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Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs. [ABSTRACT FROM AUTHOR]

The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes. [ABSTRACT FROM AUTHOR]

The Chinese tongue sole (Cynoglossus semilaevis) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation mechanisms receives a great deal of attention. Forkhead Box O (FoxO) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that foxo genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six Csfoxo members (Csfoxo1a, Csfoxo3a, Csfoxo3b, Csfoxo4, Csfoxo6-like, and Csfoxo1a-like) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4. The siRNA-mediated knockdown of the Csfoxo1a, Csfoxo3a, and Csfoxo3b genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of foxo's function and provide valuable data for studying the male differentiation of tongue sole. [ABSTRACT FROM AUTHOR]

Simple Summary: Chinese tongue sole (Cynoglossus semilaevis) typically displays female-biased sexual size dimorphism (SSD), but its epigenetic regulatory mechanisms are poorly understood, especially the role of circRNAs. To explore the function of circRNAs in Chinese tongue sole SSD, we firstly identified differentially expressed circular RNAs (DE circRNAs) in female, male, and pseudo-male C. semilaevis. Secondly, the ceRNA network containing DE circRNAs, miRNAs, and mRNAs in the three genders was constructed. Among the ceRNA network, several circRNAs such as novel_circ_004374 and novel_circ_014597 may regulate hipk2 expression by sponging miR-130-x. It is also worth exploring whether or how novel_circ_008696 regulates setd2 by binding to novel-m0387-3p. The present study provided the cirRNA and its ceRNA network that potentially regulate C. semileavis female-biased SSD for the first time. Sexual size dimorphism (SSD), which is the sexual differences in body size, has been widely reported in various species including fishes. For Chinese tongue sole (Cynoglossus semilaevis), a flatfish exhibiting typically female-biased SSD, little is known for its epigenetic regulation mechanism, especially the role of circRNAs. Here, we identified the differently expressed abundances of circRNAs in females, males, and pseudo-males to explore the potential functions of circRNAs in Chinese tongue sole SSD. In total, 14,745 novel circRNAs were screened, among which 1461 DE circRNAs were identified from the brain, gonad, liver, and muscle in female, male, and pseudo-male individuals. The ceRNA network was subsequently constructed, including 10 circRNAs, 26 mRNAs, and 11 miRNAs. These DE mRNAs were mainly related to the mRNA surveillance pathway, metabolic pathways, and cellular senescence. Importantly, the ceRNA network has revealed that several circRNAs such as novel_circ_004374 and novel_circ_014597 may regulate homeodomain interacting protein kinase 2 (hipk2) expression by sponging miR-130-x. It is also worth exploring whether or how novel_circ_008696 regulates SET Domain Containing 2, histone lysine methyltransferase (setd2), which in turn affects the epigenetic patterns of different sexual individuals. The present study not only enriches the knowledge on the potential roles of circRNA in the physiological process, but also provides new clues for the explanation of fish SSD. In future studies, the precise function and involvement of circRNAs in female-biased SSD will require more efforts. [ABSTRACT FROM AUTHOR]

Simple Summary: In fish, it is obvious that the asynchronous development of the gonads and sexual dimorphism limit the development of aquaculture, so the research into sex-differentiation and gonadal growth is very important. Due to the sexual reversal phenomenon (genetic females becoming phenotypic males), the Chinese tongue sole (Cynoglossus semilaevis) is a great model for investigating sex-differentiation. Herein, we report one gene involved in sex-differentiation and gonadal growth of the Chinese Tongue Sole. The gene cyp26b1 (cytochrome P450 family 26 subfamily b member 1) is a metabolizing Retinoic Acid (RA) enzyme. Since it regulates RA to control sex determination and differentiation, cyp26b1 is considered a critical part of mammals' ovary-antagonizing and testis-determining downstream passageway of Sry (sex-determining region Y) and Sox9 (sry-box transcription factor 9). In fish, the related research is reported only on the Japanese flounder (Paralichthys olivaceus) and zebrafish (Danio rerio). In the current investigation, the identification and expression pattern of the cyp26b1 gene in the Chinese tongue sole suggested that cyp26b1 might impact sex-differentiation and gonadal development. As an RA-metabolizing enzyme, cyp26b1 has a substantial impact on RA-signaling pathways. The cyp26b1 gene from the Chinese tongue sole was cloned and identified in this investigation. The cyp26b1 ORF was 1536 bp in length and encoded a 512 amino acid protein. A quantitative real-time PCR (qPCR) indicated that the cyp26b1 expression is no significant sexual dimorphism in the gonads at the 80 days post-hatching (dph) stages. After 4 months post-hatching (mph), the expression of cyp26b1 showed sexual dimorphism and lower level of expression in the ovaries than in the testes. An in situ hybridization demonstrated that cyp26b1 mRNA was primarily located in the testis. Interestingly, the cyp26b1 mRNA probe was also detected in the ovaries. These results suggested that cyp26b1 participates in the sex-differentiation and gonadal development of the Chinese tongue sole. [ABSTRACT FROM AUTHOR]

Simple Summary: Sexual dimorphism is a phenomenon commonly existing in animals. Chinese tongue sole Cynoglossus semilaevis is an economical marine fish with obvious female-biased size dimorphism. So, it is important to explore the molecular mechanism beyond gonadal development for sex control in aquaculture industry. RNA-binding protein Ewing Sarcoma protein-like (ewsr1) gene is important for mouse gonadal development and reproduction, however there are limited studies on this gene in teleost. In this study, two ewsr1 genes were cloned and characterized from C. semilaevis. The ewsr1-w gene, located in W chromosomes, showed female-biased expression during C. semilaevis gonadal development. In addition, knock-down effect and transcriptional regulation of Cs-ewsr1-w further suggested its essential role in ovarian development. This study broadened our understanding on ewsr1 function in teleost, and provided genetic resources for the further development of sex control breeding techniques in C. semilaevis aquaculture. Ewsr1 encodes a protein that acts as a multifunctional molecule in a variety of cellular processes. The full-length of Cs-ewsr1-w and Cs-ewsr1-z were cloned in Chinese tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of Cs-ewsr1-w was 1,767 bp that encoded 589 amino acids, while Cs-ewsr1-z was 1,794 bp that encoded 598 amino acids. Real-time PCR assays showed that Cs-ewsr1-w exhibited significant female-biased expression and could be hardly detected in male. It has the most abundant expression in ovaries among eight healthy tissues. Its expression in ovary increased gradually from 90 d to 3 y with C. semilaevis ovarian development and reached the peak at 3 y. After Cs-ewsr1-w knockdown with siRNA interference, several genes related to gonadal development including foxl2, sox9b and pou5f1 were down-regulated in ovarian cell line, suggesting the possible participation of Cs-ewsr1-w in C. semilaevis ovarian development. The dual-luciferase reporter assay revealed that the -733/-154 bp Cs-ewsr1-w promoter fragment exhibited strong transcription activity human embryonic kidney (HEK) 293T cell line. The mutation of a MAF BZIP Transcription Factor K (Mafk) binding site located in this fragment suggested that transcription factor Mafk might play an important role in Cs-ewsr1-w basal transcription. Our results will provide clues on the gene expression level, transcriptional regulation and knock-down effect of ewsr1 gene during ovarian development in teleost. [ABSTRACT FROM AUTHOR]

Background: Infectious diseases have caused huge economic loss and food security issues in fish aquaculture. Current management and breeding strategies heavily rely on the knowledge of regulative mechanisms underlying disease resistance. Though the intestinal microbial community was linked with disease infection, there is little knowledge about the roles of intestinal microbes in fish disease resistance. Cynoglossus semilaevis is an economically important and widely cultivated flatfish species in China. However, it suffers from outbreaks of vibriosis, which results in huge mortalities and economic loss. Results: Here, we used C. semilaevis as a research model to investigate the host-microbiome interactions in regulating vibriosis resistance. The resistance to vibriosis was reflected in intestinal microbiome on both taxonomic and functional levels. Such differences also influenced the host gene expressions in the resistant family. Moreover, the intestinal microbiome might control the host immunological homeostasis and inflammation to enhance vibriosis resistance through the microbe-intestine-immunity axis. For example, Phaeobacter regulated its hdhA gene and host cyp27a1 gene up-expressed in bile acid biosynthesis pathways, but regulated its trxA gene and host akt gene down-expressed in proinflammatory cytokines biosynthesis pathways, to reduce inflammation and resist disease infection in the resistant family. Furthermore, the combination of intestinal microbes and host genes as biomarkers could accurately differentiate resistant family from susceptible family. Conclusion: Our study uncovered the regulatory patterns of the microbe-intestine-immunity axis that may contribute to vibriosis resistance in C. semilaevis. These findings could facilitate the disease control and selective breeding of superior germplasm with high disease resistance in fish aquaculture. 8F1qVDdq8rkntrj1jj62Gc Video Abstract [ABSTRACT FROM AUTHOR]

