginine methyltransferase to catalyze the symmetrical dimethylation of arginine residues (sDMA) in Vasa. However, Prmt5 is involved in regulating spermatogenesis, but Vasa is not. To date, the functional genetic study on dichotomous spermatogenesis in the lepidopteran model Bombyx mori has been limited, thus the underlying mechanism remains largely unknown. In this study, we report that both BmPrmt5 and BmVasa act as essential components in the regulation of dichotomous spermatogenesis in Bombyx mori. The loss-of-function mutants of BmPrmt5 ({Delta}BmPrmt5) and BmVasa ({Delta}BmVasa) derived from CRISPR/Cas9-based gene editing show similar male and female-sterile phenotypes. Through immunofluorescence staining analysis, we found that the morphs of both{Delta} BmPrmt5 and{Delta} BmVasa sperm show severe defects, indicating an essential role for both BmPrmt5 and BmVasa in the regulation of dichotomous spermatogenesis. RNA-seq analyses indicate that the defects in dichotomous spermatogenesis observed for{Delta} BmPrmt5 and{Delta} BmVasa mutants could be attributed to the reduced expression of the spermatogenesis-related genes including Sex-lethal (BmSxl), implying that BmSxl may act downstream of Prmt5 and Vasa in regulating apyrene sperm development. These findings suggest that BmPrmt5 and BmVasa constitute an integral regulatory module essential for dichotomous spermatogenesis in Bombyx mori, in which BmPrmt5 may promote BmVasa activity through sDMA modification. Author SummaryDichotomous spermatogenesis is a notable feature in Lepidoptera insects, in which a single male produces nucleated and anucleated dimorphic sperm. Both sperm morphs are essential for fertilization, as the eupyrene sperm fertilizes the egg, while the apyrene sperm assists in transporting the eupyrene sperm to the female sperm storage organs. To date, the mechanism of dichotomous spermatogenesis is still unclear, as very limited components participating in this process have been reported. Here we defined the function of BmPrmt5 (Protein arginine methyltransferase 5) and its substrate BmVasa in dichotomous spermatogenesis in the lepidopteran insect Bombyx mori. Our genetic analyses revealed that both BmPrmt5 and BmVasa are essential for dichotomous spermatogenesis. Moreover, RNA-seq analyses, in conjunction with the genetic and immunofluorescence staining analyses, suggest a potential regulatory pathway consisting of the BmPrmt5-BmVasa module in dichotomous spermatogenesis in Bombyx mori. This study expands the knowledge of reproduction biology in insects, and may provide potential gene targets for pest control.