The Intersex Gene Is Crucial for Female Fertility and Embryonic Viability in the Red Flour Beetle, Tribolium castaneum

The intersex (ix) plays a pivotal role in the sex determination pathway of Drosophila melanogaster. The Drosophila Ix protein interacts with female-specific Doublesex protein to regulate female sexual differentiation. In this study, we functionally characterized the ix homologue (Tcix) in the red flour beetle, Tribolium castaneum. The Tcix gene comprises of four exons and three introns, and encodes a protein of 166 amino acids that contains a conserved 'Mediator complex subunit 29' (Med-29) domain. The pre-mRNA of Tcix splices in a non-sex-specific manner and is expressed across all the developmental stages of T. castaneum. RNA interference-mediated knockdown of Tcix in adult beetles led to a significant reduction in its expression in both sexes. Knockdown males did not show any phenotypic changes, but knockdown females displayed compromised ovarian development, resulting in reduced fecundity and failure of eggs to hatch. Interestingly, the expression of vitellogenin-1 (Vg-1), a key gene involved in oocyte development, was significantly downregulated in these females. Further, parental RNAi of Tcix demonstrated the failure in the hatching of eggs, despite no significant effects on the number of eggs laid. The phylogenetic analysis of Tcix revealed its close clustering with other coleopterans, which supports its evolutionary conservation. Overall, our study establishes the critical role of Tcix in female fertility and embryonic development in T. castaneum.