Blind-side hypermelanosis has emerged as a major concern in commercial rearing environments of the flatfish aquaculture industry. To date, the underlying molecular mechanisms are not well understood. To fill this gap, in this study, whole transcriptomic sequencing and analyses were performed using normal skins and hypermelanic skins of the blind side of Chinese tongue sole (Cynoglossus semilaevis). Differentially expressed long non-coding RNAs (DElncRNAs), miRNAs (DEmiRNAs), and differentially expressed genes as well as their competing endogenous RNA (ceRNA) networks were identified. A total of 34 DElncRNAs, 226 DEmiRNAs, and 610 DEGs were identified. Finally, lncRNA–miRNA–mRNA regulatory networks (involving 29 DElncRNAs, 106 DEmiRNAs, and 162 DEGs) associated with blind-side hypermelanosis were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of 162 DEGs in ceRNA networks identified DEGs (e.g., oca2, mc1r, and ihhb) in pigmentation-related biological processes and DEGs (e.g., ca4, glul, and fut9) in nitrogen metabolism, glycosphingolipid biosynthesis, and folate biosynthesis pathways, as well as their corresponding DElncRNAs and DEmiRNAs to potentially play key regulatory roles in blind-side hypermelanosis. In conclusion, this is the first study on the ceRNA regulatory network associated with blind-side hypermelanosis in flatfish. These new findings expand the spectrum of non-coding regulatory mechanisms underpinning blind-side hypermelanosis, which facilitates the further exploration of molecular regulatory mechanisms of malpigmentation in flatfish. [ABSTRACT FROM AUTHOR]

As intracellular pathogen recognition receptors (PRRs), nucleotide‐binding domain, leucine‐rich repeat containing receptors (NLRs, NOD‐like receptors) are involved in innate immune responses in vertebrates. However, there is no systemic study on NLRs in Chinese tongue sole (Cynoglossus semilaevis), a popular maricultured fish in China. In the present study, a genome‐wide survey of NLRs was performed in C. semilaevis, with the identification of 29 NLRs, including five genes from the NLR‐A subfamily (referred to as CsNOD1‐5), two genes from the NLR‐B subfamily, 18 genes from the NLR‐C subfamily (referred to as CsNLR‐C1 to 18) and four other NLR genes. Phylogenetic analysis implied that CsNOD1‐5 contained conserved functional domains and had orthologous relationships with human NOD1‐5. Moreover, CsNLR‐C genes all possessed the FISHNA domain, which is a fish‐specific NACHT subdomain. Expression analysis showed that CsNOD1‐5 and CsNLR‐C1/2 were ubiquitously expressed in various normal tissues. Bacterial infection with Vibro harveyi revealed distinct expression patterns of all the tested CsNLRs in gill, intestine, trunk kidney, liver and spleen. In particular, CsNOD1‐4 and CsNLR‐C2 were significantly upregulated in gills at 48 h post bacterial infection. In addition, CsNOD3 and CsNOD4 were significantly elevated in infectious intestine, trunk kidney, liver and spleen, revealing that their expressions were more sensitive to bacterial infection than other CsNLRs. Together with the computational protein–protein interaction network of CsNLRs, it was suggested that individual NLR genes had different roles in the innate immune cascades of C. semilaevi against bacterial infection. This study provides valuable information for further studies on CsNLR immune function. [ABSTRACT FROM AUTHOR]

The sustainable development of aquaculture has been impeded by infectious diseases worldwide. However, the genomic architecture and the genetic basis underlying the disease resistance remain poorly understood, which severely hampers both the understanding of the evolution of fish disease resistance traits and the prevention of these diseases in the aquaculture community. Cynoglossus semilaevis is a representative and commercially-important flatfish species. Here we combined genome-wide association study and Fst and nucleotide diversity filtration to identify loci important for the disease resistance. Based on 1,016,774 single-nucleotide polymorphisms (SNPs) identified from 650 Gb genome resequencing data of 505 individuals, we detected 33 SNPs significantly associated with disease resistance and 79 candidate regions after filtration steps. Both the allele frequencies and genotype frequencies of the associated loci were significantly different between the resistant and susceptible fish, suggesting a role in the genetic basis of disease resistance. The SNP with strongest association with disease resistance was located in Chr 17, at 145 bp upstream of fblx19 gene, and overlapped with the major quantitative trait locus previously identified. Several genes, such as plekha7 , nucb2 , and fgfr2 , were also identified to potentially play roles in the disease resistance. Furthermore, the expression of some associating genes were likely under epigenetic regulations between the bacterial resistant and susceptible families. These results provide insights into the mechanism that enable variation of disease resistance to bacterial pathogen infection. The identified polymorphisms and genes are valuable targets and molecular resources for disease resistance and other traits, and for advanced breeding practice for superior germplasm in fish aquaculture. [ABSTRACT FROM AUTHOR]

DNA methylation, the most widely studied and most well-understood epigenetic modification, has been reported to play crucial roles in diverse processes. Although it has been found that DNA methylation can modulate the expression of immune-related genes in teleosts, a systemic analysis of epigenetic regulation on teleost immunity has rarely been performed. In this research, we employed whole-genome bisulfite sequencing to investigate the genome-wide DNA methylation profiles in select disease-resistant Cynoglossus semilaevis (DR-CS, family 14L006) and disease-susceptible C. semilaevis (DS-CS, family 14L104) against Vibrio harveyi infection. The results showed that following selective breeding, DR-CS had higher DNA methylation levels and different DNA methylation patterns, with 3,311 differentially methylated regions and 6,456 differentially methylated genes. Combining these data with the corresponding transcriptome data, we identified several immune-related genes that exhibited differential expression levels that were modulated by DNA methylation. Specifically, DNA methylation of tumor necrosis factor–like and lipopolysaccharide-binding protein-like was significantly correlated with their expression and significantly contributed to the disease resistance of the selected C. semilaevis family. In conclusion, we suggest that artificial selection for disease resistance in Chinese tongue sole causes changes in DNA methylation levels in important immune-related genes and that these epigenetic changes are potentially involved in multiple immune responses in Chinese tongue sole. [ABSTRACT FROM AUTHOR]

The Chinese tongue sole (Cynoglossus semilaevis) is a typical female heterogamete species that exhibits female-biased sexual size dimorphism, which has severely hindered the sustainable development of the species in aquaculture. In the present study, four important somatotropic and reproductive tissues including brain, pituitary, liver, and gonad from 15 females and 15 males were used for transcriptome analysis via RNA-seq. A mean of 37,533,991 high-quality clean reads was obtained from each library and 806, 1482, 818, and 14,695 differentially expressed genes in female and male were identified from the brain, pituitary, liver, and gonad, respectively (fold change ≥ 2 and q < 0.05). Enrichment analyses of GO terms and KEGG pathways showed that nucleic acid-binding transcription factor activity, G-protein-coupled receptor activity, MAPK signaling pathway, steroid biosynthesis, and neuroactive ligand-receptor interaction may be involved in the sexual growth differences. Furthermore, via weighted gene co-expression network analyses, two modules (yellowgreen and salmon4) were identified to be significantly positive-correlated with female-biased sexual size dimorphism. An illustrated network map drawn by these two modules enabled the identification of a series of hub genes, including nipped-B-like protein A (nipbla), transcriptional activator protein Pur-beta-like (purb), and BDNF/NT-3 growth factors receptor (ntrk2). Detailed functional investigation of these networks and hub genes will further improve our understanding of the underlying molecular mechanism of sexual size dimorphism in fish. [ABSTRACT FROM AUTHOR]

