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Spermatid development relies not only on testis\u2010specific proteins but also on post\u2010translational modifications such as phosphorylation, yet such targets and their regulatory pathways remain uncharacterized in the diamondback moth,\n                      <jats:italic>Plutella xylostella<\/jats:italic>\n                      , a major worldwide pest characterized by rapid reproduction.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>RESULTS<\/jats:title>\n                    <jats:p>\n                      The testis\u2010specific serine\/threonine kinase 3 (TSSK3) is firstly identified in\n                      <jats:italic>P. xylostella<\/jats:italic>\n                      . CRISPR\/Cas9\u2010mediated\n                      <jats:italic>TSSK3<\/jats:italic>\n                      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\n                      <jats:italic>TSSK3<\/jats:italic>\n                      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\n                      <jats:italic>PxTSSK3<\/jats:italic>\n                      downregulates 2307 phosphorylation sites (1254 proteins), whereas 1608 sites (854 proteins) showed no change in protein expression.\n                      <jats:italic>PxTSSK3<\/jats:italic>\n                      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, \u03b2tubulin, histone methyltransferase (MLL5) etc.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>CONCLUSION<\/jats:title>\n                    <jats:p>\n                      This study highlights the crucial role of\n                      <jats:italic>PxTSSK3<\/jats:italic>\n                      and it\u2010mediated broad phosphorylation in promoting spermatogenesis and sustaining male fertility in\n                      <jats:italic>P. xylostella<\/jats:italic>\n                      , as well as its prospective benefits as a target for gSIT\u2010based pest control. It offers valuable insights that could be integrated into area\u2010wide pest management strategies for the suppression of this agricultural pest. \u00a9 2026 Society of Chemical Industry.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/ps.70590","type":"journal-article","created":{"date-parts":[[2026,1,30]],"date-time":"2026-01-30T12:01:29Z","timestamp":1769774489000},"page":"4757-4770","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Impacts of testis\u2010specific serine\/threonine protein kinase 3 on eupyrene spermatogenesis and contributes to male sterility in\n                    <scp>\n                      <i>Plutella xylostella<\/i>\n                    <\/scp>"],"prefix":"10.1002","volume":"82","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3314-7634","authenticated-orcid":false,"given":"Lu","family":"Peng","sequence":"first","affiliation":[{"name":"State Key Laboratory 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