The zig-zag eel (Mastacembelus armatus) is a unique economic fish species in China and exhibits significant dimorphism of male and female phenotypes. Cultivating all-male seedlings can significantly improve production efficiency. To investigate sex differentiation and gonadal development in M. armatus, high-throughput sequencing technology was used to analyze the transcriptomes of male and female gonads at different developmental stages, both before and after sex differentiation. We identified key genes involved in sex differentiation, male-specific differentially expressed genes (DEGs), including dmrt1, amh, sox9, gsdf, and dmrt2b, and female-biased DEGs, including foxl2, rspo1, gdf9, bmp15, and wnt4. GO and KEGG enrichment analyses revealed that signaling pathways such as MAPK, Wnt, and TGF-β play significant roles in sex differentiation in M. armatus. The expression levels of 13 sex-related genes, including dmrt1, sox9, amh, foxl2, rspo1, and wnt4, were determined by RT–qPCR in addition to RNA sequencing. RT-qPCR validation results were consistent with the transcriptomic data, confirming the reliability of our findings. This research provides valuable insights into the mechanisms of sex differentiation in M. armatus and lays a foundation for developing all-male populations in aquaculture.

Hemibagrus guttatus is a large omnivorous fish of significant economic value, listed as a Class II protected species in the National Key Protected Wildlife List in 2021 in China. To provide a theoretical foundation for the artificial breeding of H. guttatus, this study employs high-throughput transcriptome sequencing of testes and ovaries to elucidate the molecular regulatory pathways involved in sex differentiation. Because H. guttatus exhibits no obvious sexual dimorphism even during the breeding season, the distinctive contribution of this study compared with previous gonadal-transcriptomic investigations in other Siluriformes lies not only in documenting sex-biased genes but also in providing a molecular foundation for developing non-lethal sex-identification methods for this morphologically indistinguishable species. A total of 303,192,896 raw reads were obtained, with an effective data rate of 98.4%, indicating high sequencing quality. Differential expression analysis identified 8694 genes, including 6369 upregulated in testes and 2325 upregulated in ovaries. Among these, 88 genes were functionally annotated as sex-related, with 62 testis-biased genes such as spata17, sox9, and dmrt1, and 26 ovary-biased genes including cyp19a, wnt8, and sox12. KEGG pathway enrichment analysis revealed that the TGF-β signaling pathway, insulin secretion, and steroid hormone biosynthesis may play crucial roles in gonadal development and differentiation in H. guttatus. The expression patterns of key genes such as hsd11b1, amh, and insl3 were validated by quantitative real-time PCR, showing consistency with the transcriptome results. These findings lay a molecular foundation for understanding the regulatory mechanisms of sex differentiation in H. guttatus, and provide candidate genes for further investigation into the genetic basis of gonadal development, which is essential for improving artificial reproduction and selective breeding practices.