Search

Temperature tolerance is an important trait from both an economic and evolutionary perspective in fish. Because of difficulties with measurements, genome-wide selection using quantitative trait loci (QTLs) affecting Upper temperature tolerance may be an alternative for genetic improvement. Turbot Scophthalmus maximus (L.) is a cold-water marine fish with high economic value in Europe and Asia. The genetic bases of upper temperature tolerance (UTTs) traits have been rarely studied. In this study, we constructed a genetic linkage map of turbot using simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers. A total of 190 SSR and 8,123 SNP were assigned to 22 linkage groups (LGs) of a consensus map, which spanned 3,648.29 cM of the turbot genome, with an average interval of 0.44 cM. Moreover, we re-anchored genome sequences, allowing 93.8% physical sequences to be clustered into 22 turbot pseudo-chromosomes. A high synteny was observed between two assemblies from the literature. QTL mapping and validation analysis identified thirteen QLTs which are major effect QTLs, of these, 206 linked SNP loci, and two linked SSR loci were considered to have significant QTL effects. Association analysis for UTTs with 129 QTL markers was performed for different families, results showed that eight SNP loci were significantly correlated with UTT, which markers could be helpful in selecting thermal tolerant breeds of turbot. 1,363 gene sequences were genomically annotated, and 26 QTL markers were annotated. We believe these genes could be valuable candidates affecting high temperatures, providing valuable genomic resources for the study of genetic mechanisms regulating thermal stress. Similarly, they may be used in marker-assisted selection (MAS) programs to improve turbot performance.

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.

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.

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 H2S-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.

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.

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.

Myo-inositol is an essential vitamin for most animals, and it can modulate multiple physiological functions. In this study, we performed transcriptome gene expression profiling of gill tissue from turbot Scophthalmus maximus fed different concentrations of myo-inositol (0, 300, 600, 900, 1200 mg/kg). Results of expression tendency analysis, Weighted Gene Co-Expression Network Analysis (WGCNA), integrated transcriptome analyses, and KEGG annotation analysis of all differentially expressed genes (DEGs) demonstrated that the cytokine-cytokine receptor interaction played a core role in effects of myo-inositol on turbot, which was followed by the Jak-STAT signaling pathway. The results of qRT-PCR also showed myo-inositol mediated the gene expression of the cytokine-cytokine receptor interaction and the Jak-STAT signaling pathway in turbot. The ELISA assay indicated that myo-inositol affected the concentration change of interleukins (IL-2 and IL-10). Consequently, the interleukins associated with immune functions in the cytokine-cytokine receptor interaction played a core role in the effects of myo-inositol on turbot, which was followed by the Jak-STAT signaling pathway. Additionally, 10 hub genes associated with myo-inositol-traits were identified via WGCNA.

Turbot is an important flatfish widely distributed along the European coasts, whose fishery is centered in the North Sea. The commercial value of the species has boosted a successful aquaculture sector in Europe and China. Body growth is the main target of turbot breeding programs and is also a key trait related to local adaptation to temperature and salinity. Differences in growth rate and optimal growth temperature in turbot have been shown to be associated with a hemoglobin polymorphism reported more than 50 years ago. Here, we identified a Gly16Asp amino acid substitution in the β1 globin subunit by searching for genetic variation in the five functional globin genes within the whole annotated turbot genome. We predicted increased stability of the turbot hemoglobin by the replacement of the conserved Gly with the negative charged Asp residue that is consistent with the higher rate of αβ dimer assembly in the human J-Baltimore Gly16β->Asp mutant than in normal HbA. The turbot Hbβ1-Gly16 variant dominated in the northern populations examined, particularly in the Baltic Sea, while the Asp allele showed elevated frequencies in southern populations and was the prevalent variant in the Adriatic Sea. Body weight did not associate with the Hbβ1 genotypes at farming conditions (i.e., high oxygen levels, feeding ad libitum) after analyzing 90 fish with high growth dispersal from nine turbot families. Nevertheless, all data at hand suggest that the turbot hemoglobin polymorphism has an adaptive significance in the variable wild conditions regarding temperature and oxygen availability. Electronic supplementary material The online version of this article (10.1007/s10695-020-00872-y) contains supplementary material, which is available to authorized users.

Fish fraud is a problematic issue for the industry. For it to be properly addressed will require the use of accurate, rapid, and cost-effective tools. In this work, near infrared reflectance spectroscopy (NIRS) was used to predict nutritional values (protein, lipids, and moisture) as well as to discriminate between sources (farmed vs. wild fish) and conditions (fresh or defrosted fish). Samples of five whitefish species—Alaskan pollock (Gadus chalcogrammu), Atlantic cod (G. morhua), European plaice (Pleuronectes platessa), common sole (Solea solea), and turbot (Psetta maxima)—including farmed, wild, fresh, and frozen ones, were scanned by a low-cost handheld near infrared reflectance spectrometer with a spectral range between 900 and 1700 nm. Several machine learning algorithms were explored for both regression and classification tasks, achieving precisions and coefficients of determination higher than 88% and 0.78, respectively. Principal component analysis (PCA) was used to cluster samples according to classes where good linear discriminations were denoted. Loadings from PCA revealed bands at 1150, 1200, and 1400 nm as the most discriminative spectral regions regarding classification of both source and condition, suggesting the absorbance of OH, CH, CH2, and CH3 groups as the most important ones. This study shows the use of NIRS and both linear and non-linear learners as a suitable strategy to address fish fraud and fish QC.