β-catenin is a key signalling molecule in the canonical Wnt pathway, which plays a role in cell adhesion, embryogenesis and sex determination. However, little is known about its function in teleosts. We cloned and characterized the full-length β-catenin1 gene from half-smooth tongue sole (Cynoglossus semilaevis), which was designated CS-β-catenin1. The CS-β-catenin1 cDNA consists of 2,346 nucleotides and encodes a protein with 782 amino acids. Although CS-β-catenin1 was transcribed in the gonads of both sexes, the level was significantly higher in ovaries compared to testes. Furthermore, the mRNA level of CS-β-catenin1 was significantly upregulated at 160 days and constantly increased until 2 years of age. In situ hybridization revealed that CS-β-catenin1 mRNA was mainly localized in oocyte cells, especially in stage I, II and III oocytes. When CS-β-catenin1 expression was inhibited by injection of quercetin in the ovaries, levels of CS-Figla and CS-foxl2 mRNA were significantly down-regulated, and CS-dmrt1 was up-regulated, which suggested that CS-β-catenin1 is a potential upstream gene of CS-Figla and is involved in the development of the ovaries, i.e., folliculogenesis. [ABSTRACT FROM AUTHOR]

Aquaporin 1 (AQP1) is a member of the transmembrane water channel family of proteins with special structural features, and two AQP1 paralogous genes (aqp1aa and aqp1ab) are reported in teleosts. In the present study, the aqp1aa gene of half-smooth tongue sole (Cynoglossus semilaevis) was cloned and characterized. The full-length cDNA of aqp1aa is 1411 bp with a 786 bp open reading frame encoding a 261-amino acid putative protein with a characteristic structure consisting of 6 membrane-spanning α-helical domains and two highly conserved asparagine—proline—alanine motifs. Real-time quantitative PCR revealed that aqp1aa mRNA is expressed predominantly in the testis of males and pseudo-males, while its expression is low in the ovary and lowest in doublesex and mab-3-related transcription factor 1(DMRT1) knock out fish and triploid males. In situ hybridization indicated that aqp1aa mRNA is expressed mainly in the germ cells of males and pseudo-males, especially in spermatozoa and spermatids. These results suggest that the aqp1aa may play a role in spermatogenesis of C. semilaevis. [ABSTRACT FROM AUTHOR]

Phenotypic and genetic parameters for growth-related traits in the half-smooth tongue sole, Cynoglossus semilaevis, were estimated in 22 full-sib families produced by normal and neo-male breeding stocks. As phenotypic males with female genotypes, neo-males are harmful in C. semilaevis aquaculture because they reduce overall production. The present study evaluated the difference in the growth-related traits: total length (TL), body weight (BW) and square root of body weight (SQ_BW) at the age of 570 days between normal and neo-male offspring (neo-males used as male parents). The difference in the proportion of females between normal and neo-male offspring was also assessed. Based on the linear mixed model, restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP) were used to estimate various (co)variance components and estimated breeding values (EBVs) of growth-related traits. As a result, all the mean values of the three studied traits were significantly larger in normal offspring than in neo-male offspring. Additionally, the female proportion was significantly larger in normal offspring than in neo-male offspring. Heritability was 0.128±0.066 2 for TL, 0.128±0.065 5 for BW and 0.132±0.062 9 for SQ_BW, all of which were low level heritabilities. The correlation coefficients of EBVs and phenotypic values of the target traits were 0.516 for TL, 0.524 for BW and 0.506 for SQ_BW, all of which were highly significant ( P <0.01). Genetic correlations among TL, BW and SQ_BW were positive high (0.921-0.969) and higher than those of phenotype (0.711-0.748), both of which had low standard errors (0.063-0.123 for genotype, and 0.010-0.018 for phenotype). Compared with normal offspring, neo-male offspring have lower breeding values for each studied trait through EBVs comparison. Therefore, neo-male offspring should not be used as broodstock in a C. semilaevis breeding programs. [ABSTRACT FROM AUTHOR]

Testis-specific protein kinase 1 (Tesk1) is a serine/threonine kinase with unique structural features. In the present study, we cloned and characterized the tesk1 gene of tongue sole, Cynoglossus semilaevis. The full-length tesk1 cDNA consists of 1,672 nucleotides, encoding a 331 amino acid polypeptide with a characteristic structure composed of an N-terminal kinase domain and a C-terminal proline-rich domain. The tesk1 genomic sequence contains eight exons and seven introns. Real-time quantitative PCR revealed that tesk1 mRNA is expressed predominantly in the testis, though the level of expression varied throughout development. We used in situ hybridization to show that tesk1 mRNA is expressed in the spermatids of males and pseudo-males, but not in triploid males. Our results suggest that tongue sole Tesk1 may play a role in spermatogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein, including highly conserved domains of the DEAD-box protein family. The amino acid sequence also shared a high homology with other teleosts. Csvasa expression was mainly restricted to the gonads, with little or no expression in other tissues. Real-time quantitative polymerase chain reaction analysis revealed that Csvasa expression levels decreased during embryonic and early developmental stages and increased with the primordial germ cell proliferation. A typical sexually dimorphic expression pattern of Csvasa was observed during early development and sex differentiation, suggesting that the Csvasa gene might play a differential role in the proliferation and differentiation of male and female primordial germ cells (PGCs). Csvasa mRNA expression levels in neomales were significantly lower than those in normal males and females, indicating that the Csvasa gene might be implicated in germ cell development after sex reversal by temperature treatment. In addition, medaka (Oryzias latipes) PGCs could be transiently labeled by microinjection of synthesized mRNA containing the green fluorescence protein gene and 3′-untranslated region of Csvasa, which confirmed that the Csvasa gene has the potential to be used as a visual molecular marker of germ cells and laid a foundation for manipulation of PGCs in tongue sole reproduction. [Copyright &y& Elsevier]

Genetic sex determination by W and Z chromosomes has developed independently in different groups of organisms. To better understand the evolution of sex chromosomes and the plasticity of sex-determination mechanisms, we sequenced the whole genomes of a male (ZZ) and a female (ZW) half-smooth tongue sole (Cynoglossus semilaevis). In addition to insights into adaptation to a benthic lifestyle, we find that the sex chromosomes of these fish are derived from the same ancestral vertebrate protochromosome as the avian W and Z chromosomes. Notably, the same gene on the Z chromosome, dmrt1, which is the male-determining gene in birds, showed convergent evolution of features that are compatible with a similar function in tongue sole. Comparison of the relatively young tongue sole sex chromosomes with those of mammals and birds identified events that occurred during the early phase of sex-chromosome evolution. Pertinent to the current debate about heterogametic sex-chromosome decay, we find that massive gene loss occurred in the wake of sex-chromosome 'birth'. [ABSTRACT FROM AUTHOR]

Abstract: The small ubiquitin-like modifier (SUMO) pathway is an essential biological process in eukaryote, and Ubc9 is an important E2 conjugating enzyme (UBE2) for SUMO pathway and plays a critical role in cellular differentiation, development and sex modification in various species. However, the relationship between Ubc9 and sex modification and development in fish remains elusive. To elucidate the impact of Ubc9 on sex modification and development, the full length of the cDNA and genomic sequence was cloned from half-smooth tongue sole, Cynoglossus semilaevis. Real-time quantitative RT-PCR demonstrated that ubc9 was ubiquitously expressed in different tissues, and the expression levels varied in the different stages of embryonic and gonadal development. In addition, the expression level was significantly higher in the temperature-treated females than the normal females and males. Moreover, the PET-32-Ubc9 plasmid was constructed and the recombinant protein was expressed in Escherichia coli. Follistatin gene expression was initially up-regulated and FSE genes (cyp19a1a, ctnnb1, foxl2) were initially down-regulated after the injection of Ubc9 protein, prior to 96h eventually recovered to normal levels. Taken together, the results show that Ubc9 is involved in embryogenesis, gametogenesis and sex modification, and exerts an effect on gene expression. [Copyright &y& Elsevier]

