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To screen local probiotic strains to promote antibiotic-free farming, two potential probiotic strains (S3, S5) were recognized among 89 cultivable bacterial strains isolated from the intestine of healthy Cynoglossus semilaevis. The two potential probiotic isolates were analyzed in terms of their morphology, physiology, biochemistry, the similarity of 16S rDNA sequences, growth characteristics, enzyme production capacity, bacterial antagonism, and safety in C. semilaevis. The results revealed that the bacterial morphology and physiological and biochemical characteristics of S3 and S5 were similar to those of Bacillus subtilis. The 16S rDNA sequences had 99.9 % similarity to that of Bacillus subtilis MH 145363.1. Therefore, S3 and S5 were identified as B. subtilis. In addition, we found that S3 and S5 had a strong ability to secrete amylase, protease, and lipase. During the safety tests of S3 and S5 in C. semilaevis with high concentrations, C. semilaevis in immersion, injection, and feeding groups remained in good condition without falling ill or dying. Moreover, we found that S3 and S5 exhibited superior growth at 25~50°C, salinities of 10 to 40, and pH values of 5 to 9. Furthermore, S3 and S5 had significant bacteriostatic activity against Vibrio anguillarum, Aeromonas salmonicida, and Shewanella algae, which are the main pathogenic bacteria of mariculture fish. In summary, S3 and S5 showed superb inhibition of the pathogenic bacteria of marine fish, rapid growth, eurythermal and euryhaline features, and suitability for the intestinal environment of C. semilaevis. Thus, strains S3 and S5 have excellent commercial development potential. These results provide a basis for ecological disease prevention strategies and are also valuable for developing and utilizing probiotics. [ABSTRACT FROM AUTHOR]

The half‐smooth tongue sole, Cynoglossus semilaevis, is an important cultured flatfish species. Vibrio harveyi is a common pathogen to this fish, which may result in great economic loss to C. semilaevis culture industry. piRNAs, a non‐coding RNAs with 26–32 nt, have been regarded as promising biomarkers for cancer diagnosis and fish diseases. Here, we extracted the RNA from mucus of C. semilaevis and constructed the differential expression profiles of piRNAs between the sick fish (MS) and healthy fish (MC). We identified 45,696 differentially expressed piRNAs including 22,735 up‐regulated piRNAs and 22,961 down‐regulated piRNAs in MS group compared with MC group. The GO enrichment and KEGG pathway enrichment analyses of the differential piRNAs were carried out. The result showed immunity‐related target genes mainly involved in immune system process, response to stimulus, cell killing, immune system, infectious diseases and cell growth and death. The 10 most differentially expressed piRNAs were chosen to perform the qRT‐PCR, while only seven piRNAs were consistent with the sequence result. Compared with MC group, the expression levels of piR‐mmu‐72173>piR‐rno‐62831>piR‐xtr‐704880, piR‐dme‐15546979, piR‐mmu‐49941660, piR‐mmu‐29283297 and piR‐mmu‐1758399 were significantly lower, and piR‐gga‐10574 and piR‐gga‐134812 were significantly higher in MS group. These piRNAs may be potential biomarkers during the V. harveyi infection of C. semilaevis. This study could provide a new method to identify the infection status of C. semilaevis and understand better about the innate and adaptive immune system in C. semilaevis during bacterial infection. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) have been identified to be involved in half-smooth tongue sole (Cynoglossus semilaevis) reproduction. However, studies of their roles in reproduction have focused mainly on the ovary, and their expression patterns and potential roles in the brain and pituitary are unclear. Thus, to explore the mRNAs and lncRNAs that are closely associated with reproduction in the brain and pituitary, we collected tongue sole brain and pituitary tissues at three stages for RNA sequencing (RNA-seq), the 5,135 and 5,630 differentially expressed (DE) mRNAs and 378 and 532 DE lncRNAs were identified in the brain and pituitary, respectively. The RNA-seq results were verified by RT-qPCR. Moreover, enrichment analyses were performed to analyze the functions of DE mRNAs and lncRNAs. Interestingly, their involvement in pathways related to metabolism, signal transduction and endocrine signaling was revealed. LncRNA-target gene interaction networks were constructed based on antisense , cis and trans regulatory mechanisms. Moreover, we constructed competing endogenous RNA (ceRNA) networks. In summary, this study provides mRNA and lncRNA expression profiles in the brain and pituitary to understand the molecular mechanisms regulating tongue sole reproduction. [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]

Interleukin (IL)-11 is a multifunctional cytokine belonging to the IL-6 family, which plays essential roles in immune response. However, much less is known about the immunological functions of IL-11 in teleost. In this study, we investigated the immune properties of a teleost IL-11 homologue (CsIL-11) from tongue sole Cynoglossus semilaevis. CsIL-11 possesses four conserved α-helices and conserved CsIL-11 receptor binding residues L86 and R187, and shares 23.3%–80.1% identities with other IL-11 homologues. CsIL-11 expression was constitutive in tissues, with most abundant in blood and least abundant in spleen, and upregulated by bacterial challenge in blood, spleen, and head kidney. Recombinant CsIL-11 (rCsIL-11) in the native form of monomer, could bind to peripheral blood leukocytes (PBLs) membrane and enhance the activation and phagocytosis of PBLs. When administered in vivo , rCsIL-11 could markedly promote the host to defend against microbial infection. Overall, our findings show that CsIL-11 plays a pivotal role in regulating PBLs phagocytosis and antibacterial immunity. • CsIL-11 was expressed in multiple tissues and upregulated by bacterial infection. • rCsIL-11, as a monomer, bound to PBLs membrane and promoted the activation of PBLs. • rCsIL-11 enhanced the phagocytic capacity of PBLs. • In vivo rCsIL-11 could suppress the bacterial dissemination. [ABSTRACT FROM AUTHOR]

Developmental changes in enzymatic activities of alkaline phosphatase (AKP), acid phosphatase (ACP), amylase (AMY), pyruvate kinase (PK), Leucine aminopeptidase N (LAP), Lipase (LIP), Glutamic oxalo acetic transaminase (GOT), Lactate dehydrogenase (LDH), Chymotrypsin (CHY) and Trypsin (TRY) and levels of thyroid hormones and cortisol were measured in developing embryos and yolk-sac larvae of tongue sole, Cynoglossus semilaevis. During embryonic developmental periods, the fastest growth occurred in blastula stage and hatching appeared at 38.8 hours after fertilization (HAF). Besides PK which appeared in cleavage stage onwards, the other enzymes were observed in fertilized egg. Changeable patterns of enzymatic activities presented five types including upward trend for AMY and CHY, M-shaped trend for LIP and PK, V-shaped trend for LAP and GOT, W-shaped trend for LDH and TRY, and an oscillation trend for ACP and AKP. As for hormones, patterns were M-shaped trend for T3, oscillation trend for T4 and downward trend for cortisol. Furthermore, LDH, LIP, PK, TRY, ACP, GOT and T4 showed relatively high levels in blastula stage, which may be an important factor leading to the most rapid growth of embryo. Regression analysis revealed that AMY, GOT and ACP were significantly correlated with embryonic mass, which may provide an effective way to evaluate development of embryos. During yolk-sac larval periods, mouth-opening day appeared at 3 days after hatching (DAH). Besides LAP, GOT, T4 and cortisol which showed downward tendency, the other enzymes and T3 displayed upward tendency and reached the peak at 3 DAH, preparing well for upcoming exogenous digestion. [ABSTRACT FROM AUTHOR]