This study was designed to evaluate whether the administration of commensal Shewanella sp. MR-7 (MR-7) could ameliorate lipopolysaccharide (LPS)-induced intestine dysfunction in turbot. Fish (body weight: 70.00 ± 2.00 g) were randomly divided into three groups including the control group treated with dough, the LPS group treated with dough plus LPS, and the LPS+MR-7 (LMR) group treated with dough plus LPS and MR-7. These three groups with 24 fish each were force-fed with 1 g dough daily for 7 continuous days. The results revealed that MR-7 administration ameliorated LPS-induced intestinal injury, showing higher intestinal villus and microvillus height. Further results showed that MR-7 could inhibit LPS-induced activation of TLR-NF-κB signaling thus maintaining the normal expression levels of cytokines and finally ameliorate the intestinal inflammatory response in turbot. Compared with the LPS group, LMR group had less goblet cells and lower mucin-2 expression level. Moreover, MR-7 restored LPS-induced down-regulation of tight junction protein-related gene expression (zonula occluden-1, occludin, tricellulin and claudin-3). Further investigations indicated that MR-7 partially counteracted LPS-induced changes in gut microbiota composition, enhanced the beneficial bacteria Lactobacillus and reduced the Pseudomonas, thus maintaining the overall microbiota balance. Taken together, the administration of MR-7 could effectively restore LPS-induced intestine function disorder in turbot by ameliorating inflammatory response, mucosal barrier dysfunction and microbiota dysbiosis.

Myo-inositol is a major intracellular osmolyte that can be accumulated to protect cells from a variety of stresses, including fluctuations in the osmolality of the environment, and cortisol is thought to be an osmotic hormone in teleost fish. In this study, dietary myo-inositol resulted in increased Na+-K+-ATPase activity and gene expression of partial ion channel genes and prolonged survival time of turbot (Scophthalmus maximus) under low salinity. The cortisol regulated by dietary myo-inositol also was correlated with these outcomes. The optimal concentrations of cortisol stimulated gill Na+-K+-ATPase activity and increased the expression of ion channel genes to enhance low salinity tolerance, as indicated by longer survival time under low salinity. When cortisol level was suppressed, myo-inositol failed to increase the survival time of turbot under low salinity, and strong correlations between cortisol concentration and Na+-K+-ATPase activity, expression of partial ion channel genes, and survival time of turbot were detected. These results showed that myo-inositol enhanced the low salinity tolerance of turbot by modulating cortisol synthesis.

Supplying immunostimulants to aquatic feed has been an effective way to enhance the health of aquatic animals and substitute for antibiotics. In the present study, the potential effects of Astragalus polysaccharides (APS) were evaluated in turbot, Scophthalmus maximus. Two levels of APS (50 and 150 mg/kg) were added to the basal diet (CON) and a 63-day growth trial (initial weight 10.13 ± 0.04 g) was conducted. As the results showed, significant improvement on growth performance in the APS groups were observed. In addition, dietary 150 mg/kg APS significantly increased the total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and lysozyme activities in liver. Meanwhile, APS diets induced the mRNA expression of toll-like receptors (TLRs) such as tlr5α, tlr5β, tlr8 and tlr21, while reduced the expression of tlr3 and tlr22. The expression of inflammatory genes myeloid differentiation factor 88 and nuclear factor kappa b p65 and pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β were up-regulated in APS groups while the expression of anti-inflammatory cytokine transforming growth factor beta was inhibited. Taken together, the present study indicated that Astragalus polysaccharides could remarkably enhance the growth performance, antioxidant activity and maintain an active immune response in turbot.

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Background Increased inclusion of plant proteins in aquafeeds has become a common practice due to the high cost and limited supply of fish meal but generally leads to inferior growth performance and health problems of fish. Effective method is needed to improve the plant proteins utilization and eliminate their negative effects on fish. This study took a unique approach to improve the utilization of soybean meal (SBM) by fish through autochthonous plant-degrading microbe isolation and subsequent fermentation. Results A strain of Shewanella sp. MR-7 was isolated and identified as the leading microbe that could utilize SBM in the intestine of turbot. It was further optimized for SBM fermentation and able to improve the protein availability and degrade multiple anti-nutritional factors of SBM. The fishmeal was able to be replaced up to 45% by Shewanella sp. MR-7 fermented SBM compared to only up to 30% by SBM in experimental diets without adverse effects on growth and feed utilization of turbot after feeding trials. Further analyses showed that Shewanella sp. MR-7 fermentation significantly counteracted the SBM-induced adverse effects by increasing digestive enzymes activities, suppressing inflammatory responses, and alleviating microbiota dysbiosis in the intestine of turbot. Conclusions This study demonstrated that plant protein utilization by fish could be significantly improved through pre-digestion with isolated plant-degrading host microbes. Further exploitation of autochthonous bacterial activities should be valuable for better performances of plant-based diets in aquaculture.

