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Responses of compensatory growth to high temperature and feed restriction in juvenile tongue sole, Cynoglossus semilaevis (Güther, 1873), were investigated during a 56-days experiment. Fish were divided into seven groups including three high-temperature treatments which were reared at 28°C for 1, 2 and 3 weeks, respectively, then returned to 22°C (recorded as T1, T2 and T3), three feed-restricted treatments which were fed 25% satiation for 1, 2 and 3 weeks, respectively, then fed ad libitum (recorded as R1, R2 and R3) and a control (continuously reared at 22°C). The results showed that juvenile tongue sole exposed to thermal stress for 1 week exhibited overcompensatory growth. However, complete compensation occurred in T2, T3 and R1 fish, while partial compensation occurred in R2 and R3 fish. The body composition and energy content of fish were not significantly different among all treatments ( P > 0.05). The specific growth rate and feed efficiency of fish in T1 were significantly higher than other treatments during the whole experiment period ( P < 0.05). Higher feed efficiency and apparent digestion rate during recovery might account for the compensatory growth in tongue sole in the present study. Hyperphagia played an important role in compensatory growth for feed-restricted fish, but was not involved in the compensatory response in thermal stressed fish in the present study. The results from the present study suggested that a suitable thermal stress could be more feasible to elicit compensatory growth than feed restriction in juvenile tongue sole. [ABSTRACT FROM AUTHOR]

Summary: This study provides length–weight relationships (LWRs) for three flatfish species: Cynoglossus bilineatus (Lacepède, 1802), Solea elongata (Day, 1877), and Pseudorhombus elevatus (Ogilby, 1912) from Motaf fishing grounds (latitude 27°00′ to 28°03′N; longitude 51°41′ to 52°11′E), Bushehr province, Iran. A total of 103 fish specimens were collected using shrimp trawler with 40 mm (stretched mesh size) at depths of 10–30 m between August and September 2017. The LWRs for fish species were W = 0.0019L3.353 for C. bilineatus: W = 0.0093L3.174 for S. elongata, and W = 0.006L3.181 for P. elevatus, respectively. [ABSTRACT FROM AUTHOR]

Patched (Ptch) is a receptor in the hedgehog signaling pathway, essential for animal development. Our previous study showed that ptch 1 gene participates in the maintenance of the male germline and spermatogenesis in Cynoglossus semilaevis (csptch1). In this study, we identified a patched1 gene homolog (csptch1 x1). The csptch1 x1 gene is 5761 bp long, with a 4638 bp coding sequence that encodes 1545 amino acids. The Csptch1 x1 protein has 12 transmembrane regions and sterol-sensing domains and is highly homologous to the csptch1 (91 amino acids difference). Expression pattern analysis showed that csptch1 x1 is expressed in eight different tissues of adult tongue sole, and the expression is significantly higher in tissues of female than that in male tissues. The expression pattern in developmental stages was also analyzed. csptch1 x1 could be detected at the 1-cell stage and was highly expressed at the blastocyst, somite, and blastopore closing stages, implying that it participates in cell differentiation. In ovarian development, the expression of csptch1 x1 was initiated at 20 days after hatching (dah) and was significantly high at 35–50 and 95–150 dah. In situ hybridization showed that csptch1 x1 was predominantly expressed in primordial germ cells, oocytes, and follicular cells, but the expression of the gene was lower in the testis. These results suggest that csptch1 x1 may be mainly involved in female differentiation and ovarian development, different from the role of csptch1 in spermatogenesis. • In Chinese tongue sole (Cynoglossus semilaevis), a patched1 gene homolog was cloned and characterized. • Along development, csptch1 x1 mRNA could be detected from zygote to adult and showed female-biased expression. • csptch1 x1 was localized in primordial germ cells, oocytes, and follicular cells in ovary. • csptch1 x1 might participate in female differentiation and ovarian development. [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]

