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Chemical communication through olfaction is crucial for fish behaviours, mediating in socio-sexual behaviours as reproduction. Turbot, a flatfish with significant aquaculture production, possesses a well-developed olfactory system from early developmental stages. After metamorphosis, flatfish acquire their characteristic bilateral asymmetry with an ocular side facing the open water column, housing the dorsal olfactory rosette, and a blind side in contact with the sea bottom where the ventral rosette is located. This study aimed to address the existing gap in specific histological, ultrastructural, lectin-histochemical and immunohistochemical studies of the turbot olfactory rosettes and olfactory bulbs. We examined microdissected olfactory organs of adult turbots and premetamorphic larvae by using routine histological staining techniques, and a wide array of lectins and primary antibodies against G-proteins and calcium-binding proteins. We observed no discernible structural variations in the olfactory epithelium between rosettes, except for the dorsal rosette being larger in size compared to the ventral rosette. Additionally, the use of transmission electron microscopy significantly improved the characterization of the adult olfactory epithelium, exhibiting high cell density, small cell size, and a wide diversity of cell types. Moreover, specific immunopositivity in sensory and non-sensory cells provided us of essential information regarding their olfactory roles. The results obtained significantly enriched the scarce morphological and neurochemical information available on the turbot olfactory system, revealing a highly complex olfactory epithelium with distinct features compared to other teleost species, especially with regard to olfactory cell distribution and immunolabelling patterns., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida . dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

Objective: The waterless transport of live fish has changed the present situation of live-fish transport. However, the waterless transport environment may cause stress in fish. This research evaluated the effect of tea polyphenol-trehalose (TPT) coating solutions on Turbot Scophthalmus maximus during waterless transport., Methods: After cold acclimation, Turbot were coated and subsequently transported in a waterless environment for 18 h. Physiological and biochemical parameters were measured, including lysozyme (LZM) and immunoglobulin M (IgM) activities, serum creatinine (Cr) and uric acid (UA) concentrations, and nutritional flavor., Result: The results showed that the nonspecific immunity of Turbot was inhibited during the waterless transport; the LZM activity first increased and then decreased, and the serum Cr and UA concentrations significantly increased. In addition, the waterless transport promoted the breakdown of Turbot flesh proteins, leading to changes in nucleotides and free amino acids (FAAs). After waterless transport, the LZM and IgM activities in the TPT-treated Turbot were higher than those in the control group (CK), and the changes in FAA content and nucleotides were smaller than those observed in the CK group., Conclusion: This study shows that the use of TPT coating solution can reduce the impact of waterless transportation stress on the immune and metabolic functions of Turbot and can maintain the meat quality and flavor of Turbot., (© 2024 American Fisheries Society.)

Germ cell transplantation (GCT) is a promising biotechnology that enables the production of donor-derived gametes in surrogate recipients. It plays a crucial role in the protection of endangered species, the propagation of elite species with desired traits, and long-term preservation of genetic resources. This significance is particularly pronounced when GCT is synergistically employed with cryopreservation techniques. However, GCT often encounters challenges due to low colonization rates and, in some cases, complete loss of donor cells in recipients. Glial cell line-derived neurotrophic factor (GDNF) plays a pivotal role in sustaining the self-renewal of spermatogonial stem cells (SSCs) in mammals. Additionally, it has been shown to promote the proliferation of spermatogonia in vitro cultures in certain animal species. In turbot (Scophthalmus maximus), we found that the expressions of gdnf and gfrα1a were predominantly observed in spermatogonia rather than somatic cells, which differed from their expression patterns in mammals. The efficiency of exogenous spermatogonia transplantation in Japanese flounder (Paralichthys olivaceus) larvae could be substantially enhanced by incubating donor cells from turbot with 100 ng/ml GDNF prior to transplantation. This led to a noteworthy increase in the colonization rate, rising from 33%-50%-61.5%. Additionally, the addition of 20 ng/ml GDNF in cell medium could also promote the proliferation of turbot germ cells in vitro. These results demonstrated the gdnf in turbot testis expression characteristics and suggested that addition of GNDF could be an effective way to improve the GCT efficiency and promote the germ cells expansion during in vitro culture., Competing Interests: Declarations of competing interest The authors declare that no competing interests in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus., (Copyright © 2023. Published by Elsevier Ltd.)

