Doublesex regulates male sexual development in the cricket Gryllus bimaculatus.

Insects have evolved a diversity of regulatory mechanisms to determine their sex. Understanding the molecular regulation mechanisms of insect sex determination is of great significance in revealing the general law of insect sex determination and providing potential routes for the genetic manipulation of pest species. Although the sex determination cascade and doublesex (dsx) gene functions have been well described in some holometabolous insects, little is known about this cascade in hemimetabolous insects. In this study, we identified the dsx homolog in Gryllus bimaculatus, which belongs to the Orthoptera order and is an important model for developmental and evolutionary biology. We found that Gbdsx has two alternative splicing isoforms (male-specific Gbdsx^M and non-sex-specific Gbdsx^C). Using RNAi-mediated knock-down of Gbdsx^M in 6th-instar nymphs resulted in adult male forewings showing feminized vein development and abnormal external genitalia. CRISPR/Cas9 knockout of Gbdsx in embryos resulted in adult males becoming pseudofemales, with feminized forewings and abnormal external and internal genitalia. Additionally, the pseudofemales created by Gbdsx knockout demonstrated normal courtship trends and aggressive behavior but no actual mating behavior. However, the knockout and knock-down of Gbdsx in female crickets does not affect their sexual traits or fertility. Our results suggest that Gbdsx^M plays a critical role in the development of male cricket sexual traits, courtship and mating behavior, which furthers our understanding of sex determination in hemimetabolous insects.