A 30-day feeding trial was conducted to evaluate the effects of dietary glutamine supplementation on survival, growth performance, activities of digestive enzyme, antioxidant abilities and hypoxia stress tolerance of larval half-smooth tongue sole, Cynoglossus semilaevis (35 days after hatching, 10.64 ± 0.86 mg). Four isonitrogenous and isolipidic experimental diets were formulated with supplementation of graded levels (0.0, 0.5, 1.0 and 2.0% dry diet) of glutamine (with free dietary glutamine estimated to be about 0.03, 0.46, 0.91, 1.73% dry diet), and were randomly assigned to triplicate groups of post larvae (150 post larvae per tank) to apparent satiation five times daily. The results indicated that, survival and growth performance of fish fed all glutamine-supplemented diets were significantly increased compared to the control group (P < 0.05). Fish fed the diet supplemented with 0.5% or 1.0% glutamine showed significantly higher activities of trypsin in intestinal segments compare to those fed 0.0% or 2.0% glutamine diet (P < 0.05). Meanwhile, fish fed the diet with 2.0% glutamine had significantly higher activities of amylase in both intestinal segments and pancreatic segments than those fed the control diet (P < 0.05). Specific activities of alkaline phosphatase (AKP) and leucine-aminopeptidase (LAP) in brush border membranes (BBM) were significantly higher in fish fed all glutamine-supplemented diets compared to the control (P < 0.05). Fish fed the 2.0% glutamine diet showed significantly lower malondialdehyde (MDA) contents in larval whole body than those in the control group (P < 0.05). Activities of glutathione peroxidase (GPX) were significantly higher in fish fed the diet with 0.5% glutamine compared with those in fish fed the 2.0% glutamine diet (P < 0.05). No significant difference was found in activities of superoxide dismutase (SOD) and catalase (CAT) among dietary treatments (P > 0.05). Fish fed diets supplemented with 0.5–2.0% glutamine showed significantly higher GPX expression and lower CAT expression than those in fish fed the control diet (P < 0.05). Compared with the control group, median lethal dissolved oxygen levels were significantly lower in fish fed 0.5% and 1.0% glutamine diets while HSP70 expression were significantly stimulated after hypoxia stress in fish fed diets supplemented with 0.5% and 2.0% glutamine (P < 0.05). In conclusion, dietary glutamine supplementation could promote survival and growth of C. semilaevis post larvae through improving activities of digestive enzyme, enhancing antioxidant abilities and hypoxia stress resistance. The optimal dietary free glutamine level based on SGR for C. semilaevis post larvae in the present study was estimated to be approximately 0.63% dry diet. Statement of relevance The present study mainly investigated effects of dietary glutamine on growth performance, antioxidant ability and stress resistance of half-smooth tongue sole ( Cynoglossus semilaevis ) post larvae. Our findings have showed that dietary glutamine could improve growth performance of C.semilaevis post larvae, and for the first time revealed that dietary glutamine could modulate gene expression of antioxidant enzymes in C. semilaevis post larvae. The results are reliable and of both theoretical and practical importance. The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed.I have read and have abided by the statement of ethical standards for manuscripts submitted to Aquaculture. [ABSTRACT FROM AUTHOR]

B cell activating factor (BAFF) is a member of the tumor necrosis factor family that is known to play an important role in B cell activation, proliferation, and differentiation in mammals. However, studies of BAFF in teleosts are very limited and its function, in particular that under in vivo conditions, is essentially unknown. In this study, we conducted in vivo as well as in vitro functional analyses of a BAFF homologue (CsBAFF) from the teleost fish tongue sole (Cynoglossus semilaevis). CsBAFF is composed of 261 residues and shares moderate sequence identities with known BAFFs of other teleosts. CsBAFF expression was most abundant in immune organs and was upregulated during bacterial infection. Purified recombinant CsBAFF (rCsBAFF) bound to tongue sole lymphocytes and promoted cellular proliferation and survival. The results of an in vivo study showed that CsBAFF overexpression in tongue sole significantly enhanced macrophage activation and reduced bacterial infection in fish tissues, whereas knockdown of CsBAFF expression resulted in increased bacterial dissemination and colonization in fish tissues. Furthermore, vaccination studies showed that CsBAFF enhanced the immunoprotection of a DNA vaccine and augmented the production of specific serum antibodies. Taken together, these results provide the first in vivo evidence to indicate that teleost BAFF is an immunostimulator that significantly contributes to the innate antibacterial immune response and vaccine-induced adaptive immune response. [ABSTRACT FROM AUTHOR]