Sex differentiation in aquatic fish is important both for theoretical study and practical production, as growth dimorphism frequently appears in different sexes, especially in marine fish. The deciphered genome, identification of the male-determining gene dmrt1 and established genotypic sex screening method make Chinese tongue sole (Cynoglossus semilaevis) an ideal model to study sex differentiation in fish. In this study, comparative gonadal transcriptomic analyses were conducted for genetic females and males at 48, 68, and 108 days post hatching (dph), representing pre-, during- and post-gonadal differentiation stages, although the gonad is not completely differentiated and isolable in 48 and 68 dph individuals, while it is in 108 dph individuals. Altogether, 28 libraries were constructed, and a mean of 46.64 M clean reads was obtained. Differentially expressed gene (DEG) analysis revealed that 179 genes had similar expression patterns in males and females in all three stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the enriched pathways included ubiquitin-mediated proteolysis, lysosomes, and RNA degradation. Moreover, weighted gene coexpression network analyses (WGCNA) identified 14 modules, one of which was closely correlated with female differentiation, exhibiting female-biased expression in all three stages (48, 68, 108 dph). An illustrated core gene interaction network of this module identified 50 genes, most of which are on W chromosomes. Six genes, including two ubiquitin conjugating enzymes, were selected for further investigation, and their female-biased expression was confirmed in even earlier stages, at 10 and 30 dph. These data facilitate our understanding of sex differentiation in fish and provide a genomic rationale for screening candidate genes (preferentially W-linked genes) that could be involved in the female differentiation process. Transcriptomic patterns in tongue sole gonads representing pre-, during, and post-differentiation stages (48, 68, 108 days post hatching) were studied. WGCNA analysis has identified one module closely correlated with female differentiation (Panel A). GO and KEGG enrichment indicated that ubiquitin-related pathways played important role in female differentiation (Panel B and C). Furthermore, the core gene interaction network is constructed (Panel D). The network consists of 50 genes (including four ubiquitin-related genes, two of them belong to the top five centered genes, underlined in red). We have noticed that most genes in the network are on W chromosomes, suggesting that W chromosome-linked genes should be preferentially focused on screening candidates involved in female differentiation. [Display omitted] • Transcriptomic patterns in tongue sole gonads representing pre-, during, and post-differentiation stages were analysed. • 179 DEGs between male and female were shared in all three stages and enriched pathways include ubiquitin mediated proteolysis etc. • WGCNA identified a module closely related to female differentiation, the core network consists of 50 genes (most on W chromosome). [ABSTRACT FROM AUTHOR]

In addition to the typical sexual size dimorphism, considerable size differences within the female population of the Chinese tongue sole (Cynoglossus semilaevis) have become a further bottleneck of the improvement of sole aquaculture. To identify the internal mechanism, transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were employed simultaneously. Transcriptomic analyses of brain, pituitary gland, liver, gonad, and muscle tissues from two female groups with size differences identified 109, 698, 1325, 2299, and 2141 differentially expressed genes (DEGs), respectively. The results of these enrichment analyses suggest that the up-regulation of neuroactive ligand-receptor interaction, cell cycle, DNA replication, and MAPK signaling pathway in the group with larger females may be involved in the regulation of the observed growth differences. WGCNA of DEGs showed that cell cycle and DNA replication might be crucial pathways for accelerating cell growth in the groups with larger females. Finally, a series of hub genes including 6-phosphofructokinase type C (pfkp), ribosome biogenesis protein (wdr12), bleomycin hydrolase (blmh), and semaphorin-3A (sema3a) were recognized by the illustrated network map of modules. The linkage of cell cycle, DNA replication, and hub genes in the growth regulation of C. semilaevis provides further information for a better understanding of growth differences in fish. [Display omitted] • Many DEGs were identified in two female Cynoglossus semilaevis groups transcriptomics. • Cell cycle and DNA replication may be crucial for accelerating growth in large female group. • Hub genes including pfkp , blmh , and sema3a were recognized by network illustration. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum is a major pathogen in aquaculture, posing a substantial threat to the health and sustainability of tongue sole (Cynoglossus semilaevis) farming. This study investigated the genetic basis of immune response in tongue sole by comparing transcriptome profiles of liver and spleen tissues from symptomatic (susceptible) and healthy (resistant) individuals during a natural M. marinum outbreak. Transcriptomic analyses identified differentially expressed genes and enriched pathways related to immune responses. Key genes, including atp6ap1, gpi, and idh3a, were found to be crucial in immune response to M. marinum infection, involved in immune processes such as signal transduction, antigen processing, and metabolic pathways. Protein-protein interaction networks highlighted central hub genes such as nedd8, jun and junb, which play pivotal roles in immune regulation. These findings provide insights into the orchestrated immune responses to mycobacteriosis, which can inform selective breeding strategies for disease-resistant tongue sole strains. This is the first comprehensive transcriptome analysis of M. marinum natural infection in tongue sole, offering valuable data for future research and disease management in aquaculture. • First transcriptome study on M. marinum natural infection in tongue sole. • Nedd8 likely conferred disease resistance to M. marinum through neddylation. • Identified key and hub genes revealed an orchestrated immune response to M. marinum natural infection. [ABSTRACT FROM AUTHOR]

Vibriosis poses a significant threat to marine aquaculture species, including tongue sole (Cynoglossus semilaevis), where outbreaks can lead to mass mortalities and severe economic losses. This study aimed to elucidate the genetic basis of resistance to vibriosis in tongue sole by integrating genome-wide association study (GWAS) and transcriptome analysis. A significant quantitative trait locus (QTL) on chromosome 14 (123Kb region) associated with vibriosis resistance was identified, harboring 142 significant single nucleotide polymorphisms (SNPs). Among the candidate genes within this QTL, the jam2a gene was significantly upregulated in liver and spleen of infected fish, suggesting its crucial roles in conferring disease resistance to vibriosis. Notably, seven desirable homozygous genotypes within the jam2a gene were found to be highly prevalent in surviving individuals, indicating their potential as valuable markers for selective breeding programs aimed at enhancing disease resistance. While the identified QTL explains 3 % of the genetic variance, it underscores the polygenic nature of vibriosis resistance, suggesting that further studies are needed to identify additional contributing loci. Anyway, these findings provide a foundation for future development of disease-resistant strains in tongue sole and contribute to a deeper understanding of the genetic mechanisms underlying resistance to vibriosis in aquaculture species. • A significant QTL associated with resistance to vibriosis was identified. • Jam2a is likely a novel causal gene conferring resistance to vibriosis. • The integration of GWAS and RNA-seq offers a powerful approach for dissecting complex traits in aquaculture species. [ABSTRACT FROM AUTHOR]

Half-smooth tongue sole ( Cynoglossus semilaevis ) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum . It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum . [ABSTRACT FROM AUTHOR]

The protein tyrosine phosphatase SHP-1 plays pivotal roles in maintaining cellular homeostasis, proliferation, and differentiation. Although SHP-1 has been shown to have anti-inflammatory effects in mammals, its potential anti-bacterial effects and regulatory mechanisms have not been elucidated in teleost. Chinese tongue sole (Cynoglossus semilaevis), an economically important aquaculture flatfish, has been widely used as a genomics and disease model. Herein, using this fish species, we demonstrated the function of SHP-1 in vitro and in vivo and further revealed the underlying mechanism in immune response caused by Vibrio anguillarum. Overexpression of SHP-1 decreased mortality and alleviated histopathological deterioration of Chinese tongue sole, while its inhibition exhibited the opposite effects. Accordingly, SHP-1 inhibited the activation/transduction of the toll like receptor 5-myeloid differentiation primary response 88-nuclear factor kappa-B (TLR5-MYD88-NFκB) axis and the secretion of pro-inflammatory cytokines. Most importantly, we revealed for the first time that SHP-1 inhibits Nuclear Factor Kappa B Subunit 1 (NFκB1) phosphorylation and nuclear translocation by interacting with NFκB1, thereby suppressing the activation of NFκB signaling and blocking excessive inflammation caused by bacteria and lipopolysaccharide (LPS). In summary, our findings systematically dissect the functions of SHP-1 in bacterial inflammation using Chinese tongue sole, and provide mechanistic insights into the control of bacterial diseases in aquaculture. [Display omitted] • SHP-1 mitigated mortality and tissue damage of tongue sole after bacterial infection. • SHP-1 suppressed excessive immune response through TLR5-MYD88-NFκB pathways. • SHP-1 negatively regulated the NFκB signaling by interacting with NFκB1. • SHP-1 inhibited the phosphorylation and nuclear translocation of NFκB1. [ABSTRACT FROM AUTHOR]