The present study explored transcriptomics and gene regulation variations in the muscle of turbot fed with dietary taurine. A 70-day feeding trial was conducted using turbot (initial body weight: 3.66 ± 0.02 g) fed with different levels of dietary taurine: 0 % (C), 0.4 % (T2), 1.2 % (T4) and 2.0 % (T6). Two methods were used to analyze and verify the taurine effects on muscle growth: (1) real-time quantitative PCR (qRT-PCR) for the key muscle growth-related genes and (2) transcriptomic analysis by next-generation sequencing (NGS). The results showed that 1.2 % of dietary taurine supplementation significantly increased the expression of muscle growth stimulatory genes, including TauT, myoD, Myf5, myogenin and follistatin. And also, the 1.2 % level significantly decreased the expression of the muscle growth-restricting gene (myostatin). Meanwhile, transcriptomics analysis found that 1.2 % dietary taurine supplementation significantly increased the number of up-regulated genes linked to metabolic pathways. In contrast, taurine significantly enriched the actin cytoskeleton and metabolic pathways in the T4 and T2 groups, respectively. These findings align with the gene ontology (GO) analysis, which indicated a higher number of cellular component (CC) gene expressions at a 1.2 % of dietary taurine compared to a 0.4 % of dietary taurine supplementation. In conclusion, dietary taurine had positive impacts on the growth-stimulatory genes. Moreover, 1.2 % of dietary taurine supplementation is important to the metabolic pathway enrichment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Tight junction proteins (TJs) are important component proteins that maintaining the structure and function of TJs, connecting to each other to form a TJ complex between cells, maintaining the biological homeostasis of the internal environment. In this study, a total of 103 TJ genes were identified in turbot according to our whole-transcriptome database. Transmembrane TJs were divided into seven subfamilies, including claudin (CLDN), occludin (OCLD), tricellulin (MARVELD2), MARVEL domain containing 3 (MARVELD3), junctional adhesion molecules (JAM), immunoglobulin superfamily member 5 (IGSF5/JAM4), blood vessel epicardial substance (BVEs). Moreover, the majority of homologous pairs of TJ genes showed highly conserved alongside length, exon/intron number and motifs. As for phylogenetic analysis for 103 TJ genes, eight of them have undergone a positive selection and JAMB-like has undergone the most neutral evolution. The expression patterns of several TJ genes showed the lowest expression levels in blood, while the highest expression levels were detected in intestine, gill and skin, which all belong to mucosal tissues. Meanwhile, most examined TJ genes showed down-regulated expression patterns during bacterial infection, while several TJ genes exhibited up-regulated expression patterns at a later stage (24 h). At the same time, several potential candidate genes (such as CLDN-15, CLDN-3, CLDN-12, CLDN-5 and OCLD) were significantly down-regulated, which may indicate their important functions that involved in the regulation of bacterial infection. Currently, there is little research on CLDN5 in the intestine, but it is highly expressed in the intestine and has significant changes in intestinal expression after bacterial infection. Thus, we knocked down CLDN5 by the method of lentiviral infection. The result showed CLDN5 was related to cell migration (wound healing) and apoptosis, and the method of dualluciferasereporterassay showed that the functions of CLDN5 could be regulated by miR-24. The study of TJs may lead to a better understanding of the function of TJs in teleost., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ovarian fluid is essential for successful fertilization by maintaining the viability, motility, and velocity of sperm. The organic compounds and inorganic ions in ovarian fluid significantly influence spermatozoa's motility, velocity, and longevity. However, the effect of ovarian fluid on sperm performance is limited in teleost fish. In this study, the effect of ovarian fluid on sperm performance and its components in external fertilization species (Scophthalmus maximus, turbot) and internal fertilization species (Sebastes schlegelii, black rockfish) was investigated using computer-assisted sperm analysis, high-performance liquid chromatography, and metabolome analysis. The ovarian fluid had a distinct and species-specific effect on both species. In the black rockfish, the ovarian fluid from turbot significantly increased sperm motility (74.07% ± 4.09%), as well as VCL (45 ± 1.67 μm/s), VAP (40.17 ± 1.6 μm/s), and VSL (36.67 ± 1.86 μm/s), and longevity (352 ± 11.31 min) (P < 0.05). In the turbot, only the longevity (71.33 ± 5.69 min) and fertilization rate (65.27% ± 11.59%) showed significantly improvement (P < 0.05). The ovarian fluid was rich in organic compounds, suggesting enrichment in the glycolysis/gluconeogenesis pathways. The results suggest that glycometabolism plays a crucial role in improving sperm performance in teleost with internal fertilization. Thus, incorporating ovarian fluid into the sperm activation medium can enhance artificial fertilization in fish breeding., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1β, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The development and maturation of sperm entails intricate metabolic processes involving water molecules, amino acids, hormones, and various substances. Among these processes, the role of aquaporins ( aqps ) in the testis is crucial. Turbot ( Scophthalmus maximus ) is a significant marine flatfish species in China; however, natural egg laying in females is not feasible under cultured conditions. Consequently, artificial insemination becomes necessary, requiring the retrieval of sperm and eggs through artificial methods. In this study, we combined genomic, transcriptomics, RT-qPCR, computer-assisted sperm analysis (CASA), and immunohistochemistry to investigate the involvement of the aqp family in spermatogenesis in turbot. Through genomic data analysis, we identified 16 aqps genes dispersed across 13 chromosomes, each exhibiting the characteristic major intrinsic protein (MIP) domain associated with AQPs. The results from RNA-seq and RT-qPCR analysis revealed prominent expression of aqp4 , 10 , and 12 during the proliferative stage, whereas aqp1 showed primary expression during the mature stage. aqp11 displayed high expression levels during both MSII and MSV stages, potentially contributing significantly to the proliferation and maturation of male germ cells. Conversely, aqp8 showed elevated expression levels during the MSIII, MSIII-IV, and MSIV stages, suggesting its direct involvement in spermiogenesis. Immunohistochemical analysis unveiled the predominant localization of AQP1 protein in male germ cells rather than Sertoli cells, specifically concentrated in the head of sperm within cysts. Furthermore, a noteworthy decline in sperm motility was observed when sperm were subjected to treatment with either the AQP1-specific inhibitor (HgCl 2 ) or the AQP1 antibody. However, no direct correlation was found between the expression of Smaqp1 and sperm quality. Overall, these findings provide new insights into the involvement of aqps in teleost spermatogenesis. Moreover, they hold potential for improving techniques related to sperm activation and cryopreservation, offering valuable knowledge for future advancements in this field.

Apoptosis is the autonomous and orderly death of cells under genetic control to maintain the stability of the internal environment, and is a programmed cell death process with unique morphological and biochemical properties that is regulated by a variety of factors. Caspase gene family has a significant function in the process of apoptosis. However, the knowledge of caspases in turbot remains largely unknown. In present study, a total of nine turbot caspase genes were identified. The mRNA length of these caspase genes was ranged from 1225 bp (caspase-7) to 3216 bp (caspase-2), and the protein length was ranged from 281 aa (caspase-3a) to 507 aa (caspase-10). Phylogenetic analysis showed these caspase genes were divided into three subfamilies. The qRT-PCR results showed that turbot caspase genes were expressed in all the examined organs, especially the intestine, kidney, blood and gills. Meanwhile, we explored the expression patterns of caspase genes in the intestine, skin and gills after Vibrio anguillarum and Aeromonas salmonids infections. The results showed that caspase genes showed different expression patterns in mucosal tissues after bacterial infection, demonstrating the critical role of caspase genes in mucosal immune responses. In addition, protein-protein interaction analysis showed that caspase proteins interacted with immune molecules such as NLR, IL-1β, and birc. The results of interference and overexpression experiments showed that caspase-1 might play key roles in the regulation of the IL-1β production, but the detailed mechanism needs to be further studied. The results of this study provide valuable information for further study the roles of caspase genes in turbot, which could help us to further understand the inflammatory pathways in teleost., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A continuous intestine cell line from turbot (Scophthalmus maximus) designated as SMI was established utilizing the tissue explant technique. Primary SMI cell was cultured at 24 °C in a medium with 20% fetal bovine serum (FBS), then subcultured in 10% FBS after 10 passages. Impacts of medium or temperature on the growth of SMI were examined and the results indicated it grew well in DMEM supplemented with 10% FBS at 24 °C. The SMI cell line was subcultured more than 60 times. Karyotyping, chromosome number, and ribosomal RNA genotyping analysis revealed that SMI had a modal diploid chromosome number of 44 and originated from turbot. After being transfected with pEGFP-N1 and FAM-siRNA, a large number of green fluorescence signals were observed in SMI, indicating that SMI could be used as an ideal platform to explore gene function in vitro. In addition, the expression of epithelium-associated genes such as itga6, itgb4, gja1, claudin1, zo-1, and E-cadherin in SMI suggested the SMI had some characteristics of epidermal cells. The upregulation of immune-associated genes such as TNF-β, NF-κB, and IL-1β in SMI after stimulation with pathogen-associated molecular patterns suggested the SMI might exhibit immune functions similar to the intestinal epithelium in vivo., (© 2023. The Author(s).)