CC chemokines are the largest subfamily of chemokines, which are important components of the innate immune system. To date, sequences of several CC chemokines have been identified in half-smooth tongue sole ( Cynoglossus semilaevis ); however, the activities and functions of these putative chemokines remain unknown. Herein, we characterized a CC chemokine, CsCCL17, from tongue sole, and examined its activity. CsCCL17 contains a 303 bp open reading frame, which encodes a polypeptide of 100 amino acids with a molecular mass of 12 kDa CsCCL17 is phylogenetically related to the CCL17/22 group of CC chemokines and possesses the typical arrangement of four cysteines and an SCCR motif found in known CC chemokines. Under normal physiological conditions, CsCCL17 expression was detected in spleen, liver, heart, gill, head kidney, muscle, brain, and intestine. When the fish were infected by bacterial and viral pathogens, CsCCL17 expression was significantly up-regulated in a time-dependent manner. Chemotactic analysis showed that recombinant CsCCL17 (rCsCCL17) induced migration of peripheral blood leukocytes. A mutagenesis study showed that when the two cysteine residues in the SCCR motif were replaced by serine, no apparent chemotactic activity was observed in the mutant protein rCsCCL17M. rCsCCL17 enhanced the resistance of tongue sole against viral infection, but rCsCCL17M lacked this antiviral effect. Taken together, these findings indicate that CsCCL17 is a functional CC chemokine with the ability to recruit leukocytes and enhance host immune defense in a manner that requires the conserved SCCR motif. [ABSTRACT FROM AUTHOR]

The indoor recirculating aquaculture system (RAS) is a sustainable alternative to traditional aquaculture systems, and a biofilter is a crucial component for the stable operation of this system. In this study, the bacterial communities within different sections of the biofilter in a full-scale RAS for the culture of Tongue SoleCynoglossus semilaeviswere investigated using polymerase chain reaction–based 454 pyrosequencing. A total of 67,368 effective sequences of the 16S rRNA gene with V1–V3 regions were obtained from samples collected from the front section, rear section, and sludge of the biofilter. Using a 3% cut-off level and sufficient sequencing depth, a total of 5,185 operational taxonomic units (OTUs) were identified in these sequences across all samples. However, a low shared richness of 570 OTUs (11.0% of the reads) was found in all samples, indicating a high diversity of bacterial profiles even in a single biofilter. Based on the SILVA database classifier of phylogenetic taxa, Proteobacteria (average, 35.6%) and Bacteroidetes (average, 30.7%) were found to be the predominant species in all samples, followed by Nitrospirae (6.0%) in the front section, Acidobacteria (7.9%) in the rear section, andPlanctomycetes(9.7%) in the sludge. Overall, 404 genera were assigned to all samples. But among the 10 most common genera, onlyNitrospira,Owenweeksia, andMarinicellawere found in all samples. The results of the phylogenetic analysis suggest that the nitrifying groups of ammonia-oxidizing bacteria belong toNitrosomonasclusters, and the nitrite-oxidizing bacteria are related toNitrospiraandNitrospinaclusters. In summary, our results indicate that the RAS fixed-film biofilter is a highly species-rich ecosystem. For this reason, a potential precision-operated strategy to manage the microbial resources in the biological treatment section in an RAS should be adopted, with consideration being given to the peculiarities and requirements of the species being cultured. Received October 26, 2014; accepted February 5, 2015 [ABSTRACT FROM PUBLISHER]

Marine organisms comprising microbes, plants, invertebrates, and vertebrates elaborate an impressive array of structurally diverse antimicrobial products ranging from small cyclic compounds to macromolecules such as proteins. Some of these biomolecules originate directly from marine animals while others arise from microbes associated with the animals. It is noteworthy that some of the biomolecules referred to above are structurally unique while others belong to known classes of compounds, peptides, and proteins. Some of the antibacterial agents are more active against Gram-positive bacteria while others have higher effectiveness on Gram-negative bacteria. Some are efficacious against both Gram-positive and Gram-negative bacteria and against drug-resistant strains as well. The mechanism of antibacterial action of a large number of the chemically identified antibacterial agents, possible synergism with currently used antibiotics, and the issue of possible toxicity on mammalian cells and tissues await elucidation. The structural characteristics pivotal to antibacterial activity have been ascertained in only a few studies. Demonstration of efficacy of the antibacterial agents in animal models of bacterial infection is highly desirable. Structural characterization of the active principles present in aqueous and organic extracts of marine organisms with reportedly antibacterial activity would be desirable. [ABSTRACT FROM AUTHOR]

