Doublesex knockout via CRISPR/Cas9 disrupts fertility and sexual dimorphism of wings in the rice stem borer, Chilo suppressalis.

The doublesex (dsx) gene has a conserved role in sex determination in insects, controlling sexual development and mating behavior. Although dsx is known to participate in these critical functions, its role in insect sex determination remains not fully elucidated. Given the economic importance of the rice pest Chilo suppressalis, we employed this species as a model to investigate the function of its dsx homolog. We cloned and characterized the Csdsx gene, which is 1123 bp in length and encodes four sex-specific proteins: three female-specific isoforms of 252, 258, and 254 amino acids, respectively, and one male-specific isoform of 290 amino acids. Phylogenetic analysis revealed that Csdsx is highly conserved within Lepidoptera, containing two domains: DM DNA binding domain and DSX dimer domain. Analysis of transcripts produced from a mini-dsx construct transfected into human HEK293T cells indicates that the female-splicing pattern is the default mode. Quantitative real-time PCR showed that among various developmental stages, Csdsx expression peaked at the first instar larval stage and showed tissue-specific, stage-dependent patterns, with notably high levels in the larval midgut, pupal fat body, and adult thorax in both sexes. In comparison to wild-type (WT) adults, Csdsx-knockout individuals exhibited malformations in their external genitalia, and female wing patterns became masculinized. Dissections revealed that knockout females had a reduced number of mature oocytes, while knockout males displayed a smaller testis area. Furthermore, when Csdsx-knockout females were paired with wild-type males, their mating behavior was significantly impaired. RNA-seq revealed that Csdsx disruption led to sex-biased gene expression shifts, including upregulation of male-associated genes (e.g., OBPs, PBPs, trypsin) and downregulation of female-specific genes (vitellogenin, FAS), indicating partial masculinization at the transcriptional level. These findings underscore the critical role of the dsx gene in reproductive development and sexual dimorphism in C. suppressalis.