Long noncoding RNAs (lncRNAs), can regulate mRNA by targeting miRNA in a competing endogenous RNA network, have become a hot topic in the research of fish immune mechanism recent years. While in turbot (Scophthalmus maximus L.), an economically important marine fish, there are limited researches about the role of lncRNAs in its immune response to bacterial infection. In this study, a total of 184 differentially expressed lncRNAs (DElncRNAs) were systematically identified and characterized using whole-transcriptome sequencing of the liver of turbot challenged with Vibrioanguillarum at 0 h (control) and three different time points post infection (2 h, 12 h and 24 h, respectively). Subsequently, GO and KEGG signaling pathways of differentially expressed lncRNAs were analyzed to predict their function. We found that lncRNAs in our results were significantly enriched in several immune-related signaling pathways, including the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, Cytokine-cytokine receptor, MAPK signaling pathway, phagosome, PPAR signaling pathway and the regulation of autophagy. In addition, a total of 492 DE lncRNA - DE miRNA -DE mRNA networks were identified at three different time points post infection, which were consisted of 102 networks at 2 h, 122 networks at 12 h and 81 networks at 24 h post infection, respectively. Noticeably, 92 of these regulated networks were immune-related. These observations suggested that lncRNAs can regulate the expression of immune-related genes in the response to bacterial infection in turbot. Moreover, our findings would provide a new insight into the immune response of turbot to pathogen infection and lay a foundation for future study., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Long non-coding RNA (lncRNA) play vital regulatory roles in various biological processes. Intestine is one of the most sensitive organs to environmental and homeostatic disruptions for fish. However, systematic profiles of lncRNAs in the intestine of teleost in responses to pathogen infections is still limited. Turbot (Scophthalmus maximus L.), an important commercial fish species in China, has been suffering with Vibrio anguillarum infection, resulted in dramatic economic loss. Hereinto, the intestinal tissues of turbot were sampled at 0 h, 2 h, 12 h, and 48 h following V. anguillarum infection. The histopathological analysis revealed that the pathological trauma was mainly present in intestinal tunica mucosal epithelium. After high-throughput sequencing and bioinformatic analysis, a total of 9722 lncRNAs and 21,194 mRNAs were obtained, and the average length and exon number of lncRNAs were both less than those of mRNAs. Among which, a set of 158 lncRNAs and 226 mRNAs were differentially expressed (DE-lncRNAs and DEGs) in turbot intestine at three time points, related to many immune-related genes such as complement, interleukin, chemokine, lysosome, and macrophage, indicating their potential critical roles in immune responses. In addition, 2803 and 1803 GO terms were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Moreover, 127 and 50 KEGG pathways including cell adhesion molecules (CAMs), phagosome, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, and intestinal immune network for IgA production, were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Finally, qRT-PCR was conducted to confirm the reliability of sequencing data. The present study will set the foundation for the future exploration of lncRNA functions in teleost in response to bacterial infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

An eight-week feeding trial was conducted to investigate the effects of diets supplemented with three sulfur-containing amino acids (SAA), namely, methionine, cysteine, and taurine, on the intestinal health status of juvenile turbot (Scophthalmus maximus) fed high-lipid diets. Four diets were formulated, namely, a high-lipid control diet (16% lipid, HL) and three SAA-supplemented diets, which were formulated by supplementing 1.5% methionine (HLM), 1.5% cysteine (HLC), and 1.5% taurine (HLT) into the HL control diet, respectively. Each diet was assigned to triplicate tanks, and each tank was stocked with 30 juvenile fish (appr. initial weight, 8 g). The histological and morphometric results showed that dietary SAA supplementation obviously improved the intestinal morphology and integrity, in particular as reflected by higher height of microvilli and mucosal folds. Dietary SAA supplementation, in particular cysteine, up-regulated the gene expression of mucin-2 and tight junction proteins (ZO-1, Tricellilun and JAM). Dietary SAA supplementation remarkably down-regulated the gene expression of apoptosis-related factors such as p38, JNK, and Bax, expression of pro-inflammatory factors (e.g., NF-κB, AP-1 IL-1β, IL-8, and TNF-α). SAA supplementation resulted in higher antioxidative abilities in the intestine. Additionally, dietary SAA supplementation largely altered the communities of intestinal microbiota. Compared with the HL group, higher relative abundance of potential beneficial bacteria, and lower relative abundance of opportunistic pathogens were observed in SAA-supplemented groups. Dietary taurine supplementation significantly increased the relative abundance of Ligilactobacillus (in particular Lactobacillus murinus) and Limosilactobacillus (especially Lactobacillus reuteri). In conclusion, dietary sulfur-containing amino acids supplementation have promising potential in ameliorating the intestinal inflammation of turbot fed high-lipid diets. Especially dietary cysteine and taurine supplementation have more positive effects on the communities of the intestinal microbiota of turbot., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Excessive dietary carbohydrate commonly impairs the functions of liver and intestine in carnivorous fish. In the present study, a 10-week feeding trial was carried out to explore the regulation of biotin on the hepatic and intestinal inflammation and apoptosis in turbot (Scophthalmus maximus L.) fed with high carbohydrate diets. Three isonitrogenous and isolipidic experimental diets were designed as follows: the CC diet with 18.6% of carbohydrate and 0.04 mg/kg of biotin, the HC diet with 26.9% of carbohydrate and 0.05 mg/kg of biotin, and the HCB diet with 26.9% of carbohydrate and 1.62 mg/kg of biotin. Results showed that high dietary carbohydrate (HC diet) impaired the morphology of liver and intestine, however, inclusion of dietary biotin (HCB diet) normalized their morphology. Inflammation-related gene expression of nuclear factor κB p65 (nf-κb p65), tumor necrosis factor α (tnf-α), interleukin-1β (il-1β), il-6 and il-8, and the protein expression of NF-κB p65 in the liver and intestine were significantly up-regulated in the HC group compared to those in the CC group (P < 0.05), the HCB diet decreased their expression compared to the HC group (P < 0.05). The gene expression of il-10 and transforming growth factor-β (tgf-β) in the liver and intestine were significantly decreased in the HC group compared to the CC group (P < 0.05), and inclusion of dietary biotin increased the il-10 and tgf-β expression in the liver and intestine (P < 0.05). Moreover, compared to the CC group, the HC group had a stronger degree of DNA fragmentation and more TUNEL-positive cells in the liver and intestine, and the HCB group had a slighter degree of DNA fragmentation and fewer TUNEL-positive cells compared to the HC group. Meanwhile, the gene expression of B-cell lymphoma protein-2-associated X protein (bax) and executor apoptosis-related cysteine peptidase 3 (caspase-3) were significantly up-regulated and the gene expression of B-cell lymphoma-2 (bcl-2) was significantly down-regulated both in the liver and intestine in the HC group compared with those in the CC group (P < 0.05). Inclusion of dietary biotin significantly decreased the bax and caspase-3 mRNA levels and increased bcl-2 mRNA level in the liver and intestine (P < 0.05). In conclusion, high dietary carbohydrate (26.9% vs 18.6%) induced inflammation and apoptosis in liver and intestine. Supplementation of biotin (1.62 mg/kg vs 0.05 mg/kg) in diet can alleviate the high-dietary-carbohydrate-induced hepatic and intestinal inflammation as well as inhibit apoptosis in turbot. The present study provides basic data for the application of biotin into feed, especially the high-carbohydrate feed for turbot., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

An 8-week feeding trial was conducted, where turbot were fed four experimental diets, containing different LPC levels (0%, 0.1%, 0.25%, and 0.5%, named LPC0, LPC0.1, LPC0.25, and LPC0.5, respectively). The intestinal morphology results showed that there were no widened lamina propria and mixed inflammatory cells in the LPC-supplemented groups. Dietary LPC remarkably decreased the expression of TLRs (TLR3, TLR8, TLR9, and TLR22), MyD88, and signaling molecules (NF-κB, JNK, and AP-1). Similarly, diets with LPC supplementation markedly depressed the gene expression of NF-κB and JNK signaling pathway downstream genes (TNF-α, IL-1β, Bax, Caspase9, and Caspase-3). Furthermore, dietary LPC modified the intestinal microbial profiles, increasing the relative abundance of short-chain fatty acids-producers, lactic acid bacteria, and digestive enzyme-producing bacteria. Predictive functions of intestinal microbiota showed that turbot fed LPC diets had a relatively higher abundance of functions, such as lipid metabolism and immune system, but a lower abundance of functions, such as metabolic diseases and immune system diseases. The activities of intestinal acid phosphatase and alkaline phosphatase were also increased by dietary LPC. In conclusion, LPC supplementation could regulate the intestinal mucosal barrier via the TLR signaling pathway and alter the intestinal microbiota profile of turbot fed high-lipid diets.