Spatiotemporal occurrence and feeding habits of tonguefish ( Cynoglossus lighti Norman, 1925) larvae were investigated in an offshore area (>5 m in depth) of the inner part of Ariake Bay, Japan. All specimens were symmetric, free-swimming larvae. Although their seasonal abundance and distribution in the study site varied from year to year, spawning started in June and the larval abundance was high in August and September with a wide distribution in the inner part of the bay. Both present and previous study results strongly suggest that larvae may settle primarily in the estuary and near-shore areas of Ariake Bay after their wide distribution in the offshore area of the bay during the free-swimming stage. Larvae showed a clear feeding rhythm in which they fed on prey mainly during the daytime. Larvae fed exclusively on copepods, and identified prey were mostly Paracalanidae (mainly Parvocalanus crassirostris), Microsetella norvegica, and Oithona davisae. Pre-metamorphosis larvae fed primarily on Paracalanidae and O. davisae, whereas O. davisae formed a smaller proportion of the early-metamorphosis diet. In early metamorphoses, larvae fed preferentially on Paracalanidae and M. norvegica. [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 objectives of this study were to determine the reproductive cycle of largescale tonguesole Cynoglossus arel , a commercially valuable flatfish species, in coastal waters of Bandar Abbas, along the south coast of Iran in the Persian Gulf. From October 2009 to September 2010, 905 fish were collected in monthly samples, and their length, weight, sex, gonad weight, and maturity status recorded. These data revealed that ovary weight in females is low from July to September, then increases to a peak in February followed by a decrease, indicating that the peak spawning season is from February to March with some spawning lasting until June. Males showed a corresponding seasonal pattern in testis weight, although with much less pronounced seasonal differences than gonad weight in females. Five maturity classes were described based on ovarian and testicular histology, corresponding with macroscopic analysis of gonads. The spawning season in C. arel is prolonged, similar to several other tropical flatfish species, and larger adult females tended to have an even more prolonged spawning period than smaller, presumably younger adult females. Combined, our results indicated that C. arel is a winter-to-spring batch spawner with an asynchronous type of ovarian development. [ABSTRACT FROM AUTHOR]

Follistatin (FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole, Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction (RTPCR). FST mRNA was expressed in all 13 tissues analyzed, and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis, the transcript level of FST was the highest in the perinucleolus stage, decreased in the primary yolk stage, slightly increased in the tertiary yolk stage, and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone, which stimulated oocyte development. However, no significant variation was observed across all stages of testis development, although the expression level in the spermatogenesis stage was relatively low, which may result from the regulation of FST by aromatase. [ABSTRACT FROM AUTHOR]

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHβ, LHβ and CGa) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHβ, LHβ and CGa were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHβ transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHβ expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole. [ABSTRACT FROM AUTHOR]

A new ovarian cell line, CSO, was established from half-smooth tongue sole Cynoglossus semilaevis. Primary culture of CSO cells was initiated from digestion of ovarian tissues pieces by trypsin solution and cultured at 24° C in Dulbecco's modified Eagle's medium- F12 medium ( DMEM- F12, 1:1) (pH 7·0), supplemented with 20% foetal bovine serum, basic fibroblast growth factor ( bFGF), epidermal growth factor ( EGF), insulin-like growth factor-I ( IGF-I) and human chorionic gonadotropin ( HCG). The cultured CSO cells, fibroblastic in morphology, proliferated to 100% confluency 3 days later and had been subcultured to passage 80. Chromosome analyses indicated that the CSO cells exhibited chromosomal aneuploidy with a modal chromosome number of 42 that displayed the normal diploid karyotype of C. semilaevis [2 n = 42 t, fundamental number ( NF) = 42]. Reverse transcription polymerase chain reaction revealed that CSO cells could express ovarian somatic cell functional genes p450 armo, foxl2 and sox9a but not ovary germ cell marker gene vasa and male-specific gene dmrt1. Transfection experiment demonstrated that CSO cells transfected with pEGFP-N3 plasmid could express green fluorescence protein ( GFP) with higher transfection efficiency. The CSO cell line might serve as a valuable tool for studies on the mechanism of sex determination and oogenesis of ovary in flatfish. [ABSTRACT FROM AUTHOR]

Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the a-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) participate in reproduction by regulating the expression of target genes; however, the regulation of target mRNAs by miRNAs along the brain–pituitary–gonad (BPG)–liver axis in fish reproduction and ovarian maturation has not been investigated, but a better understanding of these processes would have significant value. In this study, we performed comprehensive miRNA sequencing of BPG–liver organs at ovarian stages IV, V and VI in Cynoglossus semilaevis , yielding a total of thirty-six small RNA libraries. In total, 3224 miRNAs were identified, including 1,945 known miRNAs and 1279 novel miRNAs. The number of miRNAs downregulated from stage IV to stage VI in all tissues was higher than the number upregulated. The key identified miRNAs (miR-34-x, miR-186-x, miR-2188-x, miR-153-x, miR-204-x, miR-211-x, miR-2188-y, miR-223-y, miR-724-x, miR-144-y, novel-m0050-3p, and novel-m0017-5p) were found to possibly simultaneously regulate several mRNAs and participate in different biological functions at various ovarian stages. Based on target gene relationships, we screened important mRNAs related to reproduction (igf1r, hsd3b, igf2r, pgr, ghr, sstr2, cyp19a1, lhcgr, AR, cyp17a1, sst2, gpr54, pgrmc1, esr1, and paqr7b). Through KEGG pathway analysis, we found that the following signaling pathways were enriched: steroid hormone biosynthesis, oocyte meiosis, progesterone-mediated oocyte maturation, focal adhesion, endocytosis, neuroactive ligand–receptor interaction, cytokine–cytokine receptor interaction, and Jak–STAT signaling. The expression patterns of the nine most differentially expressed miRNAs (miR-211-x, miR-153-x, miR-2188-x, miR-223-y, miR-2188-y, miR-186-x, miR-724-x, miR-221-x, and miR-21-y) were assayed with RT-qPCR. The expression trends for all the miRNAs validated by RT-qPCR were consistent with the sequencing data. Moreover, the miR-186-x binding sites in igf2r of C. semilaevis were verified for the first time. We speculated that miR-186-x directly regulates igf2r expression, modulates the reproductive endocrine system in C. semilaevis through the BPG–liver axis, and affects ovary maturation. Overall, this study reports a comprehensive analysis of transcriptomic modulation of the BPG–liver axis related to C. semilaevis ovary maturation. • MiRNAs in brain-pituitary-ovary-liver axis at three ovarian stages were identified. • Current findings suggest miRNAs regulate ovarian development and oocyte maturation. • The target relationship between miR-186-x and igf2r was verified. • Twelve miRNAs were identified as important for ovarian development and maturation. [ABSTRACT FROM AUTHOR]

Salinity is an important environmental factor that affects fish growth, development, and reproduction. As euryhaline fish, half-smooth tongue sole (Cynoglossus semilaevis) are a suitable species for deciphering the salinity adaptation mechanism of fish; however, the molecular mechanisms underlying low- and high-salinity responses remain unclear. In this study, RNA-seq was applied to characterize the genes and regulatory pathways involved in C. semilaevis gill responses to high- (32 ppt), low- (8 ppt), and control-salinity (24 ppt) water. Gills were rich in mitochondria-rich cells (MRCs) in high salinity. Compared with control, 2137 and 218 differentially expressed genes (DEGs) were identified in low and high salinity, respectively. The enriched functions of most DEGs were metabolism, ion transport, regulation of cell cycle, and immune response. The DEGs involved in oxidative phosphorylation, citrate cycle, and fatty acid metabolism were down-regulated in low salinity. For ion transport, high and low salinity significantly altered the expressions of prlr , ca12 , and cftr. In cell cycle arrest and cellular repair, gadd45b , igfbp5 , and igfbp2 were significantly upregulated in high and low salinity. For immune response, il10 , il34 , il12b , and crp increased in high and low salinity. Our findings suggested that alterations in material and energy metabolism, ions transport, cell cycle arrest, cellular repair, and immune response, are required to maintain C. semilaevis gill homeostasis under high and low salinity. This study provides insight into the divergence of C. semilaevis osmoregulation mechanisms acclimating to high and low salinity, which will serve as reference for the healthy culture of C. semilaevis. [Display omitted] • Gill transcriptome profiles revealed that C. semilaevis adapted high- and low-salinity water via adjusting material and energy metabolism, ion transport, cell cycle arrest, cellular repair, and immune response. • First revealed C. semilaevis gill adaptation mechanism to high and low salinity at the transcriptome level. [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]