A 12-week feeding trial was conducted to investigate the effect of dietary daidzein on the intestinal mucosal barrier function and the intestinal microbiota profile of juvenile turbot (Scophthalmus maximus L.). Three isonitrogenous and isolipidic experimental diets were formulated to contain 0 (FM), 40 (D.40) and 400 (D.400) mg kg−1 daidzein, respectively. Fish fed D.400 had significantly lower growth performance than fish fed D.40. Dietary daidzein significantly increased the feed efficiency, while significantly decreased the feed intake. Daidzein supplementation increased the activity of total anti-oxidative capacity and the gene expression of anti-inflammatory cytokine transforming growth factor-β1, Mucin-2 and tight junction proteins (Tricellulin, Zonula occludens-1 transcript variant 1, Zonula occludens-1 transcript variant 2 and Claudin-like and Occludin), and down-regulated the gene expression of pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in the intestine of turbot. Dietary daidzein increased intestinal microbial diversities, the abundance of several short chain fatty acids producers, and decreased the abundance of some potential pathogenic bacteria. However, D.400 had dual effects on lactic acid bacteria and increased the abundance of potential harmful bacterium Prevotella copri. Collectively, dietary daidzein at the levels of 40 and 400 mg kg−1 could enhance the intestinal mucosal barrier function and alter the intestinal microbiota of turbot. However, high dose of daidzein must be treated with caution for its unclear effects on intestinal microbiota of turbot in the present study.

This review describes and summarizes the knowledge on established and experimental vaccines developed against viral and bacterial pathologies affecting the most important farmed marine finfish species present in the Mediterranean area, namely European seabass Dicentrarchus labrax, sea bream Sparus aurata, turbot Psetta maxima and meagre Argyrosomus regius. The diseases that have been recorded in seabass, sea bream and meagre are caused by bacteria Vibrio anguillarum, Photobacterium damselae, Tenacibaculum maritimum as well as by viruses such as Viral Encephalopathy and Retinopathy/Viral Nervous Necrosis and Lymphocystic disease. The main pathologies of turbot are instead bacteriosis provoked by Tenacibaculum maritimum, Aeromonas sp. and Vibrio anguillarum, and virosis by viral hemorrhagic septicaemia virus. Some vaccines have been optimized and are now regularly available for the majority of the above-mentioned pathogens. A measurable immune protection has been conferred principally against Vibrio anguillarum, Photobacterium damselae sub. piscicida and VER/VNN.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish and is one of the most severe economic diseases in aquaculture. In Turkey, an increase in infectious pancreatic necrosis virus (IPNV) outbreaks in freshwater rainbow trout have been reported in recent years. This study aimed to analyze the VP2 gene from recent IPNV isolates from Turkey to determine whether there are epidemiological links between IPNV isolates from rainbow trout (Oncorhynchus mykiss; 62) and sea bass (Dicentrarchus labrax; 1), wild turbot (Scophthalmus maximus; 1) and the environment in order to investigate potential wild and farmed fish interactions. In this study, 62 Turkish IPNV isolates collected over 10 years (2005-2014) from rainbow trout, sea bass and turbot were genotypically characterized. The phylogenetic analysis indicated that Turkish IPNV isolates are closely related to strains from Denmark, Iran and Spain and that all Turkish IPNV isolates belong to genogroup V, serotype A2 (Sp strain). Furthermore, low genetic diversity was found among the Turkish isolates (identity, 95.5%-100% nucleotides and 97.8%-100% amino acids). The result of the analysis of the amino acid residues found at positions 217, 221 and 247 (proline, threonine and alanine, respectively) could be associated with virulence.

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.

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.

19 pages, 8 figures, 4 tables.-- This is an open access article distributed under the Creative Commons Attribution License, Rising trends in fish filleting are increasing the amount of processing by-products, such as skins of turbot, a flatfish of high commercial value. In line with circular economy principles, we propose the valorization of turbot skins through a two-step process: initial gelatin extraction described for the first time in turbot, followed by hydrolysis of the remaining solids to produce collagen hydrolysates. We assayed several methods for gelatin extraction, finding differences in gelatin properties depending on chemical treatment and temperature. Of all methods, the application of NaOH, sulfuric, and citric acids at 22 °C results in the highest gel strength (177 g), storage and loss moduli, and gel stability. We found no relation between mechanical properties and content of pyrrolidine amino acids, but the best performing gelatin displays higher structural integrity, with less than 30% of the material below 100 kDa. Collagen hydrolysis was more efficient with papain than alcalase, leading to a greater reduction in Mw of the hydrolysates, which contain a higher proportion of essential amino acids than gelatin and show high in vitro anti-hypertensive activity. These results highlight the suitability of turbot skin by-products as a source of gelatin and the potential of collagen hydrolysates as a functional food and feed ingredient., This research was funded by GAIN project (EU, Horizon 2020 Framework Research and Innovation Programme under GA n. 773330), and Xunta de Galicia (Grupos de Potencial Crecimiento, IN607B 2021/11

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.

