The Chinese tongue sole (Cynoglossus semilaevis) is a commercially important mariculture species; however, its fertilization and hatching rates under artificial conditions remain relatively low. Zona pellucida proteins (ZPs), which mediate sperm–egg binding, were previously identified as differentially expressed genes between newly differentiated ovaries and testes in C. semilaevis. In this study, we identified 25 ZPs of C. semilaevis through genomic analysis and classified them into five subfamilies. All genes possessed a conserved ZP domain, characteristic of the gene family from mammals to teleosts. Among them, nine genes were highly expressed in ovary cells, with the expression levels increasing during ovarian development, while another three genes were predominantly expressed in liver cells. Protein–protein interaction analysis predicted that 12 ZPs interacted with key reproductive regulators such as Gdf9, Arid4a, Arid4b, and Rbl, which were involved in steroidogenesis, sperm–egg recognition, and folliculogenesis. Functional analyses using RNA interference revealed that Cszpc7-1 knockdown in ovarian cells led to the downregulation of cyp19a, esr2, bmp15, and adamts-1, while the expression of rbl, gnas, adgrl1, and adgrl2 was upregulated. In contrast, Cszpax1 knockdown resulted in decreased expression of cyp19a, foxl2, arid4a, and zeb1, along with upregulation of arid4b, ogg1, and gdf9. These results suggested that ZP genes might contribute to ovarian homeostasis by regulating steroid hormone synthesis, follicular development, and ovulation. This study contributed to a deeper understanding of the reproductive mechanisms of C. semilaevis and provided evolutionary insights into the functional divergence of the ZP gene family across teleosts.

Marine fishes are important contributors to biodiversity conservation and socioeconomic sustainability. While genomic and multi-omics datasets for marine fish species have expanded exponentially in recent years, their systematic integration remains underexplored, hindering comprehensive investigations into gene regulation and biological systems. To bridge this gap, we developed the Marine Fish Database (MFDB; http://marinefishdb.cn), the first integrative genomics platform specifically designed for marine teleosts. MFDB compiles fragmented multi-omics resources across 89 species, encompassing genome assemblies, phylogenomic reconstructions, collinearity maps, pan gene sets, gene architectures, functional annotations, expression profiles, and evolutionary gene family analyses. MFDB further delivers specialized analytical modules, including tissue-specific gene co-expression networks, lineage-defining core gene repertoires, and macrosynteny-driven chromosomal evolution models. The platform features an intuitive and programmable interface, enabling multiscale queries, cross-omics data mining, and dynamic visualization of multidimensional biological interactions. These functionalities collectively empower users to decipher how genomic elements orchestrate phenotypic outcomes through multi-layered regulatory cascades. By integrating rapidly expanding omics datasets with robust analytical pipelines, MFDB establishes a scalable framework to accelerate hypothesis-driven discoveries in marine fish biology, evolutionary adaptation, and ecological resilience research, and further provides an easy-to-operate information platform for the molecular breeding of marine fish species.

Chinese tongue sole (Cynoglossus semilaevis), an economically important mariculture species in China, exhibits pronounced sexual dimorphism in growth, underscoring the importance of elucidating sex regulatory mechanisms for aquaculture development. Heterogeneous nuclear ribonucleoprotein K (hnrnpk) critically regulates mammalian reproductive development, yet its role in fish sex regulation remains elusive. Here, we systematically investigated the underlying function and mechanisms of hnrnpk in C. semilaevis through integrated molecular cloning, expression profiling, upstream regulatory analysis, functional assays, and transcriptome sequencing. We found that hnrnpk was highly expressed in the gonad and liver, with female-biased expression during gonadal development. Promoter activity assays revealed that sox2 and c-Jun enhanced hnrnpk transcription, whereas foxl2 and ar suppressed it. Additionally, hnrnpk was directly targeted by miR-460a-5p in C. semilaevis, revealing multi-level transcriptional and post-transcriptional regulation. Functional analyses showed that hnrnpk regulated cyp19a1a in a cell type-dependent and dose-sensitive manner: the expression of cyp19a1a was both upregulated in hnrnpk-knockdown ovarian cells and hnrnpk-overexpression testicular cells. Interestingly, foxl2 was upregulated in hnrnpk-knockdown ovarian cells but suppressed in hnrnpk-overexpression testicular cells, which showed the distinct regulation mechanisms in the different sexual programs. Transcriptomic analyses further revealed that several sex-related genes (sox9a with downregulation, etc.) were significantly regulated, and cell development and cycle pathways were dramatically enriched in functional enrichment analyses. This might indicate that hnrnpk overexpression drives C. semilaevis testis (CSTE) toward feminization reprogramming through sox9 switching and multi-pathway perturbations. Overall, our findings might reveal that hnrnpk, a female-biased gene regulated by miR-460a-5p and transcription factors, influences sex-related gene expression through sox9 switching. This study will offer new insights for C. semilaevis hnrnpk into sex determination and also provide a potential target for monosex breeding in aquaculture.

The red-spotted grouper nervous necrosis virus (RGNNV) exhibits high pathogenicity in larval C. sonnerati, yet the immune molecular mechanism remains unclear. Non-coding RNAs (ncRNAs) are vital in the host’s immune responses during viral infection. However, there has been no study on ncRNA research for this species to date. We systematically identified 105 DE microRNAs (miRNAs), 157 DE long non-coding RNAs (lncRNAs) and 31 DE circular RNAs between the infection group and control group. Functional enrichment analysis revealed that these differentially expressed genes were significantly enriched in pathways associated with innate immune defense, inflammatory, and cell death, such as JAK-STAT signaling pathway, NF-κB signaling pathway, apoptosis, and necroptosis. Furthermore, the lncRNA–miRNA–mRNA interaction network involving miR-93 was constructed, which may represent a promising candidate therapy target for future investigations. This study presents the first comprehensive ncRNA transcriptome dataset of C. sonnerati infected with RGNNV, identifies key antiviral defense and cell death-related genes and hub pathways, and thereby identifies miR-93-involved lncRNA–miRNA–mRNA network and key targeted genes (STAT1, TRIM25, UNC93B, IL12RB1, IRF8, CDKN1A, FCGR1A) as hub molecular regulators in immune response of this species.