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]

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]

The compensatory growth, body composition and energy budget of juvenile tongue sole, Cynoglossus semilaevis (Günther, 1873), subjected to an unfavourable temperature were investigated during 56 days experiment. Fish were divided into four groups including three groups, which were reared at 16°C for 1, 2 and 3 weeks, respectively, then returned to 22°C (recorded as A1, A2 and A3) and a control group (continuously reared at 22°C, C). At the end of the experiment, the body mass was significantly higher in A1 than that in the control ( P < 0.05), which indicated over compensatory growth occurred in A1 fish. No significant difference was found in body mass among A2, A3 and C fish ( P > 0.05), which indicated complete compensatory growth occurred in A2 and A3 fish. The underlying mechanisms for compensatory growth could be attributed to an improved energetic efficiency resulting from reduced metabolic expenditure and higher feed efficiency during the period of recovery. Furthermore, the fish exposed to lower temperature for 3 weeks showed hyperphagia. The results suggested that the juvenile C. semilaevis exposed to lower temperature for a suitable period (1-3 weeks) used feed more efficiently. This approach may be a useful rearing strategy for indoor culture of C. semilaevis. [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]

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most important flatfish species for aquaculture in China. To produce a monosex population, we attempted to develop a marker-assisted sex control technique in this sexually size dimorphic fish. In this study, we identified a co-dominant sex-linked marker (i.e., CyseSLM) by screening genomic microsatellites and further developed a novel molecular method for sex identification in the tongue sole. CyseSLM has a sequence similarity of 73%-75% with stickleback, medaka, Fugu and Tetraodon. At this locus, two alleles (i.e., A244 and A234) were amplified from 119 tongue sole individuals with primer pairs CyseSLM-F1 and CyseSLM-R. Allele A244 was present in all individuals, while allele A234 (female-associated allele, FAA) was mostly present in females with exceptions in four male individuals. Compared with the sequence of A244, A234 has a 10-bp deletion and 28 SNPs. A specific primer (CyseSLM-F2) was then designed based on the A234 sequence, which amplified a 204 bp fragment in all females and four males with primer CyseSLM-R. A time-efficient multiplex PCR program was developed using primers CyseSLM-F2, CyseSLM-R and the newly designed primer CyseSLM-F3. The multiplex PCR products with co-dominant pattern could be detected by agarose gel electrophoresis, which accurately identified the genetic sex of the tongue sole. Therefore, we have developed a rapid and reliable method for sex identification in tongue sole with a newly identified sex-linked microsatellite marker. [ABSTRACT FROM AUTHOR]

Background Half-smooth tongue sole (Cynoglossus semilaevis) is a valuable fish for aquaculture in China. This fish exhibits sexual dimorphism, particularly different growth rates and body sizes between two genders. Thus, C. semilaevis is a good model that can be used to investigate mechanisms responsible for such dimorphism, this model can also be utilized to answer fundamental questions in evolution and applied fields of aquaculture. Hence, advances in second-generation sequencing technology, such as 454 pyrosequencing, could provide a robust tool to study the genome characteristics of non-model species. Results In this study, C. semilaevis was subjected to dec novo transcriptome sequencing and characterization. A total of 749,954 reads were generated using a single 454 sequencing run in a full PicoTiter plate. These reads were then assembled into 62,632 contigs with a 10-fold average sequencing coverage. A total of 26,589 sequences were successfully annotated based on sequence similarities; among these sequences, 3,451 transcripts exhibited gene ontology terms and 2,362 showed enzyme commissions associated with 186 pathways from Kyoto Encyclopedia of Gene and Genomes pathways. A search of repetitive elements was performed, and 1,898 transposable elements were identified. Approximately 7,800 simplesequence repeats and 21,234 single-nucleotide polymorphisms were also detected. Conclusions Our data provided an integrated and comprehensive transcriptome resource for C. semilaevis. These data could be used for further research in population genetics, gene function, and tissue-specific gene expressions. [ABSTRACT FROM AUTHOR]

