Gametocyte-Specific Factor 1 Is Essential for Male Fertility and Transposon Homeostasis in Plutella xylostella

Gametocyte-specific factor 1 (Gtsf1) plays a pivotal role in the gonadal development by transposon silencing via the piRNA pathway in Drosophila melanogaster and Bombyx mori, and is implicated in the female sex determination in B. mori. However, its function remains elusive in other insect species, including Plutella xylostella, a highly destructive pest in cruciferous crops. In this study, we characterized PxGtsf1, encoding a 27.84 kDa protein containing two conserved U11-48K-like CHHC zinc-finger domains. PxGtsf1 was predominantly expressed in male pupae, males and testes. Using CRISPR/Cas9 technique, we successfully generated a homozygous PxGtsf1-knockout strain (△PxGtsf1) with 11 bp deletion. △PxGtsf1 females exhibited normal morphology in terms of external genitalia, body weight, ovariole morphology, ovariole length, oocytes number, and internal ovarian structure, and maintained the expression levels of genes related to female sex determination and fertility. In contrast, △PxGtsf1 males displayed reduced testis size and enlarged eupyrene sperm bundles, whereas apyrene sperm bundles appeared normal. Wild-type (WT) or △PxGtsf1 females mated with △PxGtsf1 males had significantly smaller bursa copulatrix distention area compared to those mated with WT males. Furthermore, WT or △PxGtsf1 females mated with △PxGtsf1 males produced significantly fewer eggs with lower hatching rates than those mated with WT males. Co-immunoprecipitation assays revealed that PxGtsf1 specifically interacts with PxPiwi. Small RNA sequencing further showed that PxGtsf1 knockout resulted in transposon dysregulation in the testes without affecting overall piRNA abundance, suggesting that PxGtsf1 functions in piRNA-mediated transposon regulation rather than global piRNA biogenesis. PxGtsf1 is first reported to be specifically required for male fertility in Lepidoptera insects but is dispensable for female sex determination., PxGtsf1 regulates transposon homeostasis without affecting global piRNA biogenesis. These findings provide new insights into the diverse functions of Gtsf1 in Lepidopteran insects.