Sexual size dimorphism (SSD), characterized by the body size difference in different sexes, has been commonly announced in various species included mammals, birds, reptiles, and fishes. The endocrine factors in the gonads has been regarded to be involved in SSD. Two oocyte secreted factors-growth differentiation factor 9 (gdf9) and bone morphogenetic factor 15 (bmp15) has been shown to be differentially expressed in the gonad of Chinese tongue sole (Cynoglossus semilaevis), a typical marine fish demonstrating female-biased SSD. To figure out their possible roles in fish SSD, gdf9 and bmp15 of C. semilaevis were firstly cloned. The subsequently phylogenetic and structural analysis revealed that gdf9 and bmp15 were clustered with other fish species and both contained TGF-beta domain in the C-terminal. Furthermore, the temporal and spatial expression by qRT-PCR showed that gdf9 and bmp15 displayed the highest expression level in the female gonad. Moreover, the highest levels of gdf9 and bmp15 transcripts were both detected in the 1.5-year-old female gonad. The in situ hybridization and immunofluorescence experiments revealed that their mRNAs and proteins were both located in the oocyte. Based on the methylome data and bisulfite sequencing PCR, the lowest DNA methylation levels for gdf9 was observed in the female gonad, mainly distributed in the upstream and genebody regions. As for bmp15 gene, the methylation level of females in the genebody region, especially the exon 1, was higher than that of males and pseudomale, while the methylation level of females in the downstream was the lowest. Finally, knock-down of gdf9 siRNA in C. semilaevis ovarian cells resulted in the down-regulation of alk4 and tgfbr1 , and up-regulation of bmpr2 , smad8 , and bmp15. Taken together, the female-gonad-biased expression of gdf9 and bmp15 may be partly attributed to their upstream or genebody DNA methylation status. Gdf9 might be involved in reproduction and growth regulation of C. semilaevis by affecting Smad signaling pathway. Further exploration for these two ovarian factors would be helpful to better understand C. semilaevis SSD. • C. semilaevis gdf9 and bmp15 showed female-biased expression in the gonad, with mRNAs and proteins located in the oocyte. • The female-biased expression of gdf9 in the gonad may be partly attributed to its hypomethylation in the upstream region. • The gdf9 knock-down by siRNA resulted in down-regulation of alk4 and tgfbr1 , and up-regulation of bmpr2 , smad8 , and bmp15. [ABSTRACT FROM AUTHOR]

IgT is a specific Ig isotype in teleosts, which plays extremely important roles in the mucosal immunity of fish. In the present study, the membrane-bound and secretory IgT of the half-smooth tongue sole (Cynoglossus semilaevis) were identified for the first time. The V-D-J-C structure of two forms of csIgT are translated by the same Cτ gene, and the secretory tail and transmembrane domain were encoded through alternative splicing at the 3′ end of the Cτ4. The CH regions of csIgT had high similarity with that of other flatfish (P. olivaceus and S. maximus). In healthy C. semilaevis , sIgT and mIgT were mainly expressed in mucus related tissues such as skin, intestine and gill. The transcript levels of sIgT and mIgT mRNA showed a significant induction in the immune-related tissues upon Vibrio Harveyi infection. A polyclonal rabbit anti-csIgT was successfully prepared using the csIgT heavy chain recombinant protein. Using this antibody, we detected the native IgT with the molecular mass at 220 kDa in skin total protein under non-reducing SDS-PAGE condition. Immunofluorescence analysis indicated that IgT+ B lymphocytes were intensively located in the skin, gill, intestine, and head kidney of C. semilaevis. These results suggest that IgT may participate in the immune response of C. semilaevis , which will facilitate the investigations of the immunoglobulins of marine fish. • The m IgT and sIgT genes of C. semilaevis were identified for the first time. • sIgT and mIgT were predominantly expressed in the mucosal related tissues. • After V. harveyi infection, the transcript levels of sIgT and mIgT mRNA were up-regulated. • Immunofluorescence showed the IgT+ B cells in skin, intestine, gill and head kidney. [ABSTRACT FROM AUTHOR]

Based on 1572 re-sequenced Chinese tongue sole (Cynoglossus semilaevis), we investigated the accuracy of four genomic methods at predicting genomic estimated breeding values (GEBVs) of Vibrio harveyi resistance in C. semilaevis when SNPs varying from 500 to 500 k. All methods outperformed the pedigree-based best linear unbiased prediction when SNPs reached 50 k or more. Then, we developed an SNP array "Solechip No.1" for C. semilaevis breeding using the Affymetrix Axiom technology. This array contains 38,295 SNPs with an average of 10.5 kb inter-spacing between two adjacent SNPs. We selected 44 candidates as the parents of 23 families and genotyped them by the array. The challenge survival rates of offspring families had a correlation of 0.706 with the mid-parental GEBVs. This SNP array is a convenient and reliable tool in genotyping, which could be used for improving V. harveyi resistance in C. semilaevis coupled with the genomic selection methods. • We developed the first SNP array for the Chinese tongue sole breeding. • The array contains 38,295 SNPs with an average of 10.5 kb interval between two SNPs. • We evaluated the accuracy of four GS methods using 500 to 500 k SNPs. • All GS methods outperformed ABLUP when 50 k or more SNPs were used for prediction. • The mid-parental GEBVs had a correlation of 0.706 with the phenotype of offspring. [ABSTRACT FROM AUTHOR]

Dmrt2 is a member of the dmrt gene family with a conserved zinc finger-like DNA-binding motif (DM domain). In the present study, CS - dmrt2 was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of CS - dmrt2 is 2834 bp in length, with a 251 bp 5′-untranslated region (UTR), a 1086 bp 3′-UTR and a 1503 bp open reading frame (ORF) that encodes a 501-amino-acid peptide. qPCR revealed that CS - dmrt2 was mainly expressed in C. semilaevis testes. In situ hybridization (ISH) showed CS - dmrt2 expression throughout early gonadal development (36 days after hatching (dah) and 86 dah), but the expression was higher in male gonads than in female gonads. CS - dmrt2 mRNA was highly expressed in male germ cells. Comparison of methylation levels between females and males demonstrated hypo-methylated levels of the CS - dmrt2 promoter in the male gonads, which is consistent with the high mRNA expression. These results suggest that the CS - dmrt2 gene may play a functional role in gonadal differentiation/development and germ cell maturation in the testis. • dmrt2 gene from Chinese tongue sole (Cynoglossus semilaevis) was cloned and characterized. • The expression profiles in different tissues and different stages of gonads were examined. • Cyto-location of dmrt2 in gonads were accomplished. • The analysis of methylation level was showed in this paper. [ABSTRACT FROM AUTHOR]

Abstract Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein that regulates the interaction between somatic cells and germ cells during gonad developmental process in mammals. In this study, the zglp1 of Chinese tongue sole, Cynoglossus semilaevis (cysezglp1), was cloned and characterized for the first time in fish. Cysezglp1 had an open reading frame with five exons and was located to chromosome 9. The open reading frame of cysezglp1 consisted of 1692 nucleotides and encoded a 583 amino acid polypeptide. The predicted protein contained two zinc finger structures (Znf1 and Znf2), one of which was highly homologous to the GATA-type zinc finger domain. Multiple sequence alignment showed that Znf1 was conserved across different species while Znf2 was more divergent. Through quantitative Real-time PCR (qRT-PCR), we found that cysezglp1 was predominantly expressed in gonads, and the expression level of the ovary was significantly higher than that of the testis. We compared expression level in different embryonic stages and found that cysezglp1 mRNAs were mainly expressed in the fertilized egg to the cleavage stage, subsequently declining in the blastula stage. Cysezglp1 expression was not detected from the gastrulation stage onward. In the ovary, cysezglp1 expression was detected at 120 days after hatching and expression gradually increased with the maturation of the ovary. In situ hybridization showed that the cysezglp1 was mainly expressed in oocytes. Taken together, our results suggest that cysezglp1 may play an important role in the process of oogenesis in Chinese tongue sole. Highlights • Zinc finger GATA like protein-1 (zglp1) was cloned and characterized for the first time in fish. • RT-PCR and ISH demonstrated that cysezglp1 is mainly expressed in oocytes. • Cysezglp1 may play an important role in oogenesis in the Chinese tongue sole. [ABSTRACT FROM AUTHOR]