The content of heavy metals (Pb, Cd, Cu, Zn) in the flesh on the two types of fish consisting pelagic fish include Black pomfret (Formio niger) whereas among other demersal fish include Tongue sand (Cynoglossus lingua), which was conducted in March-December 2013, taken from the western part of Madura Strait (Gresik-Ujung pangkah, Surabaya-Kenjeran, and Sidoarjo-Sedati). Pb, Cd, Cu, and Zn detected using Atomic Absorption Spectrometry brand Shimadzu AA-6200. The highest content of heavy metals, Pb in Tongue sand from the Sedati (5.63ppm±0.05), while the lowest of Cd in Black pomfret from the Ujung pangkah (0,20ppm±0,004). The results show that there is a correlation between heavy metal with fish but not significantly. The content of Pb, Cd, Cu, Zn in fish two species originating from all locations stated below the exposure limits for consumption. Safe limit fish consumption of Black pomfret is based on the WHO PTWI of 347.43 g.week-1 and Tongue sand 191,25 g.week-1. [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]

Background Half-smooth tongue sole (Cynoglossus semilaevis Günther) has been exploited as a commercially important cultured marine flatfish, and female grows 2-3 times faster than male. Genetic studies, especially on the chromosomal sex-determining system of this species, have been carried out in the last decade. Although the genome of half-smooth tongue sole was relatively small (626.9 Mb), there are still some difficulties in the high-quality assembly of the next generation genome sequencing reads without the assistance of a physical map, especially for the W chromosome of this fish due to abundance of repetitive sequences. The objective of this study is to construct a bacterial artificial chromosome (BAC)-based physical map for half-smooth tongue sole with the method of high information content fingerprinting (HICF). Results A physical map of half-smooth tongue sole was constructed with 30, 294 valid fingerprints (7.5 × genome coverage) with a tolerance of 4 and an initial cutoff of 1e-60. A total of 29,709 clones were assembled into 1,485 contigs with an average length of 539 kb and a N50 length of 664 kb. There were 394 contigs longer than the N50 length, and these contigs will be a useful resource for future integration with linkage map and whole genome sequence assembly. The estimated physical length of the assembled contigs was 797 Mb, representing approximately 1.27 coverage of the half-smooth tongue sole genome. The largest contig contained 410 BAC clones with a physical length of 3.48 Mb. Almost all of the 676 BAC clones (99.9%) in the 21 randomly selected contigs were positively validated by PCR assays, thereby confirming the reliability of the assembly. Conclusions A first generation BAC-based physical map of half-smooth tongue sole was constructed with high reliability. The map will promote genetic improvement programs of this fish, especially integration of physical and genetic maps, fine-mappings of important gene and/or QTL, comparative and evolutionary genomics studies, as well as whole genome sequence assembly. [ABSTRACT FROM AUTHOR]

The effects of culinary practices such as boiling, frying, and grilling on the proximate compositions and concentrations of metals (Cd, Pb, Cr, Zn, Fe, Cu, Mn, Ni, and Hg) in commonly consumed fish species from the Nigerian coastal waters were investigated. The selected fish species were Polydactylus quadratifilis, Chrysicthys nigrodigitatus and Cynoglossus senegalensis. The culinary practices lead to increased protein, fat, and ash contents and decreased moisture contents of these fish species. The culinary practices resulted significant increase in the concentrations of most of the studied metals and decrease in the concentrations of Fe, Cr, and Pb in some fish types. The concentrations and estimated dietary intakes of Cd, Pb, Cr, Zn, Fe, Cu, Mn, Ni, and Hg from consumption of the processed fish were within their statutory safe limits. The individual metal target hazard quotient ( THQ) values and the total THQs were less than 1 which indicates that no health risks would arise from the long-term consumption of these fish species. [ABSTRACT FROM AUTHOR]

