TSSKL is essential for sperm mitochondrial morphogenesis and male fertility in moths.

Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.