Abstract Rac1 and Rac2, belonging to the small Rho GTPase family, play an important role during the immune responses. In this study, a Rac1 homolog (CsRac1) and a Rac2 homolog (CsRac2) were cloned from the Cynoglossus semilaevis. The full-length of CsRac1 and CsRac2 cDNA was 1219 bp and 1047 bp, respectively. Both CsRac1 and CsRac2 contain a 579 bp open reading frame (ORF) which encoding a 192 amino acids putative protein. The predicted molecular weight of CsRac1 and CsRac2 was 21.41 kDa and 21.35 kDa, and their theoretical pI was 8.50 and 7.91, respectively. Sequence analysis showed that the conserved RHO domain was detected both from amino acid of CsRac1 and CsRac2. Homologous analysis showed that CsRac1 and CsRac2 share high conservation with other counterparts from different species. The CsRac1 and CsRac2 transcript showed wide tissue distribution, in which CsRac1 and CsRac2 exhibit the highest expression level in liver and gill, respectively. The expression level of CsRac1 and CsRac2 fluctuated in the liver and gill tissues at different time points after challenged by Vibrio harveyi. Specifically, CsRac1 and CsRac2 were significantly up-regulated at 48 h and 96 h post injection. Moreover, the knocking down of CsRac1 and CsRac2 in cell line (TSHKC) reduced the expression of CsPAK1, CsIL1-β and CsTNF-α. The present data suggests that CsRac1 and CsRac2 might play important roles in the innate immunity of half-smooth tongue sole. Highlights • Rac1 and Rac2 were cloned and characterized from the Cynoglossus semilaevis. • Their expression profiles in different tissues were examined. • CsRac1 and CsRac2 were up-regulated after challenged with Vibrio harveyi. • CsRac1 and CsRac2 affected the expression of CsPAK1, CsIL1-β and CsTNF-α. [ABSTRACT FROM AUTHOR]

Abstract Receptor interacting protein kinase 3 (RIP3) has emerged as an essential molecular regulator in human inflammation with accumulating evidence in vertebrates. However, the structure and functions of RIP3 in teleosts remains elusive. Here we identified one RIP3 homologue from half-smooth tongue sole Cynoglossus semilaevis , designated CsRIP3. The open reading frame (ORF) of CsRIP3 contained 1491 nucleotides and encoded a protein with 496 amino acids (Mw = 55.44 kDa). CsRIP3 expression was detected in various tissues in half-smooth tongue soles. CsRIP3 expression was up-regulated after pathogens i.e. Vibrio and poly (i:c) treatment in vivo , indicating its possible role in teleost immune response. Further analysis using human cells demonstrated that CsRIP3 exhibited highly conserved pro-apoptotic and pro-necroptotic functions in comparison with human RIP3. In conclusion, for the first time we reported the homologous structure and functions of CsRIP3 in inflammatory cell death, which provides novel perspectives on fish immunity studies in future. Highlights • Receptor Interacting Protein Kinase 3 (RIP3) cloned from half-smooth tongue sole Cynoglossus semilaevis was designated CsRIP3. • CsRIP3 was upregulated in response to bacterial and viral challenges in different tissues in half-smooth tongue sole. • CsRIP3 has conserved protein structure and performs homologous functions in cell death in comparison with mammal RIP3. [ABSTRACT FROM AUTHOR]

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as "cAMP signaling pathway" and "NOD-like receptor signaling pathway". Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including "complement and coagulation cascades", and "cytokine-cytokine receptor interaction", which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture. • There were differences in gene expression and DNA methylation levels between the resistant and susceptible families. • The DEGs and DMGs were enriched in multiple immune-related pathways between the resistant and susceptible families. • The expression of genes involved in immune-related pathways was regulated by DNA methylation. • DNA methylation regulated host resistance to vibriosis by regulating the inflammation level and immune homeostasis. [ABSTRACT FROM AUTHOR]

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0–24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole. • This study establishes the first Chinese tongue sole skin cell line, CSS. • The CSS cell line is transfectable and enables foreign gene expression. • The CSS cell line is a valuable tool for studying immunological molecular mechanisms in vitro. • Autophagy, GABAergic synapse, Apelin signaling, and Ferroptosis pathway may contribute to Chinese tongue sole skin cells' ongoing fight against banterial infection. [ABSTRACT FROM AUTHOR]

The nanos gene encodes an RNA-binding zinc finger protein, which is required in the development and maintenance of germ cells. However, there is very limited information about nanos in flatfish, which impedes its application in fish breeding. In this study, we report the molecular cloning, characterization and functional analysis of the 3′-untranslated region of the nanos gene ( Csnanos ) from half-smooth tongue sole ( Cynoglossus semilaevis ), which is an economically important flatfish in China. The 1233-bp cDNA sequence, 1709-bp genomic sequence and flanking sequences (2.8-kb 5′- and 1.6-kb 3′-flanking regions) of Csnanos were cloned and characterized. Sequence analysis revealed that Cs Nanos shares low homology with Nanos in other species, but the zinc finger domain of Cs Nanos is highly similar. Phylogenetic analysis indicated that Cs Nanos belongs to the Nanos2 subfamily. Csnanos expression was widely detected in various tissues, but the expression level was higher in testis and ovary. During early development and sex differentiation, Csnanos expression exhibited a clear sexually dimorphic pattern, suggesting its different roles in the migration and differentiation of primordial germ cells (PGCs). Higher expression levels of Csnanos mRNA in normal females and males than in neomales indicated that the nanos gene may play key roles in maintaining the differentiation of gonad. Moreover, medaka PGCs were successfully labeled by the microinjection of synthesized mRNA consisting of green fluorescence protein and the 3′-untranslated region of Csnanos . These findings provide new insights into nanos gene expression and function, and lay the foundation for further study of PGC development and applications in tongue sole breeding. [ABSTRACT FROM AUTHOR]

In the present study, the peripheral blood cells of half smooth tongue sole ( Cynoglossus semilaevis ) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function. [ABSTRACT FROM AUTHOR]

E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 ( neurl3 ) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis. [ABSTRACT FROM AUTHOR]

Thermal stress, encountered frequently in aquaculture, affects diverse physiological processes and behavior of fish; however, the mechanism underlying these effects may vary among species. Half-smooth tongue sole ( Cynoglossus semilaevis ) widely inhabits Asian coastal waters and has been intensively cultured in China. In this study, the transcriptomes of three tissues (gill, liver and muscle) of half-smooth tongue sole stressed at temperatures up to 35 °C were profiled and compared with those of the fish living at a normal temperature of 25 °C. Of 105,228 transcripts, 23,213 were annotated in GO terms, and 718, 1236 and 561 were found to express differentially in gill, liver and muscle from control, respectively. Of the differentially expressed genes, 119 were shared by all three tissues. Tissue-overlapping transcripts and the pathways and functions they defined may underline the primary response of fish to thermostressing. High temperature may cause directly protein mis-folding and hypoxia. Cells suffering thermostress either survive hypoxia or are eliminated by immune system. High temperature may have interacted indirectly through HSPs with HIF-1 and JAKs/STATs signaling pathways. The former up-regulates the expression of hypoxia inducible genes while the later up-regulates the expression of genes associating with insulin and NOD-like receptors signaling pathways. Insulin pathway functions to sustain homeostasis of sugar and lipids, aiding to survive cells, while NOD-like receptors signaling pathway strengthens immune, apoptotic and inflammatory responses, helping to survive cells. These understandings may help improve our culturing practice, the culture performance of half-smooth tongue sole and its breeding. [ABSTRACT FROM AUTHOR]