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]

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding. [ABSTRACT FROM AUTHOR]

Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole. [ABSTRACT FROM AUTHOR]

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two CsNF45 isoforms exist in tongue sole as a result of alternative mRNA splicing. [•] Both isoforms were expressed in multi-tissues but differed in expression patterns. [•] Expression of the two isoforms was regulated differently by bacterial and viral infection. [•] Both isoforms were localized in the nucleus and could activate mouse IL-2 promoter. [•] Overexpression of the two isoforms inhibited bacterial and viral infection to different extents. [Copyright &y& Elsevier]

Abstract: Thioredoxin (Trx) is a redox protein that plays an important role in anti-oxidative stress and redox signaling. In a recent report, we observed that the Trx of tongue sole (Cynoglossus semilaevis), CsTrx1, is a reductase and an antioxidant with immunostimulatory property. In the current work, we studied the biological effect of a truncated CsTrx1, CsTrx80, which is composed of the N-terminal 80 residues of CsTrx1. We found that recombinant CsTrx80 (rCsTrx80) purified from Escherichia coli exhibited no insulin disulfide reductase activity; however, unlike rCsTrx1, which showed no apparent chemotactic activity, rCsTrx80 was able to bind to the head kidney monocytes of tongue sole and caused cellular migration in a dose-dependent manner. Mutation of the two cysteine residues in the highly conserved CXXC motif had no effect on the chemotactic activity of rCsTrx80. Like rCsTrx1, rCsTrx80 induced the expression of immune relevant genes in the target cells, but the induction patterns differed from that induced by rCsTrx1. In vivo study showed that when tongue sole were administered with rCsTrx80 before being infected with a bacterial pathogen, bacterial dissemination and colonization in fish tissues were significantly reduced compared to control fish. Taken together, these results indicate for the first time that a Trx80 derived from fish Trx possesses chemotactic and immunoregulatory properties which promote host resistance against bacterial infection. [Copyright &y& Elsevier]

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]

The sea temperature has been observed to chronically increase during the past decades, leaving unpredictable influences to the marine biological resources. Thus, it is of vital significance to study the biological responses of ocean inhabited organisms with the artificially stimulated heat stress environment. Cynoglossus semilaevis provides us with an ideal model to study the influence of chronic heat stress on the sexual differentiation in marine teleosts for its genetic sex determination (GSD) + environmental effected (EE) sex determination system. In this study, the comparative experiment was conducted employing heated seawater (HT group) and ambient seawater (CT group) to cultivate juvenile C. semilaevis respectively. Significant differences were exhibited in growth performance and a delayed germ cell development effect was found in pseudomales formed under chronic heat stress. Using transcriptome analysis, the transcription profile of 55 days post fertilization (dpf) and 100 dpf juveniles' gonads were studied. A total of 47 libraries were constructed with an average mapping rate of 94.63% after assembling. GO and KEGG enrichment were proceeded using DEGs screened out between (1) pseudomale gonads at 55 dpf and 100 dpf in HT and CT group (2) pseudomale and female gonads at 55 dpf and 100 dpf in HT and CT group. Terms and pathways involved in steroid stimulation, reproduction ability, germ cell proliferation et al. were shed light on. The expression pattern of 29 DEGs including amh , hsp90b1 , pgr et al. were also provided to supplement the results of functional enrichment. Weighted gene co-expression networks analysis (WGCNA) was constructed and hspb8-like , histone H2A.V were exhibited to play vital roles in the heat-induced masculinization. Our findings facilitate the understanding for transcriptional variations in intensive masculinization cause by chronic heat stress of C. semilaevis and provide referable study of the influences on the teleosts in elevated sea temperature. [Display omitted] • Chronic heat stress during sex differentiation period causes intensive masculinization in Cynoglossus semilaevis. • Multiple growth statistics of juveniles in HT and CT group exhibit Significant differences. • Pseudomale formed under heat stress shows delayed germ cell development. • Chronic heat stress exaggerates the transcriptional differences between female and pseudomale juveniles' gonads. • WGCNA identifies two hub genes: hspb8 and Histone H2A.V that play key roles in the masculinization under chronic heat stress. [ABSTRACT FROM AUTHOR]