The spread of multi-drug resistant (MDR) bacteria has posed a threat to the development of aquaculture. Due to its effective bactericidal ability, phage therapy has been considered as an alternative to antibiotics to reduce infection caused by MDR bacteria. In this study, two Edwardsiella piscicida phages were newly-isolated and characterized to prevent or treat infection in aquaculture. The phages were designated as vB&#95;EpM&#95;ZHS and vB&#95;EpP&#95;ZHX belonging to Myoviridae and Podoviridae families, respectively, in terms of genome sequence and morphology analyses. The combination of vB&#95;EpM&#95;ZHS and vB&#95;EpP&#95;ZHX improved the therapeutic efficacy both in vitro and in vivo. The phage cocktail significantly inhibited bacterial growth in vitro and decreased approximately 40% of mortality rate and an order of magnitude of bacterial burden in zebrafish and turbot infected by E. piscicida. Moreover, the phage cocktail increased transcription levels of tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), interleukin-6 (IL-6), and interleukin-1β (IL-1β) and alleviated inflammatory levels in the hindgut and spleen of turbots. The results indicate that the phage has a promising potential for therapeutic use against E. piscicida as the antimicrobial alternative to antibiotics in aquaculture., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Turbot (Scophthalmus maximus) is an important cold-water economic fish. However, the production and development of turbot industry has been constantly hindered by the frequent occurrence of some diseases. Lacking full-length transcriptome for turbot limits immune gene discoveries and gene structures analysis. Therefore, we generated a full-length transcriptome using mixed immune-related tissues of turbot with PacBio Sequel platform. In this study, a total of 31.7 Gb high quality data were generated with the average subreads length of 2618 bp. According to the presence of 5' and 3' primers as well as poly (A) tails, FL (Full-length) and NFL (Non-full-length) isoforms were obtained. Meanwhile, we identified 32,003 non-redundant transcripts, 76.02% of which was novel isoforms of known genes. In addition, 12,176 alternative splicing (AS) events, 6614 polyadenylation (APA) events, 1905 transcription factors, and 2703 lncRNAs were identified. This work is a comprehensive report on the full-length transcriptome of immune-related tissues of turbot, and it also provides valuable molecular resources for future research on the adaptation mechanisms and functional genomics of turbot., Competing Interests: Declaration of competing interest All authors have no competing interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We evaluated six plasma physiological indexes (epinephrine, cortisol, alkaline phosphatase, superoxide dismutase, reduced glutathione, and blood glucose) in turbot (Scophthalmus maximus) cultured in five ammonia concentrations (30.28, 35.90, 42.56, 50.47, and 59.82) at different exposure times (4, 8, 12, 24, and 48 h). Then, we conducted additive main effects and multiplicative interaction (AMMI) and genotype main effects and genotype × environment interaction (GGE) biplot analyses to analyze the genotype × ammonia interaction effects on the plasma physiological indexes. The AMMI analysis results revealed that the contributions of ammonia effect first decreased sharply (4-8 h), then decreased slowly (8-24 h), and then increased slowly (24-48 h). The contributions of genotype effect first decreased sharply (4-8 h), then increased slowly (8-24 h), and then decreased slowly (24-48 h). The contributions of genotype × ammonia interactions showed a sharp rise (4-8 h), a sharp decline (8-12 h), a slow decline (12-24 h), and finally a slow increase (24-48 h). The GGE biplot analysis revealed that alkaline phosphatase and reduced glutathione contents/activities are reliable biomarkers that can be used to establish an online early warning system for behavior numerical simulation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Furunculosis caused by Aeromonas salmonicida in turbot farming is increasingly leading to huge economic losses. In this study, an inactivated vaccine containing a compound adjuvant of ginseng stem leaf saponins and aluminum hydroxide gel (GSLS/Alum) was developed against A. salmonicida and evaluated on turbot. As a result, serum antibody titer in vaccinated group was significantly higher than that in control group and the relative percentage survival (RPS) was up to 75.7%. Comparatively, the RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum adjuvant or an oil emulsion Montanide™ ISA 763A were 32.4%, 48.6% and 64.9%, respectively. Although the vaccine containing oil adjuvant elicited comparable IgM level as that containing the compound GSLS/Alum adjuvant, the latter had no obvious side effects. Moreover, the inactivated vaccine containing the compound adjuvant was more likely to induce a higher cellular immune response according to the expressions of some immune related genes. Most importantly, an excellent protection of the vaccine containing GSLS/Alum adjuvant was obtained when turbots were naturally infected under clinical condition. In summary, our study demonstrated that the formulation of GSLS and Alum is a potential compound adjuvant in turbot vaccine development., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aimed to analyze the morphology of the tongue, which varies among fish species and has not been studied in turbot (Scophthalmus maximus), using macro-anatomical, light and scanning electron microscopy (SEM). As research materials, the tongues of eight adult turbot, which were used for consumption, were examined. The roughly triangular-shaped tongue of the turbot consisted of three parts: apex, body, and root. The thickness of the tongue continued to increase from the apex to the root. Although papillae were not observed in the macroscopic examination of the tongue, microscopic examination revealed the presence of cone-like shaped papillae on the submucosa. The tongue was composed of mucosa, submucosa, and hyaline cartilage. By SEM the presence of the taste buds (Types I and III), taste pores and papillae on the dorsal surface were demonstrated. Taste buds are the first descriptions in S. maximus. Therefore our results could add new data to the fish tongue anatomy. HIGHLIGHTS: Morphological and morphometric data of the turbot (Scophthalmus maximus) tongue were obtained in this study and compared with other species. In addition, the dorsal surface of the turbot tongue was described for the first time using SEM., (© 2022 Wiley Periodicals LLC.)

