Combined transcriptome and function analyses of peripheral blood white cells highlight the disease-resistant mechanism of CD79a/CD79b in Chinese tongue sole (Cynoglossus semilaevis).

Genomic selection accelerates genetic improvement of tolerance to visceral nodular disease in the half-smooth tongue sole (Cynoglossus semilaevis).

Transcriptome–metabolome integration uncovers immune–metabolic defense mechanisms in Cynoglossus semilaevis against bacterial infections

Construction of a multi-tissue immune atlas for Chinese tongue sole (Cynoglossus semilaevis) reveals granulocyte functional adaptation and underlying regulatory mechanisms.

Chinese tongue sole (Cynoglossus semilaevis) is an important mariculture product in northern China, exhibiting significant sexual dimorphism: females grow 2-4 times faster than males and ultimately attain much greater body weights. As a well-known transcription factor crucial for regulating sex differentiation, foxl2 has been characterized in various mammals. Herein, we identified and characterized three foxl genes, foxl1, foxl2a and foxl2l. Three foxl genes exhibited a gonad-biased expression pattern, where foxl2a showed higher expression in ovary than in testis, while foxl1 and foxl2l exhibited higher expression in testis. All foxl genes were detected in testes and ovaries by ISH; foxl1/foxl2l were expressed in oocytes and sperm, and foxl2a in granulosa cells and sperm. Overexpression of foxl in testicular cells led to KEGG enrichment in DNA repair, MAPK, FOXO and progesterone-mediated oocyte maturation pathways. In tongue sole testicular cell line, knockdown of foxl1 and foxl2l resulted in upregulation of multiple male-related genes. In contrast, knockdown of foxl2a led to decreased expression of aromatase genes and increased expression of ctnnb1, indicating that foxl2a is more closely associated with female differentiation and maintenance. Our study investigated the functions of the foxl gene family in teleosts and offers valuable insights into their role in sex differentiation and gonadal maintenance in teleost fish.

Half-smooth tongue sole has high nutritional value due to its delicious meat and high protein content. However, its high protein content makes it highly susceptible to spoilage caused by microbial action. This study utilized plasma-activated water to pretreat half-smooth tongue sole, which was then subjected to various packaging methods: CK (air packaging), VP (vacuum packaging), MAP1 (75% CO2/5% O2/20% N2), MAP2 (20% CO2/5% O2/75% N2), and MAP3 (75% CO2/10% O2/15% N2). The packaged samples were stored at −1 °C. Preservation efficacy was assessed by monitoring changes in microbial counts and physicochemical quality indicators throughout storage. The findings revealed a progressive increase in microbial counts, a deterioration in fish quality, and a darkening of color over extended storage periods. During superchilling storage, the increase in total volatile basic nitrogen (TVB-N) and K value was markedly reduced in the MAP1 group. Regarding protein stability, the MAP1 group exhibited a slower rise in carbonyl content as well as a slower reduction in total sulfhydryl content, further confirming its superior preservation effect. Moreover, this group demonstrated excellence in maintaining the secondary and tertiary structures of myofibrillar proteins, thereby minimizing the structural damage of fish during superchilling storage. In summary, based on observed microbial and protein changes, MAP1 (75% CO2/5% O2/20% N2) was the most effective in preserving quality and extending shelf life.

Sexual size dimorphism (SSD) refers to the phenomenon where males and females of the same species exhibit differences in overall or partial body size, and it is widespread among mammals, birds, reptiles, and fish. Notably, this dimorphism is significantly influenced by the sexually dimorphic secretion of growth hormone (gh), a key pituitary-derived growth regulator. Commonly, the secretion of gh is positively regulated by glucagon family members such as growth hormone-releasing hormone (ghrh) and adenylate cyclase-activating polypeptide 1 (adcyap1). To explore the stimulators for pituitary hormones (especially gh) in the teleost, we performed genome-wide identification and functional characterization of the glucagon family on Chinese tongue sole (Cynoglossus semilaevis) that exhibits typical female-biased sexual size dimorphism. Four members of adcyap1/vasoactive intestinal polypeptide(vip)/ghrh family and ten members of their receptor family were identified. Expression pattern analysis revealed high expression of adenylate cyclase-activating polypeptide 1b (adcyap1b) and its receptors in the brain. Moreover, two alternative splice variants for the adcyap1b gene were discovered, resulting from the skipping of exon 4. Following the acquisition of the two eukaryotic recombinant protein splice variants (ADCYAP1b_tv1 and ADCYAP_tv2) from HEK 293T cells, incubation experiments were conducted using C. semilaevis pituitary cell line. The results demonstrated that both variants promoted the expression of gh, pro-opiomelanocortin (pomc), and corticoliberin (crh), but ADCYAP1b_tv1 had a significantly stronger effect and uniquely stimulated prolactin (prl) and somatolactin (sl). This study demonstrates a functional divergence between the two ADCYAP1b splice variants in teleosts, with ADCYAP1b_tv1 acting as a more potent and versatile pituitary hormone stimulator. Further research on their receptor-binding affinity and downstream signaling pathways would be valuable for exploring the mechanism underlying sexual size dimorphism.