Alike other flat fish, marine turbot has the particularity that changes from larvae with bilateral symmetry to adult with asymmetry, in terms of the position of the eyes. As expected, the skin configuration of this species is also affected by the development and transformation suffered by fish during metamorphosis. In this context, changes in the epidermis of marine turbot were studied using conventional staining and histochemical techniques using six lectins (UEA-I, PNA, RCA-I, WGA, Con A and SBA). During development from larvae to juvenile (3–300 days post-hatching), the epidermis increased in both thickness and the number of cell layers. In fact, the simple cuboidal epithelium observed in larvae at day 3 already became stratified at days 10–12, which sequentially increase in thickness with fish development. Turbot epidermis is composed basically of four cell types: epithelial and mucous or secretory cells that are present through the development, and pigmented cells and a type that the authors described as club-like cells that appear during and post-metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method revealed the presence of neutral glycoconjugates in mucous and club-like cells at post-metamorphic stages of fish. Accordingly, lectin analysis showed mucous cells containing glycoproteins rich in fucose (UEA-I labelling) and glycoconjugates rich in the sequence galactose-N-acetyl galactosamine (PNA and RCA-I labelling) when this cell type appears. Interestingly, melanophores were observed in the dorsal epidermis of post-metamorphic juveniles. This type of cell contains a black-to-brown pigment that provides the skin the typical colour of this fish species. Changes in mucous coat composition were observed during fish development, which was attributed to different roles of the glycoconjugates. Ministerio de Ciencia e Innovación | Ref. CGL 2009-07904 Universidade de Vigo/ CISUG

In mammals, forkhead box O3 (foxo3) plays important roles in liver immune system. The foxo3 can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on foxo3 in fish have been reported. The present study characterized the foxo3 in turbot (Scophthalmus maximus L.). Lipopolysaccharide (LPS) incubated in vitro (hepatocytes) and injected in vivo (turbot liver) were used to construct inflammatory models. The foxo3 was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of foxo3 was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that foxo3 of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of foxo3 was in muscle. Immunofluorescence result showed that foxo3 was expressed in cytoplasm and nucleus. Knockdown of foxo3 significantly increased mRNA levels of tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), interleukin-6 (il-6), myeloid-differentiation factor 88 (myd88), cd83, toll-like receptor 2 (tlr-2) and protein level of c-Jun N-terminal kinase (JNK) in sifoxo3 + LPS (siRNA of foxo3+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed foxo3 significantly decreased mRNA levels of tnf-α, il-6, nuclear transcription factor-kappa B (nf-κb), cd83, tlr-2 and the protein level of JNK in vitro. In vivo analysis, foxo3 knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed foxo3 significantly decreased levels of GPT and GOT in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in vivo. Foxo3 knockdown significantly increased mRNA levels of tnf-α, il-1β, il-6, nf-κb, myd88 and protein level of JNK in vivo in sifoxo3+LPS group compared with NC+LPS group in turbot liver. Overexpressed foxo3 significantly decreased mRNA levels of il-1β, il-6, myd88, cd83, jnk and protein level of JNK in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that foxo3 might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and tlr-2/myd88/nf-κb pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.

9 pages, 3 figures, 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, This work was financially supported by the Ministerio de Economía, Industria y Competitividad of Spain (BIO2017-82851-C3-2-R). Our laboratory is funded by the Interreg VA Spain-Portugal cooperation program (POCTEP) 2014–2020, 0474_BLUEBIOLAB project, co-funded by FEDER, and IN607B 2019/01 from Consellería de Economía, Emprego e Industria (GAIN), Xunta de Galicia. Patricia Pereiro and Raquel Lama wish to thank the Axencia Galega de Innovación (GAIN, Xunta de Galicia) for their postdoctoral (IN606B-2018/010) and predoctoral contracts (IN606A-2017/011), respectively

Oral vaccination is the most convenient method for disease protection in aquaculture due to possibility of adding vaccines as part of fish diet. However, low protective efficiency is the major problem of oral vaccination owing to some reasons, especially for antigens degradation. In this study, we developed an oral inactivated vaccine based on a chitosan/aluminum adjuvant system, and investigated immune response induced by the vaccine. As a result, Th1 and Th2 cells mediated immune responses were observed after the vaccination according to the upregulation of IL-6, IL-12, IFNγ and IgM genes expression that were deemed as the driver cytokines for triggering the responses. Moreover, ratio of IgT+/IgM+ B cells was elevated in intestine after vaccination, while IgM antibodies were also observed in the sera of vaccinated fish, suggested that both local and systemic immune responses were induced by the inactivated vaccine. Totally, this oral vaccine can be used for V. anguillarum protection and this chitosan/aluminum system is an efficient candidate adjuvant for developing more vaccines in the future.