The ontogenesis of catabolic abilities and energy metabolism during endogenous nutritional periods of tongue sole was investigated. In this work, trypsin‐like proteases (TRY) and triglyceride lipase (LIP) activities were measured to assess the capacities to catabolize proteins and lipids, respectively. Meanwhile, specific enzymes including pyruvate kinase (PK), glutamic oxalo acetic transaminase (GOT) and glutamate dehydrogenase (GDH), and hydroxyacyl CoA dehydrogenase (HOAD) as well as their ratios were assayed to evaluate the abilities to use energy substrates of carbohydrates, amino acids and fatty acids, respectively, for energy production. In addition, activities of citrate synthase (CS) and lactate dehydrogenase (LDH) and LDH/CS ratio were calculated to analyse the evolution of aerobic and anaerobic pathways. The study found that hatching occurred at 38.8 h after fertilization (HAF), mouth‐opening day of eleuteroembryo appeared at 3 days after hatching (DAH), and the most rapid embryonic growth was observed in blastula stage before hatching. Enzymatic assay revealed that except for PK which appeared in cleavage stage onwards, all the other enzymes functioned after fertilization, preparing well for the coming embryogenesis of tongue sole. By comparing the average specific activity of enzyme in each period, it can be found that the highest value occurred at 3 DAH (for TRY, LIP, PK and LDH), 2 DAH (for GDH), fertilized egg (for GOT) and segmentation stage (for HOAD and CS), and the lowest value occurred at fertilized egg (for HOAD, CS and GDH), cleavage stage (for TRY, PK and LDH), gastrula stage (for GOT) and hatching day (for LIP). Based on the changeable patterns of metabolic enzymatic activities and ratios, it is concluded that metabolic capacities on three energy substrates displayed stage‐specific traits, and the dominant energy substrate was fatty acids before segmentation stage, amino acids until hatching day and carbohydrate during eleuteroembryo period. As for energy production mode, aerobic pathway appeared to increase greater in fertilized egg and gastrula stage, whereas anaerobic pathway played a predominant role during cleavage stage, blastula stage, segmentation stage and eleuteroembryo stage. These results are valuable to elucidate the nutritional requirements of embryonic stages in tongue sole and to further understand their energy metabolic mechanisms. [ABSTRACT FROM AUTHOR]

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%–67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16. • CsIL-16 expression occurred in various tissues and regulated by bacterial pathogen. • rCsIL-16, as a homodimer, bound to peripheral blood leukocytes (PBLs). • rCsIL-16 was involved in enhancing the migration and activation of PBLs. • In vitro rCsIL-16 could inhibit intracellular survival of E. tarda. • In vivo rCsIL-16 could regulate the inflammatory products, and suppress the bacteria invasion. [ABSTRACT FROM AUTHOR]

Cynoglossus semilaevis shell is a by-product of the Cynoglossus semilaevis, a species of fish mainly distributed along the west coast of Korea. As its skin is very tough and difficult to process, it is not useful as food. For this reason, most of it is discarded except for a small amount that is used as feed, which results in environmental pollution. Considering this, there is a need for research on the development of functional materials using Cynoglossus semilaevis shell. This study focused on the mechanism of in vitro expression function of Cynoglossus semilaevis shell extract (CSE) for skin tissue in human dermal fibroblasts that induced or did not induce wrinkles by UV-B irradiation and aims to use it as a functional material for human skin beauty or wrinkle improvement through extraction and purification. According to the ELISA results using human dermal fibroblast cells, CSE reduced MMP-1 and elastase activity by up to 21.89% and 12.04%, respectively, in a concentration-dependent manner, and increased PIP synthesis by up to 62.24% in a concentrationdependent manner. The RT-PCR test results using mRNA showed the MMP-1, 2, and 3 expression levels were suppressed in the CSE-treated group compared to the UVB-induced group and caused a concentration-dependent increase in TIMP-1 in the CSE-treat group. These results suggest that CSE can maintain and improve skin tissue conditions through MMP/TIMP balancing in human dermal fibroblast cell lines and indicate its potential as a functional material for improving skin diseases and suppressing photo-aging. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole. • Dietary bile acid (BA) can significantly improve the growth performance and survival of tongue sole. • BA inclusion can significantly enhance intestinal non-specific immune response, antioxidant capability and digestive ability. • BA inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes. • Dietary BA distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. [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]

Annexin A2 (AnxA2), belonging to the annexin family, plays a crucial role in immune responses. In this study, the cDNA of the AnxA2 gene was identified in half-smooth tongue sole, Cynoglossus semilaevis. The transcript of AnxA2 gene in C. semilaevis (CsAnxA2) showed broad tissue distribution, with the highest expression level observed in the gut. CsAnxA2 expression was significantly up-regulated in the intestine, spleen, and kidney tissues following exposure to Shewanella algae. Immunohistochemical staining revealed that CsAnxA2 was predominantly expressed in epithelial cells and significantly elevated after S. algae challenge. Subcellular localization showed that CsAnxA2 was primarily localized in the cytoplasmic compartment. Moreover, proinflammatory cytokines (IL-6, IL-8 and IL-1β) exhibited significant upregulation after CsAnxA2 was overexpressed in vivo. One hundred and fifty-eight CsAnxA2-interacting proteins were captured in the intestinal tissue, showing the top two normalized abundance observed for actin beta (ACTB) and protein S100-A10 (p11). Fifty-four high abundance CsAnxA2-interacting proteins (HIPs) were primary enriched in ten pathways, with the top three significantly enriched pathways being Salmonella infection, glycolysis/gluconeogenesis, and peroxisome proliferator-activated receptor (PPAR) signaling pathway. These results provide valuable information for further investigation into the functional mechanism of AnxA2 in C. semilaevis. • The full length of CsAnxA2 is 1014 bp, encoding 337 amino acids. • CsAnxA2 exhibits the highest expression level in gut. • CsAnxA2 was up-regulated in gut, liver and spleen following S. algae infection. • Overexpression of CsAnxA2 promoted the expression of IL-6, IL-8 and IL-1β. • One hundred and fifty-eight CsAnxA2-interacting proteins were captured in gut. [ABSTRACT FROM AUTHOR]

Antioxidant responses of juvenile sole exposed to seawater acidification (SA) and Cd were investigated. SA increased lipid peroxidation (LPO) in the fish, independent of Cd concentrations. Cd at medium and high levels inflated LPO under no or moderate SA conditions. This effect was absent under high SA levels, due to SA effect exceeding and obscuring Cd effect. SA and Cd collaborated to provoke LPO, with SOD and CAT being stimulated to defend against oxidative stress, while those related to GSH redox cycle were inhibited under SA exposure. Responses of GSH-related antioxidants to Cd impact varied contingent on their interactions with SA. This defensive strategy was insufficient to protect fish from increased LPO. Antioxidants responded more sensitively to SA than Cd exposure. GSH, GR, SOD and CAT are sensitive biomarkers for SA conditions. The findings offer insights into assessing fish's antioxidant defense strategy under Cd and SA circumstances in natural habitats. [Display omitted] • Both SA and Cd induce oxidative stress and boost LPO in tongue sole. • SA amplifies oxidative stress and intensifies LPO in fish by interacting with Cd. • Antioxidants exhibit a higher sensitivity to SA compared to Cd exposure. • Escalating levels of stressors enhance the integrated antioxidant response. • Sensitive biomarkers for oxidant stress by SA and Cd exposure are identified. [ABSTRACT FROM AUTHOR]