Testis-specific protein kinase 1 ( tesk1 ) represents a conserved gene family functioning in many cellular processes. In this study, we cloned and characterized an autosome-localized tesk1 gene ( Altesk1 ) from Cynoglossus semilaevis. The open reading frame consists of 2088 nucleotides and encodes a 665 amino acid polypeptide. Phylogenetic analyses show that vertebrate Tesk1s are divided into two clusters based on protein length and AlTesk1 belongs to “long-type” group. Semi-quantitative PCR reveals that Altesk1 is predominantly expressed in ovary, despite of relatively low detection in some other tissues. Among different development stages, Altesk1 transcripts are only observed in ovary samples of 210-day and 1-year fish. In situ hybridization analyses have further confirmed its majo r localization in oocyte cells. Comparison of methylation patterns in different sexual genotypes reveals the low methylation level of Altesk1 promoter in female, which is consistent with Altesk1 high expression level in female. Taken together, this is the first time that tesk1 gene has been found to show female-biased expression and in view of this, we postulate that AlTesk1 might be involved in some cellular processes specific in ovary, e.g. oogenesis. [ABSTRACT FROM AUTHOR]

The tumor necrosis factor receptor-associated factor 6 (TRAF6) can act as a fundamental adaptor protein in a chain reaction of signal transduction and cascade events to finish off immune defenses. However, immunomodulatory research on TRAF6 gene is still limited in fish. In this study, a novel miRNA, Cse -miR-33 was identified from the whole genome of Chinese tongue sole (Cynoglossus semilaevis). After separate infections with three different Vibrio strains (V. harveyi , V. anguillarum , V. parahemolyticus) and one virus (nervous necrosis virus, NNV), the expressions of CsTRAF6 and Cse -miR-33 displayed significant time-dependent changes in immune related tissues and the trends were opposite in general. Through target gene prediction and dual luciferase reporter assay, Cse -miR-33 was proven to regulate CsTRAF6 by combining with 3′-UTR sequence of the gene. The results of qRT-PCR and western blotting (WB) analyses showed that Cse -miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level, rather than degrading the target mRNA. Further experiment indicated that Cse -miR-33 inhibitor largely reduced the death rate of Chinese tongue sole caused by V. harveyi and NNV. The expressions of CsTRAF 6-associated immune genes (such as CsIL-1R , CsMYD88 , CsIRAK1 , CsTNFα , CsIL6 and CsIL8) were also significantly changed in response to Cse- miR-33 agomir and inhibitor. The study suggested that Cse -miR-33 affected the immune response via targeting CsTRAF6 in C. semilaevis , which would provide us deep insights into miRNA-mediated regulatory network and help improve the immunity in fish. • Expressions of Cse -miR-33 and CsTRAF6 changed in response to Vibrio and virus. • Cse -miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level. • Cse -miR-33 affected V. harveyi - and NNV-caused death rate of Chinese tongue sole. • Cse -miR-33 modulated CsTRAF6 -associated immune signaling pathways. [ABSTRACT FROM AUTHOR]

• C. semilaevis dhcr7 and sc5d displayed female liver and gonad biased expression. • E2F1 and C/EBPα significantly regulated the transcriptional activity of C. semilaevis dhcr7 and sc5d promoter, respectively. • The knockdown of dhcr7 and sc5d in C. semilaevis cells reduced the expression of shh and bmp2b. The typical sexual size dimorphism (SSD) phenomenon of Chinese tongue sole (Cynoglossus semilaevis) seriously restricts the sustainable development of the fishing industry. Previous transcriptome analysis has found a close relationship between the steroid biosynthesis and C. semilaevis SSD. The 7-dehydrocholesterol reductase (dhcr7) and lathosterol 5-desaturase (sc5d) are two genes in the steroid biosynthesis pathway, playing important roles in lipid synthesis, cellular metabolism, and growth. The present study assessed their roles in the mechanism of C. semilaevis SSD. The quantitative polymerase chain reaction (qPCR) results showed that C. semilaevis dhcr7 was mainly expressed in female livers, and C. semilaevis sc5d was highly expressed in female livers and gonads. Dual-luciferase experiment showed that dhcr7 and sc5d promoters had strong transcriptional activity. The transcription factors E2F transcription factor 1 (E2F1), and CCAAT enhancer binding protein alpha (C/EBPα) significantly regulated the transcriptional activity of dhcr7 and sc5d promoters, respectively. Furthermore, small interfering RNA (siRNA) knockdown results showed that expression levels of several genes [ SREBF chaperone (scap), membrane-bound transcription factor peptidase, site 1 (mbtps1), fatty acid synthase (fasn), sonic hedgehog (shh), bone morphogenetic protein 2b (bmp2b) and AKT serine/threonine kinase 1 (akt1)] were suppressed. Protein subcellular localization results indicated that Dhcr7 and Sc5d were both specifically distributed in the cytoplasm, with co-localization been observed. The present study provides evidence that dhcr7 and sc5d might regulate C. semilaevis sexual size dimorphism by involving in energy homeostasis and cell cycle, or by affecting PI3K-Akt and Shh signaling pathways. The detailed roles of these steroid biosynthesis genes regulating C. semilaevis SSD needed more information. [ABSTRACT FROM AUTHOR]

Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi ) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5′-untranslated region (UTR), a 164 bp 3′-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa and Shewanella putrefaciens . The research revealed that CsCfi plays an important role in C. Semilaevis immunity. [ABSTRACT FROM AUTHOR]

The csdazl gene is a sex related gene of half-smooth tongue sole ( Cynoglossus semilaevis ). Our research group have cloned full length cDNA of csdazl , and studied its expression pattern. To get the further information of csdazl , we constructed a prokaryotic expression plasmid, pET-32a-CSDAZL, expressed in Escherichia coli BL21 and purified the fusion protein by His Trap. In order to detect the biological activity of the fusion protein, we injected the protein with liposome into fish, and detected other sex-related genes’ mRNA expression. The results showed that the expression levels of half-smooth tongue sole female-related genes Cyp19a and Foxl2 significantly decreased between 6 and 24 h; however, both genes’ expressions returned to their normal levels 72 h after injection, indicating that recombinant CSDAZL protein could down-regulated the expression of female-related genes, Foxl2 and Cyp19a genes, implying that the fusion protein has biological activity and csdazl plays a role in sex differentiation by regulating sex related genes’ expression. [ABSTRACT FROM AUTHOR]

Highlights: [•] This is the first miRNA identification in Cynoglossus semilaevis immune tissues. [•] Normal and Vibrio anguillarum–infected library deep sequencing identified 452 miRNAs. [•] Many C. semilaevis miRNAs were differentially regulated upon pathogen exposure. [•] Selected miRNA expression profiles were consistent between sequencing and qPCR data. [•] These are clues to understanding the role of miRNAs in C. semilaevis innate immunity. [ABSTRACT FROM AUTHOR]

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine flatfish that shows typical female-biased Sexual Size Dimorphism (SSD) and sex reversal from female to pseudomale. Previous studies have shown that the insulin-like growth factor-binding protein (igfbp) genes are differentially expressed between sexes of C. semilaevis. The igfbp family is one of the important members in the IGF system, modulating somatic growth, development and reproduction. In the study, totally fifteen igfbp genes were obtained from C. semilaevis (designated as Csigfbps). Their open-reading frame sequences varied from 561 to 1131 bp and encoded 186–376 amino acids. According to phylogenetic and structural analyses, CsIGFBPs were clustered into ten groups and 6 of them were renamed. Interaction network prediction revealed that CsIGFBPs could interact with IGF1, IGF2, PAPPA, IGFALS, FN1a and other key molecules involved in growth and development. In addition, Csigfbp transcripts exhibited different abundances in pituitary, brain, gonad and liver from female and male C. semilaevis and most of these genes showed the highest abundance in male pituitary. The mRNA abundances of Csigfbp1a and Csigfbp3b were negatively associated with their hypomethylated ratios in the upstream areas, whereas the abundance and the hypomethylation within the gene body of Csigfbp8a showed a positive correlation. 17β-estradiol and testosterone differentially modulated the mRNA abundances of Csigfbps in brain cell lines. This study provided a foundation for further functional characterizations of Csigfbp genes and might be helpful to understand the growth and reproductive mechanisms of C. semilaevis. • Fifteen Cynoglossus semilaevis igfbp (Csigfbps) genes were obtained and clustered into ten groups. • Csigfbps exhibited different abundances in pituitary, brain, gonad and liver from female and male C. semilaevis. • CsIGFBPs were predicted to interact with IGF1, IGF2, PAPPA, IGFALS, FN1a and other key molecules involved in growth and development. • Sex steroids (17β-estradiol and testosterone) differentially modulated the mRNA abundances of Csigfbps in brain cell lines. [ABSTRACT FROM AUTHOR]