The wnt gene family encodes a group of highly conserved secreted glycoproteins that play essential roles in vertebrate development, including tissue patterning, cell differentiation, and gonadal regulation. However, the genomic organization, evolutionary dynamics, and functional roles of Wnt signaling components in flatfish remain poorly understood. In this study, we performed a comprehensive genome-wide identification, evolutionary characterization, expression profiling, and functional analysis of wnt genes in Cynoglossus semilaevis, a flatfish species exhibiting ZW/ZZ sex determination and temperature-induced sex reversal. A total of 20 wnt genes were identified and classified into 13 subfamilies, displaying conserved structural organization and phylogenetic relationships consistent with other teleosts. Chromosomal mapping revealed lineage-specific WNT clusters, including a unique wnt3-wnt7b-wnt5b-wnt16 block, as well as syntenic associations with reproduction-related genes (e.g., adipor2, sema3a, nape-pld, erc2, lamb2), suggesting coordinated genomic regulation. Tissue transcriptome analysis demonstrated strong sex- and tissue-biased expression patterns, with wnt5a predominantly expressed in ovaries and wnt5b specifically upregulated in pseudo-male testes. Functional assays revealed that knockdown of wnt5a or wnt5b induced testis-specific genes (sox9b, tesk1) and suppressed ovarian markers (foxl2, cyp19a1a), indicating antagonistic regulatory roles in gonadal fate determination. Promoter analysis identified yy1a as a selective repressor of wnt5b, but not wnt5a, providing a mechanistic basis for paralog divergence. Furthermore, pull-down combined with LC-MS/MS analysis showed that WNT5b interacts with proteins enriched in ribosome biogenesis and ubiquitin-mediated proteolysis, suggesting a role in translational regulation and protein turnover during spermatogenesis. Together, these findings establish WNT5 signaling-particularly wnt5b-as a key driver of testicular development in C. semilaevis and provide new insights into the molecular mechanisms underlying sex differentiation and sex reversal in flatfish.

Transforming growth factor β (TGF-β) superfamily members are critical in teleost sex determination and differentiation. Tgfb2b is an important TGF-β ligand gene exhibiting dominant expression in the ovary of Chinese tongue sole (Cynoglossus semilaevis), yet its function in sex regulation remains unclear. In the present study, the gene expression pattern, transcriptional regulation, and knockdown effect were examined. Its expression persisted and showed a gradual increase throughout ovarian development from 3 months to 1.5 years post-hatching. In situ hybridization (ISH) revealed that the gene was distributed across oocytes at stages I–III, while scarcely detectable in the testis. The transcriptional factors CCAAT/enhancer binding protein α (C/EBPα) and Jun proto-oncogene AP-1 transcription factor subunit (c-Jun) could repress the activity of tgfb2b promoter. In vitro knockdown of tgfb2b in C. semilaevis ovarian cells led to downregulation of its downstream genes (e.g., smad1 and smad2) as well as other sex-related genes (e.g., foxl2 and esr2b). Moreover, multi-omics analysis indicated that, in C. semilaevis gonads, a miRNA named novel-m0083-3p showed an opposite expression pattern with tgfb2b and might have a binding site with the gene. By dual-luciferase assay, tgfb2b was validated to be directly targeted and suppressed by the miRNA. These results demonstrate that tgfb2b plays a significant role in ovarian differentiation and development. Further functional and molecular studies on the interplay between tgfb2b and the foxl2–cyp19a–esr axis will help elucidate the regulatory network underlying sex development in teleost.

Chinese tongue sole (Cynoglossus semilaevis) is an important marine aquaculture species that exhibits pronounced female-biased sexual size dimorphism, which limits the economic value of male stocks. The F4 generation of genome-edited dmrt1-/- ZZ males carried a stable 8 bp deletion in dmrt1, developed ovarian lamella-like gonads and were completely sterile, as in vitro fertilization with their gonads produced no viable embryos. From 12 to 15 months post-hatch (mph), their growth rate was significantly higher than that of wild-type males and ultimately comparable to that of females. They weighed 3.2-fold heavier and measured 1.38-fold longer than their 15 mph wild-type male counterparts. Muscle nutritional composition and most texture traits of dmrt1-/- ZZ males did not differ from wild-type females. However, their fat content was significantly lower than that of wild-type males, while their Omega6 content was significantly higher. Metabolomic analysis identified 1262 metabolites and revealed differential enrichment of pathways related to amino acids, energy, and antioxidant and neuromuscular metabolism, without evidence of detrimental shifts. Overall, dmrt1 editing yields a fast-growing, functionally sterile male line whose flesh quality is similar to that of wild-type tongue sole, supporting its potential use in sex-control breeding in Chinese tongue sole and providing a valuable model for studying sexual size dimorphism.

