Characteristic and functional study of intersex, a gene involved in sexual development in Gryllus bimaculatus

As a gene required for sexual development, intersex (ix), functions in concert with the female-specific product of doublesex (dsx) at the end of the hierarchy to facilitate the sex-specific differentiation of sexually dimorphic characters in female Drosophila melanogaster. In the present study, we initially identified the ix homolog in Gryllus bimaculatus, with the detection of a single isoform expressed in both sexes. Phylogenetic analyses and multiple sequence alignment revealed that Gbix exhibited conservation. Here we employed RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9), respectively, to analyze the functions of Gbix. Our findings indicated that Gbix played a crucial role in the normal development of the embryo and nymph, as well as in the regulation of wing morphogenesis in both sexes. RNAi-mediated knockdown of Gbix resulted in poorly developed ovaries, whereas testicular development was not significantly affected. Nevertheless, CRISPR/Cas9-mediated knockout of Gbix resulted in internal genital defects in both sexes, which ultimately led to a reduction in reproductive capacity in female and male individuals. Our results provide insights into the pleiotropic functions of Gbix in embryogenesis and sexual development, while also advancing our comprehension of sex determination in a hemimetabolous insect species.