Sex‐specific alternative splicing of doublesex contributes to sexual dimorphism and enables larval sex identification in an alpine moth (Lymantriinae: Gynaephora )

BACKGROUND: The alpine grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) are major pests in alpine meadows of the Qinghai-Tibetan Plateau, exhibiting striking sexual dimorphism with winged males and apterous females. The genetic basis of this dimorphism is crucial for understanding their adaptation and developing targeted control strategies. This study investigated the role of the conserved sex-determination gene doublesex (dsx) in the sexual dimorphism. RESULTS: We identified and characterized the doublesex homolog in Gynaephora (Gyndsx), which produced three sex-specific splice variants (two female-specific and one male-specific) encoding conserved protein domains. This enabled the development of a robust polymerase chain reaction (PCR)-based assay for sex identification in larval stages. Expression of Gyndsx peaked during the pupal stage, coinciding with the key period of sexual dimorphism formation. RNA interference-mediated knockdown of Gyndsx resulted in severe, sex-specific defects: males developed wrinkled, curled wings, while females exhibited incomplete eclosion, malformed ovipositors, and significant pupal lethality. Particularly, we found that Gyndsx knockdown correlated with altered expressions of four genes in the ecdysone signaling pathway: USP, E93, and br-c were significantly down-regulated, while EcR exhibited a sexually dimorphic expression shift. CONCLUSIONS: Our findings suggest that sex-specific alternative splicing of dsx contributes to sexual dimorphism in Gynaephora and point to a potential link between sex determination and the ecdysone signaling pathway that warrants further investigation. The molecular sexing method developed here provides a critical tool for monitoring field populations. This work advances the molecular understanding of insect development and identifies Gyndsx as a potential, specific target for the sustainable management of these alpine pests. © 2026 Society of Chemical Industry.