Comparison of toxic effects of phenanthrene and perfluorooctane sulfonate on Chinese tongue sole Cynoglossus semilaevis larva in a muti-biomarker approach.

EED226 treatment targets key immune genes to suppress LPS response in Chinese tongue sole splenocytes.

SHP-1 regulates T cell-mediated early adaptive immunity in Chinese tongue sole (Cynoglossus semilaevis) infected with Mycobacterium marinum.

Genome-wide characterization and immune-modulatory roles of PPAR genes in Cynoglossus semilaevis: insights into inflammation regulation and host defense.

BackgroundChinese tongue sole (Cynoglossus semilaevis) is now a commercially important flatfish species widely farmed in China. In recent years, frequent outbreak of vibriosis has caused high mortality rates and enormous economic losses to the aquaculture industry of Chinese tongue sole. The intestinal microbiome plays a crucial role in host immunity and protection against pathogen invasion. However, the interactions between the intestinal microbiome and host genes in vibriosis remain poorly understood.MethodsIn this study, we investigated the changes in intestinal histopathology, intestinal microbiome and host gene expression in resistant and susceptible individuals at 7 days post infection with Vibrio harveyi, and identified the host gene-microbe correlations. Histopathological sections were conducted to detected the histopathological changes in the posterior intestinal tissues of resistant and susceptible individuals. 16S rRNA sequencing was performed to characterize the changes in intestinal microbial community. RNA-seq was used for the identification of host differentially expressed genes (DEGs). The associations between intestinal microbes and host genes were illustrated by perform an integrated analysis of the differential intestinal microbes and host DEGs.ResultsObvious histopathological differences were observed between the resistant and susceptible groups in terms of inflammatory cells infiltration, and tissue dissociation of mucosal layer. 16S rRNA sequencing analysis indicated that Vibrio increased but Stenotrophomonas, Chryseobacterium, Delftia, and Salinivibrio decreased in the susceptible group. Compared to the control group, 1,986 differentially expressed genes (DEGs) were detected in the susceptible group, significantly more than the 310 DEGs found in the resistant group. DEGs in the susceptible group were significantly enriched in immune-related GO terms, such as antigen processing and presentation, MHC protein complex, and immune response, and pathways, including antigen processing and presentation, phagosome, and proteasome. Through an integrative analysis of differential intestinal microbes and host DEGs, 207 strong gene-microbe correlations were identified.ConclusionsThe results of this study indicate that V. harveyi infection severely damages the intestinal tissue and substantially alters the composition of intestinal microbiome and the expression of host genes, especially in susceptible individuals. Strong gene-microbe correlations may imply that the intestinal microbiome may interact with host genes to collectively regulate the vibriosis resistance in Chinese tongue sole, but the causality between them remains unestablished and requires further validation.

Ftz-f1 is involved in gonadal development by transcriptionally regulating the cyp19a1 gene in Chinese tongue sole (Cynoglossus semilaevis).

Identification of functional ncRNAs in the liver reveals a novel regulatory mechanism associated with the dynamic balance of growth and reproduction in Cynoglossus semilaevis

The anti-Müllerian hormone (amh) and its receptor, amhr2, along with the downstream bone morphogenetic protein receptors (bmprs), have been recognized as the central regulators in teleost sex determination (SD) and differentiation. However, their evolution and function in reproduction among diverse teleost lineages may represent species-specific patterns and still need more explanation. In this study, systematic investigations of amh signaling genes, including amh, amhy (Y-linked paralog of amh), amhr2, bmpr1, and bmpr2, were conducted among teleost species. The results revealed generally conserved gene copy number, phylogeny, structure, and synteny, among teleost amh signaling genes. Notably, significantly accelerated evolutionary rates (dN/dS) were found in teleost amhy compared to amh, and amh exhibited faster molecular evolution in amhy-SD teleosts than in non-amhy-SD teleosts, suggesting their enhanced evolutionary plasticity in teleosts. Expression profiling identified testis-biased expression of the most amh signaling genes in fish species with different SD genes and mechanisms, including Lateolabrax maculatus and Dicentrarchus labrax from Order Perciformes, Cynoglossus semilaevis and Paralichthys olivaceus from Order Pleuronectiformes, and Salmo salar and Oncorhynchus mykiss from Order Salmoniformes, with ovary-biased expression also found in Salmoniformes. A weighted gene co-expression network analysis further uncovered strong species-specific functional interactions between amh signaling components and genes of germ-cell development, the meiotic process, etc. Collectively, the integrated evidence from this study supports the hypothesis that amh signaling provides the key molecules governing sex differentiation in a species-specific manner in diverse teleost lineages, independent of its SD role, and interacts with functions of both testis and ovary development.

Role of the cell cycle-related gene cdk2 and its associated ceRNA network in sexual size dimorphism of Cynoglossus semilaevis.

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.