Aquaculture plays important roles in global food security. Prized as a valuable commercial species, the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis) has faced unprecedented challenge due to bacterial diseases, including vibriosis caused by Vibrio anguillarum. Alternative splicing (AS) is an important layer of post-transcriptional regulation, being involved in almost all biological processes, including development, reproduction, and immunity. However, its roles in immunity in half-smooth tongue sole remain elusive. In this study, therefore, we profiled the changes of alternative splicing in half-smooth tongue sole after infection with different concentrations of V. anguillarum , and investigated their functions by both enrichment analysis and targeted gene transcript overexpression analysis with half-smooth tongue sole macrophages. Intriguingly, differentially alternatively spliced (DAS) genes were found to be involved in RNA splicing for each bacterial treatment and for the shared gene set, consistent with previous studies that splicing factor themselves often underwent alternative splicing. A set of 12 immune-related shared DAS genes were identified, including hp , c5 , and traf2b. Targeted gene analysis using the shared DAS gene, haptoglobin (hp), showed that alternative splicing resulted in changes of protein domains, and overexpression analysis demonstrated that two transcripts of hp exhibited contrary roles in stimulating or reducing the expression of immune factors in response to lipopolysaccharides (LPS), reflecting the vital roles of alternative splicing in immunity of tongue sole. DAS events were further verified by qRT-PCR (20 primer pairs for 10 DAS events) to confirm the reliability of our bioinformatics analysis. This study, for the first time, revealed the involvement of alternative splicing in immune responses of half-smooth tongue sole using both omics data and targeted gene functional analysis, setting a solid foundation for further analysis and providing clues for potential usage in controlling bacterial diseases in aquaculture. • RNA splicing was enriched for DAS genes in each comparison and the shared gene set. • A set of 12 immune-related genes were identified in 104 shared DAS genes. • Two hp splicing isoforms exhibited contrary roles in stimulation of immune factors. [ABSTRACT FROM AUTHOR]

Since the 20th century, as research on the fluctuating asymmetry (FA) of otoliths in sedentary fish has deepened in a gradual way, many scholars have focused on exploring the ability of their otolith FA to indicate different environmental pressures. In this study, a typical benthic fish – Cynoglossus joyneri from three artificial habitats (artificial reef area (ARA), oyster reef area (ORA) and kelp cultivation area (NCA)) and a natural habitat (natural area (NA)) of Haizhou Bay (Lianyungang, Jiangsu Province, China) was selected as the research object, of which four otolith characters (length, width, perimeter, and area) were used to detect FA based on the squared coefficient of asymmetry variation (CV2 a). The results showed that the CV2 a of otolith in C. joyneri tended to decrease as their body length increased. In terms of indicating environmental pressure, the CV2 a of otolith length in NA was significantly higher than those in the other area (P < 0.05); the CV2 a of otolith width in NA was significantly higher than those in ARA and ORA (P < 0.05), and those in NCA were significantly higher than those in ORA (P < 0.05); and the CV2 a of otolith area in NA was significantly higher than those in ARA (P < 0.05), and those in NCA were significantly higher than those in ORA (P < 0.05). Generally, the otolith FA of C. joyneri in artificial habitats has lower CV2 a than that in natural habitats, indicating that the environmental pressure in artificial habitats is lower than that in natural habitats. We believe that C. joyneri can serve as an indicator species for environmental pressure between different habitats, which could be considered as a groundbreaking milestone in the study of otolith FA in fish. The findings not only advance the ability to assess environmental pressure in sedentary fish, which provide new insights into evaluating environmental pressure in artificial and natural habitats. • to explore the environmental pressure using otolith fluctuating asymmetry. • to indicate artificial and natural habitats using otolith fluctuating asymmetry. • to expand the database of fluctuating asymmetry of coastal water. [ABSTRACT FROM AUTHOR]

Salinity, being an indispensable abiotic factor crucial for the survival of marine organisms, has demonstrated diverse alterations globally in response to the current trend of global warming. In this study, the effect of chronic low salinity stress on teleosts' sex differentiation was investigated using Cynoglossus semilaevis , an economically important fish with both genetic and environmental sex determination system. The cultivation experiment was conducted employing artificially simulated seawater of 20 ppt and ambient sea water of 30 ppt to rear juveniles C. semilaevis. Throughout the experiment, the growth performance was assessed and the histology of gonadal development was examined, a significantly lower masculinization rate was observed in LS group. To gain further insights, transcriptome analysis was conducted using raw reads obtained from 53 libraries derived from gonads of 55 days post fertilization (dpf) and 100 dpf juveniles in both LS and CT groups. GO/KEGG enrichment were further proceeded, Terms and pathways involved in reproduction ability, germ cell proliferation, immune function, steroid metabolism etc., were illuminated and a possible crosstalk between HPI and HPG axis was proposed. WGCNA was conducted and two hub genes, hspb8-like and Histone H2A.V were exhibited to be of great significance in the changes of masculinization rate. Our findings provided solid reference for sex differentiation study of GSD + ESD species in a constantly changing ocean environment, as well as practice guiding significance for the environmental management for the culture of C. semilaevis. [Display omitted] • Low salinity stress caused decreased pseudomale rate of Cynoglossus semilaevis. • Growth was affected whereas gonadal differentiation status was not after salinity stress. • Multiple genes/pathways contributed to the rescue effect were identified. • WGCNA identifies hspb8-like and Histone H2A.V as key hub genes in the rescue effect. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) have been reported to be involved in multiple biological processes. However, the roles of lncRNAs in the reproduction of half-smooth tongue sole (Cynoglossus semilaevis) are unclear, especially in the molecular regulatory mechanism driving ovarian development and ovulation. Thus, to explore the mRNA and lncRNA mechanisms regulating reproduction, we collected tongue sole ovaries in three stages for RNA sequencing. In stage IV vs. V, we identified 312 differentially expressed (DE) mRNAs and 58 DE lncRNAs. In stage V vs. VI, we identified 1,059 DE mRNAs and 187 DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DE mRNAs were enriched in ECM-receptor interaction, oocyte meiosis and steroid hormone biosynthesis pathways. Furthermore, we carried out gene set enrichment analysis (GSEA) to identify potential reproduction related-pathways additionally, such as fatty metabolism and retinol metabolism. Based on enrichment analysis, DE mRNAs with a potential role in reproduction were selected and classified into six categories, including signal transduction, cell growth and death, immune response, metabolism, transport and catabolism, and cell junction. The interactions of DE lncRNAs and mRNAs were predicted according to antisense , cis -, and trans- regulatory mechanisms. We constructed a competing endogenous RNA (ceRNA) network. Several lncRNAs were predicted to regulate genes related to reproduction including cyp17a1 , cyp19a1, mmp14, pgr , and hsd17b1. The functional enrichment analysis of these target genes of lncRNAs revealed that they were involved in several signaling pathways, such as the TGF-beta, Wnt signaling, and MAPK signaling pathways and reproduction related-pathways such as the progesterone-mediated oocyte maturation, oocyte meiosis, and GnRH signaling pathway. RT-qPCR analysis showed that two lncRNAs (XR_522278.2 and XR_522171.2) were mainly expressed in the ovary. Dual-fluorescence in situ hybridization experiments showed that both XR_522278.2 and XR_522171.2 colocalized with their target genes cyp17a1 and cyp19a1 , respectively, in the follicular cell layer. The results further demonstrated that lncRNAs might be involved in the biological processes by modulating gene expression. Taken together, this study provides lncRNA profiles in the ovary of tongue sole and further insight into the role of lncRNA involvement in regulating reproduction in tongue sole. [ABSTRACT FROM AUTHOR]