Neutrophils can capture and kill pathogens by releasing neutrophils extracellular traps (NETs), which play critical roles in anti-microbial infection in mammals; however, the mechanisms involved in NETs formation and its role in anti-bacterial infection in teleost fish remains largely unknown. In this study, to explore the function of NETs in turbot, we established an in vitro bacterial infection model in head kidney derived neutrophils, and found that the haemolysin over-expressed Edwardsiella piscicida (ethA + ) could induce a robust phenotype of NETs, compared with that in wild type or ethA mutant (ethA +  -ΔethA) strains. Besides, the NETosis was mediated by ethA +  -induced pyroptosis, and arms the ability of bacterial killing in neutrophils of turbot. Moreover, we found that neutrophils elastase (NE) might involves in this pyroptotic signaling, rather than inflammatory Smcaspase. Taken together, this study reveals the important role of pyroptosis in NETs formation in turbot neutrophils, suggesting that NETs formation is a critical immune response during bacterial infection in teleost fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The diseases triggered by Vibrio anguillarum infection have created huge economic losses to the turbot (Scophthalmus maximus) farming industry. However, the immune mechanism of turbot to V. anguillarum infection has not been deeply investigated. To better understand the immune response of turbot to V. anguillarum infection, transcriptome analysis of the head kidney and liver of turbot was performed. A total of 15,948 and 11,494 differentially expressed genes (DEGs) were obtained from the turbot head kidney and liver, respectively. Transcriptome analysis revealed that the head kidney and liver of turbot have some differences in the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs for the different functions of these two organs. Although there are many uncertain factors in this immune process, such as the occurrence of alternative splicing (AS) events and the differences in the protein structure of the DEGs, the NFκB signalling pathway, MKK-dependent AP-1 activation, JAK-STAT signalling pathway, the signal transmission of MHC Ⅰ and a series of DEGs including HSP90 driving NLRP3 to produce inflammatory factors (IL-1β, IL-8, TNFα, etc.) were possible important immune response pathways for turbot to V. anguillarum infection. Overall, our research has conducted a preliminary exploration of the immune mechanism of turbot in response to V. anguillarum infection., (© 2022 John Wiley & Sons Ltd.)

Seasonal reproduction is generally controlled by the hypothalamus-pituitary-gonadal (HPG) axis in fish. Previous studies have demonstrated that the kisspeptin (Kiss)/kisspeptin receptor (Kissr) system, a positive regulator of the HPG axis, mediates the responses to environmental cues. Turbot ( Scophthalmus maximus ), a representative species of Pleuronectiformes, is one of the most commercially important fish species cultured in Europe and North China. However, the mechanisms by which the Kiss/Kissr system regulates the reproductive axis of turbot according to seasonal changes, especially photoperiod, have not been clearly characterized. In the current study, the cDNA sequences of kiss2 / kissr2 , along with kiss1 / kissr3 which was thought to be lost in flatfish species, were cloned and functionally characterized. The kiss1 , kiss2 , and kissr3 transcripts were highly detected in the brain and gonad, while kissr2 mRNA was only abundantly expressed in the brain. Moreover, kiss / kissr mRNAs were further examined in various brain areas of both sexes. The kiss1 , kissr2 , kissr3 mRNAs were highly expressed in the mesencephalon, while a substantial degree of kiss2 transcripts were observed in the hypothalamus. During annual reproductive cycle, both kiss and kissr transcript levels declined significantly from the immature to mature stages and increased at the degeneration stage in the brains of both sexes, especially in the mesencephalon and hypothalamus. The ovarian kiss1 , kiss2 , and kissr2 mRNA levels were highest at the vitellogenic stage (mature stage), while expression of kissr3 was highest at the immature stage. The testicular kiss and kissr transcripts were highest in the immature and degeneration stages, and lowest at the mature stage. In addition, intraperitoneal injection of Kiss1-10 and Kiss2-10 significantly stimulated mRNA levels of pituitary lhβ , fhsβ , and gthα . In summary, two Kiss/Kissr systems were firstly proven in a flatfish species of turbot, and it has a positive involvement in controlling the reproduction of the Kiss/Kissr system in turbot. The results will provide preliminary information regarding how the Kiss/Kissr system controls seasonal reproduction in turbot broodstock., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Wang, Liu, Feng, Xu, Wang, Liu and Li.)