A range of perfluoroalkyl substances (PFASs) was analysed in marine fish, farmed fish, crustaceans, bivalves and European eel caught in (mostly) Dutch waters, or purchased at Dutch markets (approximately 250 samples, collected between 2012 and 2018). ΣPFAS levels were highest in eels collected from rivers and lakes (average 43.6 ng/g and max 172 ng/g), followed by shrimps collected near the Dutch coast (average 6.7 and max. 33 ng/g ww), and seabass (average 4.5 and max. 9.4 ng/g ww). Most of the farmed fish (e.g. trout, catfish, turbot, salmon, tilapia, pangasius) were among the lowest contaminated samples in this study (averages ranged from 0.06 to 1.5 ng/g ww). Geographically, levels in marine fish from the northern North Sea (e.g. haddock, whiting, herring) were lower than in the central and southern North Sea (e.g. cod and flatfish). Concerning eel, no substantial geographical differences were found (apart from two distinct locations). The contamination pattern was similar in all species, where PFOS mostly dominated the profile, and other long-chain PFASs being frequently detected. Short-chain PFASs were rarely found. PFOS concentrations in eel varied from 3.3 ng/g (close to the North Sea) to 67 ng/g ww in eel caught from Ghent-Terneuzen canal. The majority of detected PFOS levels in eels (93%) and 1 shrimp sample from Eems-Dollard exceeded the EU Environmental Quality Standard (EQS) for surface water of 9.1 μg/kg ww. Other samples (e.g. shrimps, bivalves, flounder), subject to the EQS, did not exceed this level.

A 12-week feeding trial was conducted to investigate the effect of citric acid on the involvement of TLRs in the soybean meal induced inflammatory response and tight junction disruption in the distal intestine of juvenile turbot (Scophthalmus maximus L.). Four isonitrogenous and isolipidic practical diets were formulated: fish meal-based diet (FM); 40% fish meal protein in FM replaced with soybean meal protein (SBM); SBM + 1.5% citric acid and SBM + 3% citric acid. Compared to the FM, diet SBM significantly increased the gene expression of TLRs (TLR2, TLR3, TLR5b, TLR9, TLR21, TLR22) and MyD88, as well as TLR related molecules (NF-κB, IRF-3, p38 and JNK), which were remarkably reduced by dietary citric acid. Similarly, citric acid supplementation in SBM markedly depressed gene expression of pro-inflammatory cytokines (TNF-α and IFN-γ) and pore-forming tight junction protein Claudin-7, and enhanced gene expression of the anti-inflammatory cytokine TGF-β1 and TJ proteins related to the decrease in paracellular permeability (Claudin-3, Claudin-4, Occludin, Tricellulin and ZO-1). Compared to the SBM, the concentration of IgM and C4 in serum was significantly reduced by dietary citric acid. In brief, dietary citric acid could synchronously inhibit TLRs-dependent inflammatory response regulated by NF-κB and IRF3, as well as cause TLRs-dependent tight junction disruption modulated by p38 and JNK. Therefore, citric acid could function on mitigating soybean meal induced enteropathy in the distal intestine of juvenile turbot.

This is the accepted manuscript of the following article: Folgueira I, Lamas J, De Felipe AP, Sueiro RA, Leiro JM. (2019). Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite. Fish Shellfish Immunol. 2019 Jul 5;92:802-812. doi: 10.1016/j.fsi.2019.07.008 Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbot This study was financially supported by grants from the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional -FEDER- (European Union) (AGL2017-83577-R) and from the Xunta de Galicia (ED431C2017/31) and also by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429 SI

Forkhead box O1 (foxo1) is a transcription factor and plays important roles in glucose metabolism. In the present study, foxo1 in turbot Scophthalmus maximus was cloned and characterized. The siRNA of foxo1 was used to investigate the functions of foxo1 in turbot hepatocytes glucose metabolism. After that, a 10-week feeding trial with two different dietary carbohydrate levels (15% and 21%, respectively) was conducted to analyze the function of foxo1 in glucose metabolism in vivo. Results showed that the foxo1 was identified as 2176 bp (base pair) with a 2025 bp open reading frame, which encoded 675 amino acids. Sequence analysis showed that foxo1 of turbot was highly homologous to most of fishes. Tissue distribution analysis revealed that the highest expression of foxo1 was in liver. After in vitro analysis, foxo1-specific small interfering RNA (sifoxo1) treatment significantly decreased the expressions of cytosolic phosphoenolpyruvate carboxykinase (cpepck) and glucose-6-phosphatase1(g6pase1) in primary hepatocytes. Expression of mitochondrial phosphoenolpyruvate carboxykinase (mpepck) was not significantly inhibited. In contrast, the expression of glucose-6-phosphatase2 (g6pase2) increased significantly. After the in vivo study (feeding trial), with the decreased expression of foxo1 in turbot due to high dietary carbohydrate level (21%), the expression of g6pase2 was significantly upregulated. However, the expression of glucokinase (gk) was not changed significantly. These increased the level of blood glucose and hepatic glycogen. In conclusion, data from both in vitro (primary hepatocytes) and in vivo (feeding trial) showed that downregulated foxo1 in turbot could not result in significant depression of gluconeogenesis and activation of glycolysis. This could be one of the reasons why intake of high level of carbohydrate resulted in prolonged hyperglycemia in turbot.