Extracellular vesicles play a regulatory role in intracellular and intercellular transmission through a variety of biological information molecules, including mRNA, small RNAs and proteins. piRNAs are one kind of regulatory small RNAs in the vesicles at the post transcriptional level. Hereby, we isolated the extracellular vesicles from skin mucus and screened the piRNA profiles of these vesicles, aiming at developing biomarkers related to bacterial infections in Cynoglossus semilaevis. The different profilings of piRNAs in mucous extracellular vesicles of C. semilaevis were compared through small RNA sequencing, between fish infected with Vibrio harveyi and healthy ones. The number of clean reads on the alignment of exosome sick (ES) group was 105, 345 and that of exosome control (EC) group was 455, 144. GO and KEGG pathway enrichment analysis showed that most of the target genes were involved in cellular process, response to stimulus, biological regulation, immune system process and signal transduction, signal molecular and interaction, transport and catabolism. The 45 final candidate piRNAs related to immunity or infectious diseases included 20 piRNAs with high expression in the ES group and 25 piRNAs with a low expression in the ES group. After verification by qRT-PCR, there was significant difference of five piRNAs expression level between infected fish and healthy fish, in line with the sequencing. The expression level of piR-mmu-16401212, piR-mmu-26829319 and piR-gga-244092 in infected fish were significantly lower than that of control group, while piR-gga-71717 and piR-gga-99034 were higher, which implying that these piRNAs in mucous extracellular vesicles can be used to identify diseased fish from normal ones. This work supplied a novel class of biomarker for infection diagnosis in fish, and it will be benefit for screening disease resistant breeding of C. semilaevis. • Profilings of piRNAs in mucous extracellular vesicles were presented on Vibrio harveyi infection in C. semilaevis. • Differential piRNAs in extracellular vesicles from skin mucus were developed as biomarkers relating to Cynoglossus semilaevis bacterial infection. • Novel biomarkers for infection in fish will be benefit for screening disease resistant breeding of C. semilaevis. [ABSTRACT FROM AUTHOR]

This study provides length‐weight relationships (LWR) that had not yet been reported for four fish species belonging to four different families: Chrysichthys johnelsi Daget, 1959, Cynoglossus senegalensis (Kaup, 1858), Citharichthys stampflii (Steindachner, 1894) and Pseudotolithus typus, Bleeker, 1863. Data were collected in the Fatala estuary, republic of Guinea, each month between January 1993 and March 1994, using alternately a purse seine net (length 250 m, height 20 m, 14 mm side mesh) and gillnets (side mesh sizes of 10, 12.5, 15, 15.5, 20, 22.5, 25, 30, 35 and 40 mm). For each fish specimen, fork or total length and total weight were measured with precision to 0.1 cm and 0.1 g respectively. A wider size range, compared to the size range available in FishBase, was obtained for Pseudotolithus typus. The present study also provides a new maximum total length for Citharichthys stampflii. The determination coefficient (r²) between length and total body weight ranged between 0.958 in Citharichthys stampflii and 0.989 in Cynoglossus senegalensis. The allometric coefficient b varied from 2.9552 (Chrysichthys johnelsi) to 3.7288 (Citharichthys stampflii). The value of LWRs for the four fish species should assist fisheries scientists and managers to complement their further studies of population parameters to improve management decisions in Fatala estuary and, more widely, in West Africa. [ABSTRACT FROM AUTHOR]

Fisheries and Oceans Canada conducts an annual multidisciplinary survey in the Estuary and northern Gulf of St. Lawrence. The objectives of this survey are varied; assess the biodiversity of species found near the bottom; estimate the abundance of groundfish and invertebrates; assess physical and biological (phytoplankton and zooplankton) oceanographic conditions; monitor the pelagic ecosystem; and collect samples for various research projects. In 2020, the survey was conducted between August 12 and September 5 on board the CCGS Teleost. Due to the context of the Covid-19 pandemic, the number of days at sea and the number of scientists on board the ship had to be reduced. The survey successfully carried out 147 trawl tows as well as 66 CTD water column casts, and 34 zooplankton samples. This report presents the results of the 147 tows. In total, 78 fish taxa and 206 invertebrate taxa were identified during the mission. Historical perspectives (catch rates, spatial distribution and length frequency) are presented for 25 taxa. These commercial fishery-independent data will be used in several stock assessments including cod (Gadus morhua), redfish (Sebastes spp.), Greenland halibut (Reinhardtius hippoglossoides), Atlantic halibut (Hippoglossus hippoglossus), witch flounder (Glyptocephalus cynoglossus) and northern shrimp (Pandalus borealis). A preliminary analysis of water temperature data collected in 2020 shows that conditions have warmed at 150 m and deeper, reaching new records since 1915 at 200 and 300 m. The cold intermediate layer was warmer in 2020 than in 2019 except in the Estuary where it remained stable. Surface waters were also warmer than normal, by 1.5°C, in July-August. [ABSTRACT FROM AUTHOR]

Cynoglossus semilaevis blood biochemical parameters and antioxidant indexes that respond to fishing stress were measured under the influence of different feed taurine levels. With taurine levels of 0%, 0.5%, 1% and 1.5%, 4 groups of test feeds were prepared. After 28 days of feeding. Serum was collected before stress and at 0, 4, 12, 24, 48, 96 h after stress to determine the content of total protein, cholesterol, GPT, GOT, nitric oxide, lysozyme, blood sugar and the activity of glutathione (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA) and antioxidant capacity (AOC), the results show: Feeding a feed with a taurine content of 0.5% has a better inhibitory effect on cholesterol and GOT in Cynoglossus semilaevis after fishing stress, and has obvious antistress effect on lysozyme and protein. The 0.5% group had the lowest MDA content 12 h after fishing stress. The results suggested that feeding the feed with a taurine content of 0.5%, the Cynoglossus semilaevis blood biochemical parameters and antioxidant indexes to fishing stress has a better response. [ABSTRACT FROM AUTHOR]

Many early studies of ribosomal RNA gene (rDNA) suggested that rDNA tandem repeats within species are homogeneous. However, increasing number of reports have found intra-individual rDNA polymorphism across a range of taxa. Here, we reported a high level of intra-individual polymorphism of 18S-ITS1-5.8S rDNA in the genome of Cynoglossus melampetalus (Pleuronectiformes: Cynoglossidae), indicating a non-concerted evolution manner. Sequence alignments found two distinct types of 18S and 5.8S (Type A and B) and five types of ITS1 sequence (Type A - E) coexisted in the genome differing in length, GC content, secondary structure stability and minimum free energy. Based on the unique features of pseudogene and comparison of the conserved 18S rDNA sequence and 5.8S secondary structure of 22 flatfishes revealed that Type B sequences of 18S, 5.8S and their linked ITS1 were putative pseudogenes. So far, detection of rRNA pseudogenes from the multiple rDNA copies has been an intricate puzzle. Our results, as a result, provide a new ideal for rRNA pseudogene identification. • Five types of ITS1-5.8S-ITS2 sequence were found, indicating a non-concerted evolution pattern. • Type B sequences of 18S, 5.8S and their linked ITS1 were putative pseudogenes. • Variations in the conserved 18S and 5.8S revealed the putative pseudogenes. • ►These findings provide a new ideal for rRNA pseudogene identification. [ABSTRACT FROM AUTHOR]