The pattern recognition receptors (PRRs) can recognize the conserved molecular structures of pathogens to active the innate immune responses, and subsequently induce the antigen-specific adaptive immune responses for the clearance of infected pathogen. Among the PRRs, Toll-like receptors (TLRs) are the first and best characterized PRRs across all the species. Among the TLR members, TLR7 showed significant conservation across the vertebrates, with the lowest rate of evolution for its LRR domains from primates to fishes. In the current study, one TLR7 (SmTLR7) gene was captured in turbot, with a 3144 bp open reading frame (ORF), that encoding 1047 amino acid residues. Following multiple sequence comparison, SmTLR7 was found to have the highest similarity and identity both to Paralichthys olivaceus with 91.9% and 85.9%, respectively. In phylogenetic analysis, SmTLR7 was firstly clustered with Japanese flounder, and then clustered with fugu, rainbow trout, and zebrafish. In addition, SmTLR7 was widely expressed in all the examined tissues with the highest expression level in spleen, followed by skin, while the lowest expression level was detected in blood. Following both Edwardsiella tarda and Vibrio anguillarum challenge, SmTLR7 was significantly down-regulated in gill and intestine, and up-regulated in skin. Moreover, SmTLR7 was significantly up-regulated in head kidney macrophages following LPS, LTA, PGN and polyI:C stimulation, as well as showed the strongest binding ability to LPS, followed by PGN, LTA, and polyI:C in a dose-dependent manner. Finally, following RNAi of SmTLR7, MyD88 and IL-1β were slightly up-regulated, while TRAF6 and IL-8 were significantly down-regulated. The characterization of TLR7 can expand our understanding of the PRRs in teleost fishes, and eventually aid the exploration of interactions between host and pathogen., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Synthetic pyrethroid pesticide is commonly used in agricultural activities in the Black Sea region during reproduction period of turbot. In this sense, in vivo and in vitro studies have shown that cypermethrin (CYP) could be one of the environmental factors affecting decreasing turbot stocks. In this study, effects of in vivo and in vitro administration of CYP, a synthetic pyrethroid, on sperm kinematics motility (MOT), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straight line velocity (STR), amplitude of lateral head (ALH), beat cross frequency (BCF), oxidative stress biomarkers malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO) and also histopathological alterations in gonads were investigated in spermatazoa of turbot (Schopthalmus maximus). Broodstock was supplied from culture origin and used in spawning season, additionally, two (0, 0.187 and 0.218 ppb) and three (0, 1.025, 2.05 and 4.1 ppb) different CYP concentrations were performed for in vivo and in vitro studies, respectively. In vivo and in vitro studies, significant reductions were found in sperm MOT, PM, VCL, VSL, VAP, LIN, and ALH properties depend on the increase in CYP concentrations (p < 0.05). Besides, activities of GSH, GPx, SOD, and CAT increased. In terms of histological alterations, no difference was observed among groups (0, 0.187 and 0.218 ppb) in the maturity stage of the germ cells. According to obtained results, sperm kinematics was affected significantly with increased the dose levels of CYP (p < 0.05)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Propolis is non-hazardous resinous substance mixture containing bioactive ingredients such as polyphenols, flavonoids and organic acid. It has been widely used as food supplement and immune adjuvant due to its benefits in anti-microbial and immunomodulation. Edwardsiella piscicida is a kind of threatening pathogen which could cause high mortality in turbot. However, whether propolis could enhance the innate immune response against E. piscicida infection in turbot remains unknown. In this study, we found dietary propolis addition could improve the expression of anti-oxidative stress related enzymes, e.g., SOD, CAT and GPT, and relieved the histopathological changes of juvenile turbot after E. piscicida infection. Moreover, propolis addition increased the expression of cytokines such as il-1β, il-6 and tnf-α in different organs of juvenile turbot. Importantly, rescued survival and decreased bacterial loads were observed in propolis feeding group. Taken together, these findings suggest that the important roles of propolis in protecting juvenile turbot from E. piscicida infection, indicating propolis might be applied as a promising immunopotentiator candidate in aquaculture., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A nine-week feeding trial was conducted to comprehensively investigate the effects of different levels of dietary lipid on intestinal physiology of juvenile turbot. Three diets with different lipid levels (8%, 12% and 16%) were formulated, which were designated as the low-lipid group (LL), medium-lipid group (ML) and high-lipid group (HL), respectively. Each diet was fed to six replicate tanks, and each tank was stocked with 35 fish. The results revealed that medium dietary lipid (12%) increased the activities of intestinal digestive enzymes and brush border enzymes. Excessive dietary lipid (16%) decreased the intestinal antioxidative enzyme levels and increased the lipid peroxidation pressure. In addition, HL stimulated the occurrence of intestinal inflammation and significantly up-regulated the mRNA expression level of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β). Dietary LL and HL induced the apoptosis of intestinal epithelial cells. Sequencing of bacterial 16 s rRNA V4 region indicated that the abundance and diversity of intestinal microflora in fish fed with medium lipid diet (12%) were significantly higher than those in other groups, indicating the intestinal microflora ecology in group ML was more balanced. MetaStat analysis indicated that both low- and high-lipid diets significantly reduced the relative abundance of intestinal beneficial bacteria. In conclusion, results of this study demonstrated the sensitivity of intestinal health and microbiota to dietary lipid levels. From the perspective of microecological balance, medium dietary lipid (12%) was more conducive to maintaining the intestinal microflora stability of turbot., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The class A scavenger receptors play important roles in innate immunity and are distributed on plasma membrane of macrophages and other cell types. Notably, the class A scavenger receptor 4 (SCARA4) contains a typical C-type (calcium-dependent) lectin domain, which belongs to the collectin family of pattern recognition receptors and is involved in the immune response against infection. Here, one turbot SCARA4 gene was identified with a 2,292 bp open reading frame (ORF) encoding 763 amino acid residues. Multiple sequence analysis and phylogenetic analysis confirmed that SmSCARA4 gene was more close to that of P. olivaceus. Gene structure and syntenic analysis showed conserved exon/intron organization pattern and syntenic pattern across selected vertebrate species. Tissue distribution analysis showed SmSCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. Following both E. tarda and V. anguillarum challenge in vivo, SmSCARA4 was significantly repressed in gill and intestine. Remarkably, SmSCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. Knockdown and overexpression of SmSCARA4 revealed its interactions with the two pro-inflammatory cytokines, TNF-α and IL-1β. Finally, repression of SmSCARA4 via combined treatment of LPS and overexpression of SmSCARA4 construct in turbot head kidney macrophages further indicated an inhibitory role of SmSCARA4 in LPS-stimulated inflammation. Taken together, turbot SmSCARA4 plays an important role in turbot immunity, especially in the mucosa-related systems; SmSCARA4 possesses strong binding specificity to LPS, and exerts protective roles in response to LPS infection by reducing the release of pro-inflammatory cytokines. The mechanisms of inhibitory role of SmSCARA4 in LPS-elicited inflammation await further investigation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Seven immune factors, lysozyme, hepcidin, heat-shock protein (HSP) 70, HSP90, immunoglobulin M, C-type lectin, and Lily-type lectin, were measured by PCR in the livers, spleens, and head kidneys of turbot infected with Vibrio anguillarum. Additive main effects and multiplicative interaction (AMMI) and genotype main effects and genotype × environment interaction (GGE) biplot analysis were used to analyze genotype × tissue interactions for immunological factors. The AMMI analysis revealed that immune factor expression was significantly affected by genotype, tissue, and genotype × tissue interactions. Genotype (65.85%) was the major contributor to the total variation in immune factor expression in comparison to tissue effects (7.54%) and genotype × tissue interactions (12.52%). GGE biplot analysis revealed differences in the ranking of the seven immune factors in the three tissues; head kidney possessed the strongest ability to distinguish the seven immune factors. The test tissue locations were divided into liver-spleen and head kidneys regions; HSP70 was expressed the highest in the liver-spleen regions, and lysozyme had the highest expression in the head kidney region. Overall, HSP70 and HSP90 had the best expression and stability in the three tissues., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Soya-saponins represent key anti-nutritional factors that contribute to soybean meal-induced enteritis, and glutamine is an effective fish intestine protectant that combats the negative effects of soya-saponins. Nuclear transcription factor-kappa B (NF-кB) systems are involved in the interactions between soya-saponins and glutamine, and the goal of the present work was to clarify the related molecular mechanisms used by the NF-кB kinase (IKK)/inhibitor of NF-κB (IκB)/NF-кB system. Primary cultured turbot (Scophthalmus maximus L.) intestinal epithelial cells were concurrently administrated with 1 mg/mL of soya-saponins and several levels of glutamine (0, 0.5, 1.0 and 2.0 mM) for 12 h and then subjected to real-time PCR and Western blot assays. Compared with cells treated with soya-saponins alone, glutamine significantly decreased the expression of interleukin-1 beta, interleukin 8 and tumor necrosis factor α genes, significantly reduced nuclear and cytosolic NF-κB p65 abundance levels in a dose-dependent manner, increased the IκBα protein level but decreased its phosphorylation, and down-regulated the IKKα/β and phosphorylated IKKα/β levels. In conclusion, this in vitro work confirmed that glutamine attenuated soya-saponin-induced inflammatory responses in turbot intestines. Moreover, it identified molecular pathways in which glutamine first decreased the p65 level and then prevented its nuclear translocation. In addition, glutamine reduced IκBα phosphorylation and maintained its level. Finally, glutamine decreased IKK expression and phosphorylation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that belongs to the interleukin-1 (IL-1) family of cytokines. As occurs with IL-1β, it is synthetized as an inactive precursor peptide that is mainly processed by the cysteine protease caspase-1 in the inflammasome complex. In mammals, and in collaboration with IL-12, it has been described as an important cytokine controlling the Th1-mediated immune responses through the induction of IFN-γ. Although its function in mammals is well stablished, the activity of this cytokine in teleost remains to be elucidated. This could be due, among other things, to the absence of this gene in the fish model species zebrafish, but also to its complex regulation. As it was observed for rainbow trout and human, il18 splicing variants were also found in turbot, which could represent a regulatory mechanism of its bioactivity. In the case of turbot, three splicing variants were observed (SV1-3), and one of them showed an insertion of 10 amino acids in the middle of the potential caspase-1 cleavage position, reflecting that this is probably a form resistant to the processing by the inflammasome. Phylogenetic and three-dimensional analyses of turbot Il18 revealed that it is relatively well-conserved in vertebrates, although only a partial conservation of the gene synteny was observed between fish and mammals. As it was expected, turbot il18 splicing variants were mainly expressed in immune tissues under healthy conditions, and their expression was induced by a bacterial challenge, although certain inhibitions were observed after viral and parasitic infections. In the case of the viral challenge, il18 downregulations did not seem to be due to the effect of type I IFNs., (Copyright © 2021. Published by Elsevier Ltd.)

