Molecular characterization of <i>transformer</i> , <i>transformer‐2</i> , and <i>doublesex</i> genes in the carambola fruit fly, <i>Bactrocera carambolae</i> , revealing potential for genetic pest management

Globalization and climate change may be driving the spread of the quarantine fruit fly, Bactrocera carambolae, highlighting the need for ecofriendly control methods like the Sterile Insect Technique (SIT), which relies on releasing sterile males to reduce wild populations. However, developing effective strains through genetic manipulation of sex-determination genes remains challenging due to limited knowledge of this pathway in B. carambolae. In this study, the structures of three key orthologous genes-transformer (tra), transformer-2 (tra-2), and doublesex (dsx)-were characterized and compared with those of other Bactrocera species. These genes were found to share conserved structures, following a bottom-up pattern from Bcardsx to Bcartra-2 and Bcartra, with intron retention observed in Bcartra as a structural variation. Developmental expression analysis revealed that the splicing patterns of Bcartra first appeared at 6 hours post oviposition (hpo), with sex-specific patterns established by 10 hpo. The expression profiles in B. carambolae were similar to those in the closely related species B. dorsalis, although gene expression occurred later in B. carambolae. RNAi knockdown of Bcartra resulted in nearly all phenotypic males, supporting the role of BcarTRA in controlling the sex-specific splicing of Bcartra itself and the downstream genes doublesex and fruitless. Additionally, in silico predictions of protein interactions (TRA^F, TRA-2, and RBP1) and putative cis-regulatory elements on Bcartra pre-mRNA suggested specific binding events occur at these conserved sites. These findings contribute to the development of potential genetic tools for pest control and provide insights into the evolutionary relationships among these orthologous genes.