• Here we firstly isolated the exosomes from mucus in Cynoglossus semilaevis , the first report of exosomes from flatfish mucus. • miRNAs profiling of mucous exosomes between healthy and Vibrio harveyi infectious fish were compared for differential miRNAs screening and three candidate miRNAs were verified for signatures by qRT-PCR. • The screened miRNAs in mucous exosomes is not only significant for infection diagnosis, but also has practical value for screening disease resistant breeding of Cynoglossus semilaevis. Exosomes are important mediators of vesicle transportation and contain microRNAs (miRNAs) that mediate transcriptional gene knockout and silencing in biological processes. Moreover, exosomic miRNAs are promising biomarkers for disease diagnosis and physiological status indication in many species, including fish. The impact of the Vibrio harveyi pathogen on Cynoglossus semilaevis aquaculture is becoming more and more serious as the industry expands. To overcome this challenge, miRNAs in mucous exosomes were screened by small RNA sequencing and verified by quantitative real-time PCR to develop biomarkers. This is the first capture of exosomes from flatfish mucus coupled with miRNA profiling. The results revealed significant differences in expression levels of some miRNAs between infected and healthy fish. Three unique miRNAs were identified for V. harveyi infection diagnosis; expression levels of dre-miR-205-5p and dre-miR-205-5p in infected fish were significantly lower than controls, while dre-miR-100-5p expression was higher. These miRNAs in mucous exosomes could be used to differentiate diseased and healthy fish in an early screening method with practical value for breeding disease-resistant C. semilaevis. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvβ, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8β, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish. • First report on iTRAQ proteome in the skin of C. semilaevis against V. vulnificus infection. • Eleven DEPs were validated by qRT-PCR. • Sixteen significant immune pathways were involved in skin immune responses. • Significantly pathological changes were observed in gills, intestine and skin after V. vulnificus infection. [ABSTRACT FROM AUTHOR]

Half‐smooth tongue sole Cynoglossus semilaevis is a high‐value marine fish species. The intensive farming of this flatfish species has been suffered from disease outbreak. Poly‐β‐hydroxybutyrate (PHB) is a carbon storage compound accumulated by a wide variety of prokaryotes and is considered biocontrol agent applied in aquaculture. In this study, the dietary effect of PHB‐accumulating Halomonas (HM.PHB) was investigated on survival, growth, resistance on Vibrio anguillarum and intestine microbial composition of C. semilaevis juveniles. Halomonas was fermented, which contained 76% PHB on cell dry weight. The fish juveniles were fed formulated feeds containing 1% HM.PHB, 3% HM.PHB and 5% HM.PHB, and 3% particle PHB (Par.PHB) and 3% Halomonas containing no PHB (HM) for 6 weeks. The basal feed feeding was considered as the control. The results revealed that both Halomonas‐PHB and particle PHB significantly improved the survival and growth of the juveniles than the control (p <.05), while HM was in between. When challenged with V. anguillarum, the survival rate in 1% HM.PHB, 3% HM.PHB and 5% HM.PHB was significantly improved than the control. Moreover, the increased intestine fold, epithelium height and surface were also observed in 3% HM.PHB. PCoA based on high‐throughput sequencing of 16S rRNA gene fragment indicated that dietary Halomonas‐PHB and particle PHB modified and stabilized the intestine microbial community of the fish. Our study demonstrated that Halomonas‐PHB is an effective feed additive for C. semilaevis juvenile culture. The outcome of this study facilitates the application of Halomonas‐PHB in C. semilaevis and other marine fish aquaculture. [ABSTRACT FROM AUTHOR]

The Ocellated tonguesole Cynoglossus quadriocellatus n. sp. is described from off Exmouth Gulf and Dampier Archipelago, Western Australia, eastern Indian Ocean, based on specimens collected at depths of 1-48 metres. The new species is characterised within the Cynoglossus ogilbyi species group by the snout bluntly rounded; head length 19-28% of SL, snout length 4-8% of SL; eyes not contiguous; nostrils 2; rostral hook not extending to below eye; lateral lines on eyed side three, on blind side one; mid-lateral line scales 73-88; scales between dorsal and mid-lateral lines 11-14; scales on both eyed and blind sides ctenoid; dorsal-fin rays 96-112; anal-fin rays 88-101; caudal-fin rays 10; vertebrae 8 + 40-44; two ocelli on tail. A key to the species of the Cynoglossus ogilbyi species-group is presented, and variation within the group is discussed. Other species in the group include C. broadhursti Waite 1905, C. maccullochi Norman 1926, C. ogilbyi Norman 1926, C. macrophthalmus Norman 1926, C. maculipinnis Rendahl 1921. Cynoglossus maccullochi and C. ogilbyi are recorded from Western Australia for the first time. [ABSTRACT FROM AUTHOR]

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. alga e infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae -infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish. • The protein expression was compared in intestine of normal and S. algae -infected C. semilaevis. • Seventy differentially expressed proteins (DEPs) were identified. • Protein-protein interaction analysis indicated that 29 DEPs were interrelated. • The immune defense was activated and metabolic processes were disturbed in intestine. [ABSTRACT FROM AUTHOR]

The ninth complement component (C9) is a terminal complement component (TCC) that is involved in creating the membrane attack complex (MAC) on the target cell surface. In this study, the CsC 9 (C9 of Cynoglossus semilaevis) cDNA sequence was cloned and characterized. The full-length CsC 9 cDNA measured 2,150 bp, containing an open reading frame (ORF) of 1,803 bp, a 5′-untranslated region (UTR) of 24 bp and a 3′-UTR of 323 bp. A domain search revealed that the CsC9 protein contains five domains, including two TSP1s, an LDLRA, an EGF, and a MACPF. Quantitative real-time PCR analysis showed that CsC 9 at the mRNA level was expressed in all the tested tissues, with the highest expression being observed in the liver. CsC 9 expression is significantly upregulated in the tested tissues after challenge with Vibrio anguillarum. To further characterize the role of CsC9, peripheral blood mononuclear cells of C. semilaevis were used for transcriptome analysis after incubation with recombinant CsC9 (rCsC9) protein. A total of 3,775 significant differentially expressed genes (DEGs) were identified between the control and the rCsC9-treated group, including 2,063 upregulated genes and 1,712 downregulated genes. KEGG analyses revealed that the DEGs were enriched in cell adhesion molecules, cytokine-cytokine receptor interactions, T cell receptor signaling pathways, B cell receptor signaling pathways and Toll-like receptor signaling pathways. The results of this study indicate that in addition to participating in MAC formation, CsC9 might play multiple roles in the innate and adaptive immunity of C. semilaevis. • The Cynoglossus semilaevis complement component 9 gene was first identified in flatfish. • CsC 9 mRNA is ubiquitous and highly expressed in the liver. • CsC 9 was upregulated in immune tissues by infection with Vibrio anguillarum. • Gene expression and immune pathways in leukocytes were influenced by the rCsC9 protein. [ABSTRACT FROM AUTHOR]