MicroRNAs could not only regulate posttranscriptional silencing of target genes in eukaryotic organisms, but also have positive effect on their target genes as well. These microRNAs have been reported to be involved in mucosal immune responses to pathogen infection in teleost. Therefore, we constructed the immune-related miRNA-mRNA networks in turbot intestine following Vibrio anguillarum infection. In our results, 1550 differentially expressed (DE) genes and 167 DE miRNAs were identified. 113 DE miRNAs targeting 89 DE mRNAs related to immune response were used to construct miRNA-mRNA interaction networks. Functional analysis showed that target genes were associated with synthesis and degradation of ketone bodies, mucin type O-Glycan biosynthesis, homologous recombination, biotin metabolism, and intestinal immune network for IgA production that were equivalent to the function of IgT and IgM in fish intestine. Finally, 10 DE miRNAs and 7 DE mRNAs were selected for validating the accuracy of high-throughput sequencing results by qRT-PCR. The results of this study will provide valuable information for the elucidation of the regulation mechanisms of miRNA-mRNA interactions involved in disease resistance in teleost mucosal immune system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aeromonas salmonicida is an aquatic pathogen that can infect a variety of fish. Phage therapy has been applied to treat bacterial infections. In this study, we obtained three A. salmonicida subsp. masoucida phage isolates from sewage, and one phage (vB&#95;AsM&#95;ZHF) exhibited the best antibacterial effect, based on in vitro kinetics experiments. Sequencing indicated that the vB&#95;AsM&#95;ZHF genome is 161,887 bp (41.24% C+G content) with 237 predicted open reading frames. No antibiotic resistance or virulence genes were detected in the complete genome, which is a requirement for phage therapy safety. Intraperitoneal injection of phage vB&#95;AsM&#95;ZHF into turbot at 8 × 10 4 PFU/fish rescued turbot from A. salmonicida subsp. masoucida injection and reduced the bacterial burden by 1 order of magnitude. Injection of vB&#95;AsM&#95;ZHF also decreased levels of inflammatory cell infiltration in muscle tissue, cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) in serum and the expression of the inflammatory factors IL-1β, IL-6, IFN-γ, transforming growth factor β, TNF-α, and hepcidin in the liver, spleen, and head kidney of turbot. Phage vB&#95;AsM&#95;ZHF demonstrated antibacterial ability in vitro and in vivo and significantly reduced mortality in turbot challenged by A. salmonicida subsp. masoucida . This study revealed that phage vB&#95;AsM&#95;ZHF can effectively treat the infection caused by A. salmonicida subsp. masoucida in turbot. IMPORTANCE A. salmonicida is an aquatic pathogen that can infect different fish and causes economic loss to the global aquaculture industry. Clinical strains of A. salmonicida have developed multidrug resistance, and phage therapy is being evaluated for controlling bacterial infections. Phages are biological antibacterial agents and have the potential to be therapeutic agents against multidrug-resistant bacteria. In this study, three A. salmonicida subsp. masoucida phages were isolated from sewage, and their biological behaviors were characterized. The newly isolated phage vB&#95;AsM&#95;ZHF could inhibit A. salmonicida subsp. masoucida infection in vitro and in vivo , suggesting that it may be an alternative strategy to antibiotics for protecting fish against multidrug-resistant A. salmonicida subsp. masoucida in the aquaculture industry.

Background: Feeding-induced cell signaling and metabolic responses affect utilization of dietary nutrients but are rarely taken advantage of to improve animal nutrition., Objectives: We hypothesized that by modulating postprandial kinetics and signaling, improved dietary utilization and growth performance could be achieved in animals., Methods: Juvenile turbot (Scophthalmus maximus L.) with an initial mean ± SD weight of 10.1 ± 0.01 g were used. Two feeding frequencies (FFs), either 1 or 3 meals/d at a fixed 2.4% daily body weight ration, and 2 diets that were or were not supplemented with 1% crystalline leucine (Leu), were used in the 10-wk feeding trial. At the end of the trial, a 1-d force-feeding experiment was conducted using the aforementioned FF and experimental diets. Samples were collected for the analysis of postprandial kinetics of aminoacidemia, mechanistic target of rapamycin (mTOR) signaling activities, protein deposition, as well as the mRNA expression levels of key metabolic checkpoints at consecutive time points after feeding., Results: Increased FF and leucine supplementation significantly enhanced fish growth by 7.68% ± 0.53% (means ±SD) and 7.89% ± 1.25%, respectively, and protein retention by 4.01% ± 0.59% and 4.44% ± 1.63%, respectively, in feeding trial experiments. The durations of postprandial aminoacidemia and mTOR activation were extended by increased FF, whereas leucine supplementation enhanced mTOR signaling without influencing the postprandial free amino acids kinetics. Increased FF and leucine supplementation enhanced muscle protein deposition 21.6% ± 6.85% and 22.3% ± 1.52%, respectively, in a 24-h postfeeding period., Conclusions: We provided comprehensive characterization of the postprandial kinetics of nutrient sensing and metabolic responses under different feeding regimens and leucine supplementation in turbot. Fine-tuning of postprandial kinetics could provide a new direction for better dietary utilization and animal performances in aquaculture., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus, and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1β release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Soy saponins, as thermo-stable anti-nutrients in soybean meal (SBM), are the primary causal agents of SBM-induced enteritis, which represents a well-documented pathologic alternation involving the distal intestines of various farmed fish. Our previous work showed that soy saponins might lead to SBM-induced enteritis, destroy tight junction structure and induce oxidative damage in juvenile turbot. Glutamine, as a conditionally essential amino acid, is an important substrate utilized for the growth of intestinal epithelial cells. An 8-week feeding trial was carried out to determine whether glutamine can attenuate the detrimental effects of soy saponins. Three isonitrogenous-isolipidic experimental diets were formulated as follows: (i) fish meal-based diet (FM), considered as control; (ii) FM + 10 g/kg soy saponins, SAP; and (iii) SAP + 15 g/kg glutamine, GLN. The results showed that dietary soy saponins significantly increased the gene expression levels of inflammatory markers (IL-1β, IL-8 and TNF-α) and related signaling factors (NF-кB, AP-1, p38, JNK and ERK), which were remarkably attenuated by dietary glutamine. Compared to SAP group, GLN-fed fish exhibited significantly higher expression levels of tight junction genes (CLDN3, CLDN4, OCLN, Tricellulin and ZO-1). Glutamine supplementation in SAP diet markedly suppressed the production of reactive oxygen species, malondialdehyde and protein carbonyl, and enhanced the activities of antioxidant enzymes as well as the mRNA levels of HO-1, SOD, GPX and Nrf2. Furthermore, GLN-fed fish had a remarkably lower number of autophagosomes compared to SAP-fed fish. In conclusion, our study indicated that glutamine could reverse the harmful effects of soy saponins on intestinal inflammation, tight junction disruption and oxidative damage, via attenuation of NF-кB, AP-1 and MAPK pathways and activation of Nrf2 pathway. Glutamine may have the function of controlling autophaghic process within an appropriate level of encountering inflammation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches., Results: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD., Conclusions: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: As flatfish, turbot undergo metamorphosis as part of their life cycle. In the larval stage, turbot live at the ocean surface, but after metamorphosis they move to deeper water and turn to benthic life. Thus, the light environment differs greatly between life stages. The visual system plays a great role in organic evolution, but reports of the relationship between the visual system and benthic life are rare. In this study, we reported the molecular and evolutionary analysis of opsin genes in turbot, and the heterochronic shifts in opsin expression during development., Results: Our gene synteny analysis showed that subtype RH2C was not on the same gene cluster as the other four green-sensitive opsin genes (RH2) in turbot. It was translocated to chromosome 8 from chromosome 6. Based on branch-site test and spectral tuning sites analyses, E122Q and M207L substitutions in RH2C, which were found to be under positive selection, are closely related to the blue shift of optimum light sensitivities. And real-time PCR results indicated the dominant opsin gene shifted from red-sensitive (LWS) to RH2B1 during turbot development, which may lead to spectral sensitivity shifts to shorter wavelengths., Conclusions: This is the first report that RH2C may be an important subtype of green opsin gene that was retained by turbot and possibly other flatfish species during evolution. Moreover, E122Q and M207L substitutions in RH2C may contribute to the survival of turbot in the bluish colored ocean. And heterochronic shifts in opsin expression may be an important strategy for turbot to adapt to benthic life.

