PSI/Masc-Dsx Regulatory Network in Silkworm Sex Determination Pathway

In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), the sex determination cascade is regulated by the cooperative actions of P-element somatic inhibitor (BmPSI) and Masculinizer (BmMasc), which control the sex-specific splicing of doublesex (Bmdsx). However, the functional interplay between these two critical factors remains poorly understood. In this study, we investigated the cooperative roles of BmPSI and BmMasc through transgenic overexpression and pull-down assays coupled with mass spectrometry. Co-overexpression of BmPSI and BmMasc resulted in female-specific lethality, whereas overexpression of either gene alone caused no lethality. Pull-down coupled with mass spectrometry identified 52 common interacting proteins of BmPSI and BmMasc, including Lethal(3)neo18, a mitochondrial respiratory chain Complex I core subunit, suggesting a potential link between sex determination and mitochondrial function. Transcriptome analysis of previously generated Bmdsx knockout mutants (Bmdsx^M, Bmdsx^F, and Bmdsx^C) revealed 703 commonly dysregulated genes, which were predominantly expressed in gonads and enriched in cytoskeletal and metabolic pathways. Notably, the oxidative phosphorylation pathway was enriched in both the Bmdsx mutant transcriptome and the BmPSI/BmMasc common interactome. Collectively, these findings indicate that BmPSI and BmMasc cooperate to regulate sex determination, and raise the possibility that synergistic disruption of mitochondrial function may underlie the female-specific lethality observed upon their co-overexpression. This study provides new insights into the molecular crosstalk between sex determination and mitochondrial metabolism in insects.