Light is a key environmental factor that synchronizes various life stages from embryo development to sexual maturation in fish. For turbot, light spectra have the most influence at the larval and juvenile stages. In the current study, differences in the development of embryos and the performance of newly hatched turbot larvae exposed to five different spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), green (LDG, peak at 533 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm), were examined. At 62.8 h post fertilization, a higher number of embryos exposed to short-wavelengths (LDG and LDB) had developed a heartbeat in comparison with embryos exposed to other wavelengths. Larvae exposed to the green spectrum had higher malformation rates than larvae exposed to the other spectra, indicating that larvae exposed to green light may have significantly reduced survival rates. The results of non-specific immunity parameters showed that the mRNA expression levels of cathepsin D (CTSD), cathepsin F (CTSF), catalase (CAT) and metallothionein (MT) in larvae exposed to LDB were significantly higher than those exposed to other spectra, but CAT activity in larvae exposed to LDB was significantly lower than larvae exposed to the other spectra. There was no significant difference in MT activity in larvae exposed to the five different spectra. The mRNA expression level of lysozyme (LZM) in larvae exposed to LDR was significantly higher than other spectra, while there was no significant difference in LZM activity observed in larvae exposed to LDR, LDG, LDB and LDF. The difference of the enzyme activity of total superoxide dismutase (T-SOD) was not significant among larvae exposed to the five spectra. mRNA expression of the heat shock protein 70 (HSP70) was significantly higher in newly hatched larvae exposed to LDB, LDR and LDG, indicating that larvae exposed to LDB, LDG and LDR exhibited a stress response. The mRNA expression level of the insulin-like growth factor-1 (IGF-1) and growth parameters in the newly hatched larvae exposed to the different spectra were not significantly different. The results of the present study indicate that LDO and LDF should be used for embryo incubation and newly hatched larvae when rearing turbot. This study provides a theoretical basis for optimizing the incubation light environment for fertilized turbot eggs, promoting immunity and reducing stress responses in newly hatched larvae.

Gonadotropins (GtHs) play a pivotal role in regulating the reproductive axis and puberty. In this study, full-length sequences coding for common glycoprotein α subunit (CGα) and luteinizing hormone β (LHβ) were isolated from female turbot (Scophthalmus maximus) pituitary by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. Results showed that the two cDNAs consisted of 669 and 660 nucleotides encoding 129 and 139 amino acids, respectively. CGα and LHβ manifested typical characteristics of glycoprotein hormones, high homologies with the corresponding sequences of available teleosts, and high homology with that of Hippoglossus hippoglossus. CGα, FSHβ, and LHβ mRNAs were abundant in the pituitary, but less expressed in extra-pituitary tissues. The cgα, fshβ, and lhβ were detected at 1-day post-hatching (dph) and peaked simultaneously at early-metamorphosis (22 dph). cgα and fshβ mRNA levels were significantly increased at pre-metamorphosis, peaked in early metamorphosis, and then gradually decreased until metamorphosis was completed. Conversely, lhβ mRNA levels gradually decreased at pre-metamorphosis, dramatically peaked at early metamorphosis, and then decreased during metamorphosis. In addition, the mRNA levels of cgα were significantly higher than those of fshβ and lhβ during turbot larval metamorphic development, whereas no significant difference was found between fshβ and lhβ. These results suggested (i) an early activation of the GtHs system after hatching, which was the highest expression at early metamorphosis, and (ii) FSHβ and LHβ were together involved in the establishment of the reproductive axis during larval development in turbot. These findings contribute to further understanding the potential roles of GtHs during fish larval development.

Galectins are a family of galactoside-binding proteins with an affinity for β-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.

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)., Ege University Scientific Research Projects Coordination [16SUF032], Funding This work was supported by the Ege University Scientific Research Projects Coordination [grant numbers 16SUF032] .