Sexual size dimorphism (SSD), whereby females and males exhibit different body sizes, are widely documented in animals. To explore crucial regulators implicated in female-biased SSD of Chinese tongue sole (Cynoglossus semilaevis), GWAS was conducted on 350 females and 59 males. Twenty SNPs and 25 genes including zbed1 , nsd3 , cdc45 , klhl29 , and smad4 with -log(p) > 7 were screened, mainly mapping to sex chromosome. The chromosome W-linked gene z bed1 attracted particular attention because it is a master key for cell proliferation. Thus, the regulatory network of zbed1 in C. semilaevis was explored by DAP-seq and 1352 peaks were discovered in the female brain. Moreover, zbed1 potentially regulated hippo signaling pathway, cell cycle, translation, and PI3k-Akt signaling pathway in C. semilaevis. These findings identify crucial SNPs and genes associated with female-biased SSD in C. semilaevis , also provide the first genome-wide survey for the zbed1 regulatory network in fish species. • GWAS of females and males Cynoglossus semilaevis identified 20 SNPs with -log(p) > 7, mainly located on sex chromosome w and z. • 25 genes including zbed1 , nsd3 , cdc45 , klhl29 , and smad4 were annotated around the region of 20 kb from the significant SNPs. • DAP-seq revealed that zbed1 potentially regulated hippo signaling pathway, cell cycle, translation and PI3k-Akt pathway. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding protein 7 (igfbp7) is a member of the insulin-like growth factor binding protein (IGFBP) superfamily, which is involved in the regulation of insulin-like growth factor (IGF) and insulin signal transduction. Recently, igfbp7 was found to be female-biased expressed in the gonad of Chinese tongue sole (Cynoglossus semilaevis), a marine flatfish exhibited female-biased sexual dimorphism. This study aimed to explore the roles of igfbp7 in the growth and sex differentiation of C. semilaevis. Firstly, C. semilaevis igfbp7 was cloned, and the coding sequence (CDS) was 666 bp encoding 221 amino acids. The phylogenetic and structural analyses revealed that C. semilaevis igfbp7 was clustered with Atlantic halibut (Hippoglossu shippoglossus), sailfin molly (Poecilia latipinna) , and so on. The subsequent temporal and spatial expression analyses showed that igfbp7 was mainly expressed in female gonad. During the different gonad developmental stages, the expression of igfbp7 displayed the highest level at 6-month. Methylome data showed that female igfbp7 exhibited lower methylation levels than male and pseudomale across the whole gene region in the gonad. Dual-luciferase experiment revealed that C. semilaevis igfbp7 promoter exhibited strong transcription activity. In addition, pituitary-specific positive transcription factor 1 (POU1F1) and steroid hormone-testosterone significantly enhanced the transcriptional activity of the igfbp7 promoter. In vitro transfection experiment revealed the activation of igfbp7 on the nuclear factor kappa B (NF-κB) signaling pathway. Finally, the activation of insulin-like growth factor 1 receptor (igf1r), serine/threonine kinase 1 (akt) and heat shock 60 protein 1 (hspd1) was observed in C. semilaevis ovary cells transfected by igfbp7 small interfering RNA (siRNA). These results provided causal clues for igfbp7 in growth regulation by influencing igf1r , akt and NF-κB signal. • C. semilaevis igfbp7 displayed female-gonad-biased expression, which was negatively related to its DNA methylation level. • C. semilaevis igfbp7 promoter exhibited strong transcription activity, which was enhanced by POU1F1 or testosterone. • The activation of igf1r , akt , and hspd1 were observed in C. semilaevis oocyte cells transfected by igfbp7 siRNA. [ABSTRACT FROM AUTHOR]

Tongue sole is an economically important marine flatfish with significant sexual growth dimorphism that female with superior growth performance, and phenotypic female proportion (FP) is rather low (10%–30%) under rearing conditions. Knowledge of whether genetic variation exists for FP is needed for determining the feasibility of including this desirable trait into breeding goal in tongue sole. The aims of this study were to estimate the heritability of FP and genetic correlations between FP and growth traits (body weight, BW; total length, TL) by using a population of 16,240 individuals from 157 full-sib families (involving four year-classes (YCs), three consecutive pedigreed generations). At juvenile stage, fish from each family were random sampled to measure BW and TL, and then sexed by examining gonad tissues under microscope. The overall FP was 43.8% across all year-classes, and family FP ranges from 25.4% to 69.9%. Across YCs, there was a low but significant genetic variation for FP with heritability estimates of 0.02 ± 0.00 (observed scale) and 0.03 ± 0.01 (underlying scale). Within YC, heritability estimates varied from 0.00 ± 0.00 to 0.08 ± 0.01 on observed scale, and from 0.00 ± 0.00 to 0.04 ± 0.01 on underlying scale. Genetic correlations between FP and BW, and between FP and TL were both of moderately positive magnitude, which were 0.46 ± 0.09 and 0.39 ± 0.10 respectively, and significantly different from zero (P < 0.05). These results confirm the existence of genetic variation for FP and demonstrate the feasibility of joint improvement of FP and growth traits through selective breeding. • The analytic results indicate the existence of genetic variation of female proportion in tongue sole. • Low but significant heritability estimates were obtained based on two models. • Genetic correlations between female proportion and growth traits were of moderately positive magnitude. [ABSTRACT FROM AUTHOR]

• Cynoglossus semilaevis dhcr24 exhibited sex-biased expression patterns in the gonad and liver, which may be partly attributed to the different epigenetic modification in the female, male and pseudomale individuals. • C. semilaevis dhcr24 promoter displayed strong transcription activity in vitro and the putative transcription factors (e.g., ER, AR, SREBP, and POU1F1a) provided clues for its regulation mechanism. • The knock-down of dhcr24 in C. semilaevis liver cells induced the changes of steroid biosynthesis related genes (e.g., ebp) and PI3K/Akt pathway core components (e.g., pi3k). The sexual size dimorphism of the Chinese tongue sole (Cynoglossus semilaevis) has greatly obstructed its sustainable development; however, the underlying mechanism remains unclear. Based on C. semilaevis transcriptomic information, 24-dehydrocholesterol reductase (dhcr24) was identified in steroid biosynthesis, showing female-liver-biased expression. Dhcr24 has been reported to participate in various processes, such as cholesterol synthesis, oxidative stress response, neuroprotection, and cell survival. The present study assessed its role in the sexual size dimorphism in fish. First, detailed expression pattern analysis showed that dhcr24 mRNAs were extensively expressed in tissues and the highest levels were found in the liver and gonads of females. Analysis of the dhcr24 promoter region demonstrated different DNA methylation statuses in female, male, and pseudomale gonads with higher epigenetic modification in males. The confirmation of transcription activity of the dhcr24 promoter and putative transcription factors (e.g., ER, AR, SREBP, and POU1F1a) provides the foundation for studying its regulatory mechanism. Finally, dhcr24 -siRNA mediated knock-down assay using C. semilaevis liver cells showed that steroid biosynthesis related genes (e.g., ebp , dhcr7 , and sc5d), core component of PI3K/Akt pathway (e.g., pi3k), and igf1r exhibited different expression patterns. Further investigation on the interplay between steroid hormones, dhcr24 , PI3K/Akt, and IGF-1 systems will be valuable to better understand the mechanism underlying the sexual size dimorphism in C. semilaevis. [ABSTRACT FROM AUTHOR]