Doublesex regulates the development of sex‐specific organs in rice stem borer, Chilo suppressalis

The doublesex (dsx) gene is a crucial component of the insect sex determination pathway, playing a pivotal role in sexual differentiation and development. While the functions of dsx have been extensively characterized in several insect species, its molecular mechanisms and specific roles in sex determination remain largely unexplored in the lepidopteran rice stem borer, Chilo suppressalis (Walker), a significant agricultural pest. In this study, we identified two male-specific and five female-specific Csdsx transcripts in C. suppressalis. Phylogenetic analyses and multiple sequence alignments revealed that Csdsx is highly conserved among lepidopterans. To investigate its functional roles, we employed CRISPR/Cas9-mediated mutagenesis to generate C. suppressalis mutants. Specifically, target sites were designed on exons 1, 3, 4, and 5 to disrupt the common region (exon 1), female-specific exons (exon 3 and exon 4), and male-specific regions of Csdsx (exon 5). As anticipated, mutants exhibited abnormal development of both external and internal genitalia during the pupal and adult stages. Furthermore, mutations induced sex-specific sterility. Notably, knockout of sex-specific Csdsx isoforms also resulted in abnormal wing patterns and antennal morphology. This study demonstrates the conserved role of Csdsx in sexual differentiation in C. suppressalis and highlights its potential as a molecular target for environmentally friendly pest management strategies against this agricultural pest. © 2025 Society of Chemical Industry.