EcDmrt2 promotes male sexual development by regulating EcIAG expression in Exopalaemon carinicauda

The Doublesex and Mab-3 related transcription factor (DMRT) family genes are crucial for sex determination and differentiation across animals, but its genetic role in decapod crustaceans remains poorly understood. Although previous RNAi studies suggest their involvement in sexual development, definitive genetic evidence is still missing. Here, we found that EcDmrt2 in Exopalaemon carinicauda exhibits a male-biased expression pattern at the critical sexual differentiation stage and adult stage, making it a promising candidate gene for a sex regulator. To reveal its function, we employed the CRISPR-Cas9 to target the DM domain of EcDmrt2 and generate stable homozygous mutants. Through analyzing the phenotype of these homozygous mutants, we found that about 20% of EcDmrt2 mutants of genetic males exhibited external feminization, while the testicular morphology and germ cells remained unchanged. Comparative RNA-seq revealed that the depletion of EcDmrt2 led to remarkably downregulation of genes essential for testicular structural maintenance and spermatogenesis, while a comprehensive network of female development associated genes were progressively upregulated. Additionally, compared to wild-type males, genetically male EcDmrt2 mutants with external feminization showed nearly complete ablation of the insulin-like androgenic gland hormone ( EcIAG ) expression. These phenotypic and molecular evidence collectively demonstrate that EcDmrt2 can regulate the expression of EcIAG and further affect the gene expression responsible for the development of testis, partially change the secondary sex characteristics. The results highlight the functional diversity of DMRT genes in sexual development of decapods compared to other taxa, providing key insights into the sex determination mechanisms of crustaceans.