The interactions between selenium (Se) and mercury (Hg) were assessed in order to interpret public health risk, associated with dietary mercury exposure due to fish consumption. For this purpose, the mass and molar concentrations of Se and Hg were determined in the edible tissues of six species of fish, collected from the commercial fishing grounds of Turkey. The Se/Hg molar ratios and selenium health benefit values (Se-HBVs) were also calculated. The main fish species exported from Turkey to Europe were studied to determine the risks or benefits for human health. The mean Hg levels (μg g−1, wet weight) ranged from 0.01 (in turbot) to 0.45 (in Atlantic bluefin tuna). The average selenium concentrations were between 0.96 μg g−1 (in thornback ray) and 1.86 μg g−1 (in turbot). The molar ratios of Se/Hg were above 1 for all species and greater than 100 in turbot, red mullet, and whiting. Positive Se-HBVs were determined for all samples, showing health benefits. Since Se is present in molar excess of Hg in the fish muscles, organic Hg exposures from eating these fish is not a public health concern.

In Baffin Bay and Davis Strait, reports indicate that Northern Fulmars (Fulmarus glacialis) are vulnerable to bycatch in the fisheries for Greenland Halibut (Reinhardtius hippoglossoides). We modeled the potential current and future impacts the expanding halibut fishery may have on fulmar populations in the region using population viability analysis. By varying age-specific bycatch vulnerability, detectability, and the size of the at-risk population, we tested how different scenarios may influence population trajectories. From 2011 to 2015, the bycatch rate of fulmars was approximately 212 (SD ± 111) individuals per year. This could cause declines (−12%) over three generations (66 years) at the three colonies closest to the fishing grounds. However, declines could be as high as −33% over this same period if unobservable bycatch is considered, and as low as −0.4% if bycatch is distributed among a larger population. Several uncertainties we modeled could be reduced by improving how bycatch data are recorded by at-sea observer programs.

This experimental study determined internal distributions of an array of radio-elements (54Mn, 60Co, 65Zn, 134Cs, 241Am, 109Cd, 110mAg, 75Se and 51Cr) accumulated from seawater by three chondrichthyan fish species (Scyliorhinus canicula (dogfish), Raja undulata (undulate ray) and Torpedo marmorata (spotted torpedo)) and three teleost species (Scophthalmus maximus (turbot), Sparus aurata (seabream) and Dicentrarchus labrax (seabass)). The study tested the hypothesis that the chondrichthyan (cartilaginous) fish taxon and teleost (bony) fish taxon have different patterns of bioaccumulation of these radio-elements in six body components (head, digestive tract, liver, kidneys, skin and muscle), consistent with their long period of evolutionary divergence. Comparisons of body component CFs between the two taxa for each radio-element and the full array of radio-elements showed highly significant differences (p ≤ 0.001) between cartilaginous and bony fishes in each body component, confirming the existence of a strong and pervasive phylogenetic signal; however, the subset of radio-elements most determinant of these differences were unique for each body component. Partitioning between the three individual bony species and the three individual cartilaginous species also occurred repeatedly among their body components, particularly for bony fishes. Distributions of these radio-elements among body components were typically highly heterogeneous for both fish taxa.

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.

The present study aimed to assess the levels of 98 multi-class pharmaceuticals including cardiovascular drugs, antidepressants, hypnotics, antibiotics, and sulfonamides occurring in the muscle tissue of fish caught in the Baltic Sea. The following fish species were collected: perch (Perca fluviatilis), flounder (Platichthys flesus), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), cod (Gadus morhua callarias), bream (Abramis brama), crucian (Carassius carassius). Additionally, in the examined fish muscle the levels of heavy metals and trace elements were determined (As, Ag, Au, Ba, Cd, Co, Cr, Cu, Hg, Li, Mo, Ni, Pb, Sb, Se, Sn, Tl, V) as well as the levels of cholesterol and its 5 derivatives (7-ketocholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 5β,6β-epoxy-cholesterol, 5α,6α-epoxycholesterol). In the performed studies 11 out of 98 examined pharmaceuticals were detected in fish muscle. The levels of pharmaceuticals in fish muscle varied depending on the species. In the tissues of bream and crucian, no pharmaceuticals were found. Mercury, lead and arsenic were detected in the muscles of all examined fish. Based on the hazard factor for Hg, Pb, Cd, Ni (target hazard quotient, THQ &lt, 1), it was found that the consumption of the studied fish does not constitute a health risk. However, the THQ for As remained &gt, 1 indicated possible risk from those metals. In the examined fish muscle the total cholesterol oxidation products (COPs) level of oxysterols were, respectively: 6.90 (cod) μg/g–4.18 μg/g (perch), which corresponded to 0.7–1.5% of cholesterol. The main COPs evaluated were 7-ketocholesterol (0.78 ± 0.14–1.79 ± 0.06 μg/g), 7β-hydroxycholesterol (0.50 ± 0.04–3.20 ± 2.95 μg/g) and 5β,6β-epoxycholesterol (0.66 ± 0.03–1.53 ± 0.66 μg/g). The assessment of health hazards due to contaminations is necessary, which may help to introduce national legislation and global standards aimed at reducing or even eliminating the exposure to contaminants.

