RNA Interference Targeting Testis-Specific Serine/Threonine Protein Kinase 1 (TSSK1) Gene Triggers Male Infertility in Zeugodacus tau

Zeugodacus tau, a highly destructive agricultural quarantine pest causing severe economic losses to global fruit crops, urgently requires the development of male fertility-based control strategies. Here, we identified and characterized the testis-specific serine/threonine protein kinase 1 gene (ZtTSSK1) in Z. tau. The encoded protein of ZtTSSK1 is highly conserved among dipteran species. Spatiotemporal expression analysis revealed predominant expression in adult males, with specific localization to the testes. In situ hybridization further localized ZtTSSK1 transcripts to the transformation zone. Furthermore, functional characterization by RNA interference (RNAi) revealed that knockdown of ZtTSSK1 resulted in a significant 62% reduction in sperm counts. While egg numbers laid by females mated to dsZtTSSK1- versus dsGFP-injected males did not differ, hatching rates were significantly lower for eggs from dsZtTSSK1 matings. These findings establish ZtTSSK1 as a key regulator of spermatogenesis and male fertility in Z. tau, providing a theoretical foundation and candidate target for genetic-based sterile insect technique (gSIT) development.