Knowledge on population structure and genetic diversity is a focal point for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Here we used the GBS approach for the genome-wide identification of SNPs in a collection of Cynoglossus semilaevis and for the assessment of the level of genetic diversity in C. semilaevis genotypes. GBS analysis generated a total of 55.12 Gb high-quality sequence data, with an average of 0.63 Gb per sample. The total number of SNP markers was 563, 109. In order to explore the genetic diversity of C. semilaevis and to select a minimal core set representing most of the total genetic variation with minimum redundancy, C. semilaevis sequences were analyzed using high quality SNPs. Based on hierarchical clustering, it was possible to divide the collection into 2 clusters. The marine fishing populations were clustered and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. These analyses showed that genotypes were clustered based on species-related features. Differential significant SNPs were also captured and validated by GBS and SNaPshot, with linkage disequilibrium and haplotype analysis, seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. And SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. These differential SNPs could be also employed as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations in future. • The marine fishing individuals of Cynoglossus semilaevis were clustered together and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. • Differential significant SNPs were captured and validated, linkage disequilibrium and haplotype frequencies analysis were performed • seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are a group of pollutants which occur in considerable amounts in the environment and food. In this study, a meta-analysis study was conducted on PAHs concentrations in fish in different parts of the world. The results showed that the PAHs were observed and quantified in fish in the most considered studies. The maximum and minimum concentration of PAHs was found in Cynoglossus Bilineatus fish of Persian Gulf (3970 ng/kg) and Cyprinus Carpio fish of Caspian Sea (0.004 ng/kg), respectively. These values are below the maximum value currently allowed by European Union regulations (12.0 μg/kg wet weight). [ABSTRACT FROM AUTHOR]

Keywords: arachidonic acid; characterization; cloning; Cynoglossus semilaevis; pmch2; sex dimorphism EN arachidonic acid characterization cloning Cynoglossus semilaevis pmch2 sex dimorphism 3472 3477 6 07/14/20 20200801 NES 200801 Melanin-concentrating hormone (MCH), originally discovered in the teleost pituitary, is a hypothalamic neuropeptide involved in the regulation of body colour in fish (Cerdá-Reverter & Canosa, 2009; Kawauchi, Kawazoe, Tsubokawa, Kishida, & Baker, 1983; Mizusawa, Kasagi, & Takahashi, 2018; Mizusawa, Kobayashi, Yamanome, Saito, & Takahashi, 2013; Mizusawa, Yamamura, Kasagi, Cerdá-Reverter, & Takahashi, 2018). The deduced Chinese tongue sole pMCH2 contains a 24-amino acid signal peptide (MISVSSVLITLVLSSVLSSHLVVVA) and a dibasic (RR) cleavage site near the C terminus that likely is used to generate the MCH peptides, a 23-amino acid peptide (NTELDMLRCMIGRVYRCWGTSN). The functions of MCH in fish reproduction and a potential gender difference in MCH action would merit investigation. Arachidonic acid, characterization, cloning, Cynoglossus semilaevis, pmch2, sex dimorphism. [Extracted from the article]

Mitochondrial genome (mitogenome) structure and gene order are generally considered conserved in vertebrates. However, the flatfish (Pleuronectiformes) mitogenomes exhibit the most diversified gene rearrangement patterns. Here, we report a newly sequenced mitogenome of Cynoglossus melampetalus (Pleuronectiformes: Cynoglossidae). The total length of the C. melampetalus mitogenome is 16,651 bp, containing 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, a putative control region, and an L-strand replication origin. Like all previously reported tongue sole (Cynoglossinae) mitogenomes, the C. melampetalus tRNA-Gln gene is inverted from the light to the heavy strand (Q inversion), accompanied by the translocation of CR, which is downstream to the 3'-end of ND1. In addition, we observed a unique tRNA-Ile-Met-Glu (IMQ) gene order that differed from the tRNA-Glu-Ile-Met (QIM) order previously reported for other 14 Cynoglossinae mitogenomes. To our knowledge, it is the first report of two different patterns of mitogenomic gene-arrangement within the same genus in teleost. According to the Q inversion, Met pseudogene (ψ Met) and long intergenic gap (186 bp) between M and Q genes, the observed gene rearrangement pattern were presumably supported by mitochondrial recombination and tandem duplication/random loss models. The reduced trend of the intergenic gap between Q and I also suggests that the event of gene rearrangement can be traced back to early Cynoglossinae differentiation. [ABSTRACT FROM AUTHOR]

Background: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed "pseudomales" who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring. Results: To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation-related and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish. Conclusions: These signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed "pseudomales" in C. semilaevis. [ABSTRACT FROM AUTHOR]

Perfluorooctane sulfonate (PFOS) is an increasingly important environmental pollutant that is pervasive in the environment. A number of studies have focused on the toxicological effects of PFOS on model fish species (zebrafish and medaka), but little is known about the impact of PFOS on commercially important marine fish. Thus, the present study examined transcriptome responses to PFOS exposure in the liver of juvenile Cynoglossus semilaevis, an important farmed flatfish in China. Then, in response to PFOS challenges, 1695 and 5244 genes were identified as significantly increased and depressed, respectively. Significant expression changes were observed in immune‐related genes (cytokine–cytokine receptor interaction, T‐helper [Th]17 cell differentiation, and the chemokine nuclear factor‐kappa B and T‐cell receptor signaling pathways), indicating that immunotoxicity is a key aspect of the effects of PFOS on C. semilaevis. Exposure to PFOS also altered the gene expression levels of hormones (inhibin, insulin, somatostatin, and glucagon), which could lead to severe metabolic and endocrine dysfunction. As expected from previous studies, several phase I and phase II detoxification enzymes were significantly up‐regulated, which could facilitate the biotransformation and detoxification of PFOS in C. semilaevis. The present study provides new insights into the molecular toxicology of PFOS in a commercially important fish species. Environ Toxicol Chem 2020;39:556–564. © 2019 SETAC [ABSTRACT FROM AUTHOR]

Sex reversal is a complex biological phenomenon exhibited by Cynoglossus semilaevis. Some genetic females may irreversibly convert to pseudomales, thus increasing aquaculture costs because males grow much more slowly than females. In this study, an integrative analysis of transcriptome and proteome was performed to compare differences in gene and protein expression in females and pseudomales after gonad differentiation in C. semilaevis. Based on RNA-Seq results, 1893 genes showed differences in expression at the transcript level between females and pseudomales. Of these differentially expressed genes (DEGs), zona pellucida sperm-binding protein 4-like (LOC103393374 , ZP4), zona pellucida sperm-binding protein 4-like (LOC103396071, ZP4) and forkhead box L2 (foxl2) were highly expressed in females and doublesex and mab-3 related transcription factor 1(dmrt1) and doublesex and mab-3 related transcription factor 3 (dmrt3) were highly expressed in pseudomales. GO enrichment analysis results indicate that wnt signaling pathways and oocyte maturation are two terms enriched in female. At the protein level, Tandem Mass Tags analysis revealed that 324 proteins differed in their relative abundance between pseudomales and females. KEGG analysis found that pseudo-highly expressed proteins were enriched in the ubiquitin mediated proteolysis pathway. For integrative analysis, the Spearman correlation coefficient between the transcriptome and proteome was 0.59. Among 52 related genes, 46 DEGs (88%) were well matched in their levels of change in protein abundance. These findings reveal major active pathways in female and pseudomale gonads after sex reversal and provide new [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important roles in multiple biological processes and organismal immunity. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a , CsTRAF2b , CsTRAF3 , CsTRAF4 , CsTRAF5 , CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Functional analysis of CsTRAF3 , CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3 , CsTRAF4 and CsTRAF6 in HEK-293T cells. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research. • We identified seven TRAF genes in Chinese tongue sole, subsequently cloned the complete ORFs of CsTRAFs. • Bioinformatic analysis confirmed the identities and nomenclature. • The CsTRAFs are highly expressed in immune-related tissues and participate in the immune response after V. harveyi infection. • Only CsTRAF6 could strongly activated NF-кB pathway after co-transfection with NF-кB reporter plasmid in HEK-293T cells. [ABSTRACT FROM AUTHOR]

