Nap1 is essential for eupyrene spermatogenesis and migration in Plutella xylostella

Spermatogenesis is a key process for the sexual reproduction species. In lepidopteran insects, spermatogenesis produces two different types of sperms, in which eupyrene sperm carry genomic DNA and fertilize eggs, whereas apyrene sperm are necessary for eupyrene sperm to enter eggs. However, functional genetic studies of spermatogenesis in Plutella xylostella remain a longstanding puzzle even though the phenomenon in lepidoptera has been widely documented more than a century. In this study, we particularly focus on the gene Nap1 which belongs to the Nucleosome assembly protein family. Our findings revealed that Nap1 was highly expressed in the testes, and the disruption of PxNap1 induced male sterility in P. xylostella, while the fertility of mutant females was comparable to wild-type females. Additionally, through immunofluorescence staining analysis, we found that the eupyrene sperm bundles presented diffusedly scattered nuclei in PxNap1 mutant males, while the nuclei in the wild-type were clustered together presented as needle shape. We also found that PxNap1 deficiency hinders the transfer of eupyrene sperm to the bursa copulatrix and spermatheca of females. However, the apyrene spermatogenesis was not affected in the PxNap1 mutant. RNA-seq analyses indicated that the defects of eupyrene sperm in PxNap1 mutants were related to energy metabolic such as pentose and glucuronate interconversions, biosynthesis of amino acids, and pentose phosphate pathway. Our study demonstrates that PxNap1 plays crucial function in eupyrene spermatogenesis and eupyrene sperm migration. Our research provides valuable insights for the genetic factors underlying reproductive processes in Lepidopteran insects.