In fish species, the morphological structure of the eye varies depending on environmental conditions. Morphometric data about the sensory organs of fish is lacking. Therefore, this study aims to describe the morphological structure of the turbot eye using gross, light and scanning electron microscope examinations. The turbot eyeball was found to comprise three layers: the tunica fibrosa bulbi (cornea, sclera), the tunica vasculosa bulbi (choroidea, iris) and the tunica nervea bulbi (retina). The thickness of the centre of the cornea measured approximately 153.14 μm, and the peripheral thickness measured 410.81 μm. The sclera consisted of a two-part cartilage structure that was connected with elastic fibres. The choroideal rete was found in the tunica vasculosa bulbi, and its thickness measured 1.6 ± 0.1 mm. Moreover, no pigment was found in the choroidea. The lens was determined to be a very hard and transparent structure extending towards the cornea. In addition, we detected five ligaments in the equatorial plane of the eye, in which the tendon of the retractor lentis muscle attaches to the lens. Also, there were six extraocular muscles in the turbot. This study is the first to present detailed descriptions of morphological structures and morphometric data for all the layers of the turbot eye. Since the anatomical structure of the eye in fish is variable, it is thought that the data on the turbot eye will contribute to the anatomy literature., (© 2020 Wiley Periodicals LLC.)

We have completed the characterization of the turbot (Scophthalmus maximus) myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, propeptide and light and heavy chains, and also with haem cavities, a Ca +2 -binding motif and several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx expression and an increase in epx2b1 expression over time. In turbot infected experimentally with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. These results suggest that the four genes responded differently to the same stimuli. Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish and includes two sister groups. One of the groups includes turbot mpx and was only found in teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share orthologous genes with other teleosts and also with holosteans, suggesting that these genes appeared earlier on than the mpx gene., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this study, 1,800 turbot (Scophthalmus maximus) individuals from 30 full-sib families were experimentally infected with Vibrio anguillarum, and the expression levels of the immune factors lysozyme, hepcidin, heat-shock protein 70 (HSP70), HSP90, immunoglobulin M (IgM), C-type lectin and Lily-type lectin in the liver were measured by real-time PCR. Heritability values of the seven immune factors were 0.289 ± 0.087, 0.092 ± 0.024, 0.282 ± 0.043, 0.244 ± 0.027, 0.343 ± 0.081, 0.092 ± 0.011 and 0.084 ± 0.009, respectively. The ranges of phenotypic, genetic and environmental correlations were -0.889 to 0.759, -0.841 to 0.888 and -0.919 to 0.883, respectively. The heritability values of HSP70, HSP90 and IgM were moderate, and the genetic correlations between HSP70, HSP90 and IgM were moderate to highly positive, which suggests that the immunocompetence of turbot against V. anguillarum can be improved by genetically improving these three immune characters via multi-trait integrated breeding technology or indirect selection., (© 2020 John Wiley & Sons Ltd.)

In this study, the microbiological, physicochemical, and flavor changes of turbot (Scophthalmus maximus) coated with a composite active coating of locust bean gum (LBG) and sodium alginate (SA) supplemented with daphnetin emulsions (0.16, 0.32, 0.64 mg·mL -1 ) were determined during 18 days of refrigerated storage (4 ± 1 °C). Results showed that LBG-SA coatings containing 0.32 mg·mL -1 daphnetin emulsions could significantly lower the total viable count (TVC), psychrophiles, Pseudomonas spp. and H 2 S-producing bacteria counts, and inhibit the productions of off-flavor compounds including the total volatile basic nitrogen (TVB-N), trimethylamine (TMA) and ATP-related compounds. 32 volatile compounds were identified by solid phase microextraction combined with gas chromatography-mass spectrometer method (SPME-GC/MS) during refrigerated storage and the treated turbot samples significantly lowered the relative content of fishy flavor compounds. Further, the LBG-SA coatings containing daphnetin could also delay the myofibril degradation of the turbot samples. These results indicated that the LBG-SA coatings with 0.32 mg·mL -1 daphnetin were a potential alternative way to improve the quality of turbot during refrigerated storage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species. Many reports have indicated vital roles of galectins in innate immunity, especially in the mucosal tissues against infection. However, the systematic identification of galectin gene family is still lacking in teleost. Here, we characterized the galectin gene family and investigated their expression profiles post bacterial challenge in turbot (Scophthalmus maximus L.). In this study, a total of 13 galectin genes were characterized in turbot, phylogenetic analyses revealed their strong relationships to half smooth tongue sole and puffer fish, and syntenic analyses confirmed the orthology suggested by the phylogenetic analysis. In addition, the copy number of galectin genes is similar across a broad spectrum of species from fish to amphibians, birds, and mammals, ranging from 8 to 16 genes. Furthermore, the galectin genes were widely expressed in all the examined turbot tissues, and most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after Vibrio anguillarum infection in the intestine, gill and skin, suggesting that galectins were involved in the mucosal immune response to V. anguillarum infection in turbot. In addition, subcellular localization analysis showed lgals3a was distributed in the cytoplasm and nucleus. However, the knowledge of galectins are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)