The transient receptor potential vanilloid 4 (TRPV4), another Ca2+ entry channel, belongs to the vanilloid subfamily and responds to a number of different physical and chemical stimuli and exists widely in mammals. However, our understanding of the TRPV4 in fish remains poor. Therefore, we studied the TRPV4 gene from Cynoglossus semilaevis, named CsTRPV4 that encodes a putative protein of 870 amino acids common in structure and characteristic of mammalian TRPV4, including the domains of ANK repeats, six TM, TRP domain, and CaMBD. The CsTRPV4 was expressed ubiquitously in examined tissues: higher expression in the heart, spleen, testis, and eye, but lower expression in kidney and liver. Surprisingly, the expression of CsTRPV4 in lateral line was significantly higher than in many other tissues as the CsTRPV4 was expressed significantly in the free neuromasts. In addition, CsTRPV4 in the free neuromast from the larval fish was significantly expressed in the hair cells of the free neuromasts. Therefore, the free neuromasts can act as a mechano-sensor to the mechanical stimulation in molecular level in C. semilaevis, which lays a foundation for further study of the functions of the free neuromasts. [ABSTRACT FROM AUTHOR]

At present, the function and profile of long noncoding RNAs (IncRNAs) in half-smooth tongue soles Cynoglossus semilaevis remain poorly understood. Lherefore, we identified IncRNAs in the species using large-scale deep sequencing approaches. A total of 96 726 222 clean reads and 2497 IncRNAs were obtained from the compound samples. In total, 1 694 known IncRNAs and 803 novel IncRNAs were identified. Most of the novel IncRNAs distributed mainly in a range of 200–3 000 nt and contained 2–6 exons. Six novel IncRNAs were selected for expression analysis by quantitative reverse transcription-polymerase chain reaction, of which lnc_770 and lnc_150 were expressed mainly in the ovary. Lhis study provides a valuable resource for IncRNA studies of half-smooth tongue sole and improves our understanding of the function of non-coding RNA in fish. [ABSTRACT FROM AUTHOR]

Collectin 11 (CL-11, also known as collectin kidney-1, CL-K1), a new member of the vertebrate C-type lectin superfamily, plays an important role in innate immunity as a pattern recognition molecule of the lectin complement pathway. However, little is known about CL-11 in teleosts. In the present study, a CL-11 homolog was identified and characterized from half-smooth tongue sole (Cynoglossus semilaevis) (designated as CsCL -11). The full-length cDNA of CsCL -11 is 1220 bp long and includes a 5′untranslated region (5′-UTR) of 180 bp, a 3′-UTR of 218 bp and an open reading frame (ORF) of 819 bp encoding 273 amino acids. Multiple sequence alignment revealed that the deduced CsCL-11 protein has the typical modular architecture (EPN and WTD) conserved throughout vertebrates, suggesting a conserved function of CsCL-11. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed CsCL -11 to be ubiquitously distributed in tissues and highly expressed in the ovary and liver. A pattern of significant upregulation of CsCL -11 expression was observed in the blood, spleen, head kidney and gill at 6 h, 12 h and 24 h after infection with Vibrio anguillarum , and western blotting showed that natural CsCL-11 protein levels in the blood were significantly increased after V. anguillarum infection. Moreover, by binding to various bacteria, recombinant CsCL-11 (rCsCL-11) expressed in HEK-293 T cells displayed strong antibacterial activity. Taken together, these results suggest that CsCL -11 is a unique C-type lectin that is likely involved in host defense against bacterial infection. To our knowledge, this is the first study on CL-11 in marine fish. • Collectin 11 gene of Cynoglossus semilaevis (CsCL -11) was first identified in marine fish. • CsCL -11 mRNA was ubiquitously and highly expressed in the ovary and liver. • CsCL -11 was up-regulated in immune tissues by infection with Vibrio anguillarum. • rCsCL-11 protein showed antibacterial activity by directly bound to bacterial cells. [ABSTRACT FROM AUTHOR]

Interleukin-10 (IL-10) is a pleiotropic cytokine and plays a crucial role in immunity. In the current study, we examined the expression patterns and biological functions of tongue sole Cynoglossus semilaevis IL-10 (CsIL-10). CsIL-10 is composed of 186 amino acid residues and shares 46.3%–71.7% identities with other teleost IL-10. Csil-10 expression occurred in multiple tissues and was regulated by bacterial infection. Recombinant CsIL-10 (rCsIL-10) in the form of a dimer bound to a wide range of bacterial species but did not affect bacterial growth. rCsIL-10 could interact with peripheral blood leukocytes (PBL) and significantly reduce the phagocytic activity, ROS production, and apoptosis of PBL. When injected in vivo , rCsIL-10 significantly suppressed the expression of proinflammatory cytokines and promoted bacterial dissemination in tongue sole tissues. Consistently, knockdown of Csil-10 significantly inhibited bacterial infection in tongue sole. Taken together, these results indicate that CsIL-10 plays a negative regulatory role in the immune response against bacterial infection. • Csil-10 expression was found in multiple tissues and upregulated by bacterial infection. • The rCsIL-10 dimer bound to a wide range of bacteria but had no effect on bacterial growth. • Binding of rCsIL-10 to leukocytes inhibited the phagocytic activity, ROS production, and apoptosis of leukocytes. • CsIL-10 is a secreted cytokine that suppressed the production of proinflammatory cytokines. • CsIL-10 enhanced bacterial infection in fish tissues. [ABSTRACT FROM AUTHOR]

Our previous studies have indicated that dietary arachidonic acid (ARA) significantly affects the gonadal steroidogenesis in the marine teleost tongue sole Cynoglossus semilaevis, and this effect was more positive in male fish than in female fish (Aquaculture, 468, 378–385). As a following up study, the present study was further aimed at investigating the possible mechanisms in the brain mediating the effects of dietary ARA on gonadal steroidogenesis. A 70‐day feed trial was repeated with two‐year‐old tongue sole, using three experimental diets with graded levels of ARA, 0.34%, 2.53%, and 9.63% of total fatty acids. Each diet was fed to triplicate groups of 23 fish (15 males and 8 females). The results confirmed the positive effect of dietary ARA on testosterone production in male fish. The concentration of gonadotrophin‐releasing hormone (GnRH) in serum responded to dietary ARA in a similar pattern with the testosterone concentration, but the concentration of gonadotrophin in serum was not affected by dietary ARA. The response of gene expression of PKCβ, ARRB1, ARRB2, ERK2 and ATF3 in the brain to dietary ARA was in good agreement with those of GnRH and testosterone, indicating the possible involvement of PKC‐ARRB‐ERK‐ATF3 pathway in signalling transduction of GnRH. However, the PI3K/Akt and TLR/NF‐κB pathways may not be directly involved in the regulation of GnRH metabolism by ARA. This is the first study reporting the possible involvement of PKC/MAPK pathways in regulation of reproductive endocrine processes by long‐chain polyunsaturated fatty acid in brain of marine fish. [ABSTRACT FROM AUTHOR]

