Abstract

BACKGROUND The genetic-based sterile insect technique (gSIT) is a sustainable, eco-friendly pest control strategy, whose development hinges on identifying key fertility-associated molecular targets—particularly those regulating spermatogenesis. Spermatid development relies not only on testis-specific proteins but also on post-translational modifications such as phosphorylation, yet such targets and their regulatory pathways remain uncharacterized in the diamondback moth, Plutella xylostella , a major worldwide pest characterized by rapid reproduction. RESULTS The testis-specific serine/threonine kinase 3 (TSSK3) is firstly identified in P. xylostella . CRISPR/Cas9-mediated TSSK3 deletion successfully constructs two homozygous mutant strains, with mutation rates of 1.1% and 2.1%, respectively. While TSSK3 deficiency does not affect mating behavior, the absence of TSSK3 blocks testicular development and leads to complete male sterility. This was primarily attributed to chromatin condensation abnormalities during spermatogenesis, resulting in the death of eupyrene sperm and suppressing fertilization. Deletion of PxTSSK3 downregulates 2307 phosphorylation sites (1254 proteins), whereas 1608 sites (854 proteins) showed no change in protein expression. PxTSSK3 deficiency led to a significant enrichment of potential phosphorylation substrates in xxxxxx_S_DxExxx and xxxxxx_T_PPxxxx motifs, as well as spermatogenesis, male gamete generation, mitotic metaphase/anaphase transition, microtubule cytoskeleton organization pathways, including ODF, βtubulin, histone methyltransferase (MLL5) etc. CONCLUSION This study highlights the crucial role of PxTSSK3 and it-mediated broad phosphorylation in promoting spermatogenesis and sustaining male fertility in P. xylostella , as well as its prospective benefits as a target for gSIT-based pest control. It offers valuable insights that could be integrated into area-wide pest management strategies for the suppression of this agricultural pe