A Testis‐Specific Aralkylamine N ‐Acetyltransferase Regulates Dimorphic Sperm Function and Male Fertility in Moths

ABSTRACT Most Lepidopterans produce two distinct sperm types—nucleated eupyrene (fertile) and anucleate apyrene (non‐fertile), yet the genetic mechanisms governing this dimorphism remain poorly understood. Using the fall armyworm ( Spodoptera frugiperda ) as a model, we identified a Lepidoptera‐conserved, testis‐specific arylalkylamine N ‐acetyltransferase (LTNAT) indispensable for male fertility. Functional analyses showed that LTNAT is expressed in both sperm types, and its depletion disrupts mitochondrial derivatives (MDs) in eupyrene sperm and drastically impairs apyrene sperm motility. Multi‐omics analyses revealed LTNAT mutation perturbs energy metabolism, lipid homeostasis, and transmembrane transport—alterations correlating with abnormal MD structure. Furthermore, LTNAT deficiency likely compromises apyrene sperm motility by downregulating Sex‐lethal and flagellar assembly genes (e.g., dyneins and intraflagellar transport proteins ). CRISPR/Cas9‐mediated knockout of LTNAT homologs in two other lepidopteran species similarly reduced male fertility, confirming functional conservation. Mass release of LTNAT ‐deficient males significantly suppressed female fertility in caged experiments. Collectively, LTNAT is a core regulator of dimorphic sperm development in Lepidoptera, highlighting its potential as a target for innovative insecticidal or genetic pest control.