Interleukin-20 (IL-20), as an essential member of IL-10 family, plays vital roles in mammalian immunological response such as antimicrobial, inflammation, hematopoiesis, and immune diseases. In teleost, the study about immune antimicrobial function of IL-20 is largely scarce. In this article, we revealed the expression profiles and the immunological functions of the IL-20 (CsIL-20) in tongue sole Cynoglossus semilaevis. CsIL-20 is composed of 183 amino acid residues, with seven cysteine residues and a typical IL-10 domain which comprises six α-helices and two β-sheets, and shares 34.4–71.2 % identities with other teleost IL-20. CsIL-20 was constitutively expressed in a variety of tissues and regulated by bacterial invasion, and the recombinant CsIL-20 (rCsIL-20) could bind to different bacteria. In vitro rCsIL-20 could interact with the membrane of peripheral blood leukocytes (PBLs), leading to the attenuation of reactive oxygen species (ROS) production and acid phosphatase activity in PBLs. In line with In vitro results, In vivo rCsIL-20 could obviously suppressed the host immune against bacterial infection. Furthermore, knockdown of CsIL-20 in vivo could markedly enhance the host antibacterial immunity. Collectively, these observations offer new insights into the negative effect of CsIL-20 on antibacterial immunity. • The CsIL-20 expression profiles were regulated by bacterial infection. • rCsIL-20 could bind a variety of bacteria. • rCsIL-20 could interact with PBLs, resulting in the suppression of acid phosphatase activity and ROS production in PBLs. • In vivo the presence of CsIL-20 could facilitate the dissemination of bacteria. [ABSTRACT FROM AUTHOR]

Sexual size dimorphism (SSD) characterized by different body size between females and males have been reported in various animals. Gonadectomy experiments have implied important regulatory roles of the gonad in SSD. Among multiple factors from the gonad, TGF-β superfamily (especially BMP/GDF family) attracted our interest due to its pleiotropy in growth and reproduction regulations. Thus, whether BMP/GDF family members serve as crucial regulators for SSD was studied in a typically female-biased SSD flatfish named Chinese tongue sole (Cynoglossus semilaevis). Firstly, a total of 26 BMP/GDF family members were identified. The PPI network analysis showed that they may interact with ACVR2a, ACVR2b, ACVR1, BMPR2, SMAD3, BMPR1a, and other proteins. Subsequently, DAP-seq was employed to reveal the binding sites for yin yang 1 (yy1), a transcription factor involved in gonad function and cell growth partly by regulating TGF-β superfamily. The results revealed that two yy1 homologues yy1a and yy1b in C. semilaevis could regulate Hippo signaling pathway, mTOR signaling pathway, and AMPK signaling pathway. Moreover, BMP/GDF family genes including bmp2 , bmp4 , bmp5 , gdf6a , and gdf6b were important components of Hippo pathway. In future, the crosstalk among yy1a , yy1b , and TGF-β family would provide more insight into sexual size dimorphism in C. semilaevis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse -miR-722) was significantly differentially expressed after infection with Vibrio anguillarum , reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse -miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse -miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse -miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse -miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse -miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse -miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse -miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse -miR-722 regulated the expression of CsC5aR1 , CsC5a , and the inflammatory factors CsIL1-β , CsIL6 , CsIL8 , and CsTNF-α both in vitro and in vivo. In addition, Cse -miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse- miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse- miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost. • Cse -miR-722 targeted CsC5aR1 and silenced CsC5aR1 at the translation level. • Cse -miR-722 affected the phagocytic activity and respiratory burst of macrophages. • Cse -miR-722 regulated immune responses through the C5a/C5aR1 complement pathway. • Cse -miR-722 reduced mortality and body damage after Vibrio anguillarum infection. [ABSTRACT FROM AUTHOR]

The length–weight relationships (LWRs) for seven fish species belonging to five families in the Yangtze River Estuary and its adjacent waters are presented. Cynoglossus gracilis, Cynoglossus macrolepidotus, Ctenotrypauchen chinensis, Collichthys niveatus, Ophichthusapicalis and Erisphex potti were collected monthly in 2009 using trammel nets (the size of smaller mesh net was 1.5 m × 15 m × 4 panels with mesh sizes of 2.5, 3.4, 4.3 and 5.8 cm; the size of larger‐mesh net was 2.4 m × 30 m × 4 panels with mesh sizes of 5.0, 6.0, 7.0 and 8.0 cm; soaking overnight). Lophiogobius ocellicauda were collected December in 2020 using shrimp trawl nets (mesh size 2.0 cm). The precision of measurement for the fish specimens is 0.1 cm total length and 0.1 g total weight. The estimated ranges of the parameters a and b for the seven fish species were from 0.0001 to 0.0289 and 2.718 to 3.541, respectively. Two new maximum total length were recorded for Ctenotrypauchen chinensis and Ophichthusapicalis. [ABSTRACT FROM AUTHOR]

Summary: This study estimated the LWRs of six fishes from the coastal waters of Greater Accra, Ghana. Data was obtained monthly from June 2018 to July 2019 and analyzed for total length and weight measurement to the nearest 0.1 cm and 0.01 g respectively. The sampling was done using hooks and line as well as monofilament and multifilament gear types. The mesh sizes for both monofilament and multifilament gear types ranged from 0.5 to 2 inches whereas the hooks types ranged from 6 to 12. The b values for the assessed species were within the range 2.8–3.2. There was strong correlation between the length and weight of all the species (R2 >.95). This study provides LWRs for Cynoglossus senegalensis, Scorpaena histrio and Pseudotolithus senegalensis and larger maximum total length (TLmax) for Syacium micrurum, Eucinostomus melanopterus and Brachydeuterus auritus. [ABSTRACT FROM AUTHOR]

Length-weight relations were estimated for 14 fish species sampled from the coastal waters off the Gwangyang Bay in South Korea. The following species were studied: Okamejei kenojei (Müller et Henle, 1841); Muraenesox cinereus (Forsskål, 1775); Thryssa adelae (Rutter, 1897); Thryssa kammalensis (Bleeker, 1849); Tribolodon hakonensis (Günther, 1877); Inimicus japonicus (Cuvier, 1829); Chelidonichthys spinosus (McClelland, 1844); Jaydia lineata (Temminck et Schlegel, 1842); Sillago japonica Temminck et Schlegel, 1843; Pholis nebulosa (Temminck et Schlegel, 1845); Favonigobius gymnauchen (Bleeker, 1860); Pampus echinogaster (Basilewsky, 1855); Cynoglossus joyneri Günther, 1878; Takifugu niphobles (Jordan et Snyder, 1901). The length-weight relation of Thryssa adelae (Rutter, 1897), (Engraulidae) has not been previously reported. The new maximum total length of Thryssa kammalensis (18.0 cm) is now provided. The values of coefficient a ranged from 0.0007 to 0.0218, and the values of exponent b ranged from 2.82 to 3.52. [ABSTRACT FROM AUTHOR]

The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature. [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]