Soybean meal-induced enteropathy (SBMIE) is prevalent in aquaculture. The aim of this study is to evaluate the role of daidzein on SBMIE of juvenile turbot (Scophthalmus maximus L.) by feeding with fish meal diet (FM), soybean meal diet (SBM, 40% fish meal protein in FM replaced by soybean meal protein) and daidzein diet (DAID, 40 mg/kg daidzein supplemented to SBM) for 12 weeks. We found that daidzein supplementation elevated the gene expression of anti-inflammatory cytokine TGF-β, decreased gene expression of pro-inflammatory cytokines TNF-α and signal molecules p38, JNK and NF-κB. SBM up-regulated the genes expression related to oxidative stress and apoptosis, but dietary daidzein restored it to the similar level with that in FM group. Moreover, dietary daidzein up-regulated gene expression of tight junction protein, and modified the intestinal microbial profiles with boosted relative abundance of phylum Proteobacteria and Deinococcus–Thermus, genera Sphingomonas and Thermus, species Lactococcus lactis, and decreased abundance of some potential pathogenic bacteria. In conclusion, dietary daidzein could ameliorate SBM-induced intestinal inflammatory response, oxidative stress, mucosal barrier injury and microbiota community disorder of turbot. Moreover, p38, JNK and NF-κB signaling might be involved in the anti-inflammatory process of daidzein, and daidzein itself might act as an antioxidant to resist SBM-induced oxidative damage.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

Nitrate (NO3−), a potential toxic nitrogenous compound to aquatic animals, is distributed in aquatic ecosystems worldwide. The aim of this study was to investigate the effects of different NO3− levels on growth performance, health status, and endocrine function of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems (RAS). Fish were exposed to 0 mg/L (control, CK), 50 mg/L (low nitrate, LN), 200 mg/L (medium nitrate, MN), and 400 mg/L (high nitrate, HN) NO3-N for 60 d in experimental RAS. Cumulative survival (CS) was significantly decreased with increasing NO3− levels in LN, MN, and HN. The lowest CS was 35% in the HN group. Growth parameters, including absolute growth rate, specific growth rate, and feed conversion rate, were significantly different in HN compared with that in the CK. Histological survey of gills and liver revealed dose-dependent histopathological damage induced by NO3− exposure and significant differences in glutamate pyruvate transaminase and glutamate oxalate transaminase in MN and HN compared with that in the CK. The hepatosomatic index in HN was significantly higher than that in the CK. Additionally, NO3− significantly increased bioaccumulation in plasma in LN, MN, and HN compared to that in the CK. Significant decreases in hemoglobin and increases in methemoglobin levels indicated reduced oxygen-carrying capacity in HN. Additionally, qRT-PCR and enzyme-linked immunosorbent assay (ELISA) were developed to investigate key biomarkers involved in the GH/IGF-1, HPT, and HPI axes. Compared with that in the CK, the abundance of GH, GHRb, and IGF-1 was significantly lower in HN, whereas GHRa did not differ between treatments. The plasma T3 level significantly decreased in LN, MN, and HN and T4 significantly decreased in HN. The CRH, ACTH, and plasma cortisol levels were significantly upregulated in HN compared with that in the CK. We conclude that elevated NO3− exposure leads to growth retardation, impaired health status, and endocrine disorders in turbot and the NO3− level for juvenile turbot culture should not exceed 50 mg/L NO3-N in RAS. Our findings indicate that endocrine dysfunction of the GH/IGF-1, HPT, and HPI axes might be responsible for growth inhibition induced by NO3− exposure.

14 pages, 7 figures, 2 tables, supplementary material https://doi.org/10.1016/j.ygeno.2021.04.007.-- Availability of data and materials: All dataset generated during this study are included (in this article and as supplementary information files) or are available in public repositories. Turbot Genome Sequencing data are at NCBI databases (Bioproject: PRJNA631898) (https://www.ncbi.nlm.nih.gov/genome/?term=turbot) and BioProject es PRJNA649485 (https://www.ncbi.nlm.nih.gov/bioproject/649485; Accessión numbers: SRX8843737, SRX8843739, SRX8843738), 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, This work was supported by the Spanish Ministry of Economy and Competitiveness, Grant: AGL2014-57065-R, by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 81792 (AQUA-FAANG) and by Consellería de Educación, Universidade e Formación Profesional. Xunta de Galicia, Grant number: ED431C 2018/28, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

6 pages, 1 table.-- This work is licensed under a Creative Commons Attribution 4.0 International License, The increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment. Aim: To evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon. Methods: From 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae. Results: No zoonotic parasites were found in any of the fish investigated. Conclusions: The risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation, This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 634429

UV filters are potentially harmful to marine organisms. Given their worldwide dissemination and the scarcity of studies on marine fish, we evaluated the toxicity of an organic (oxybenzone) and an inorganic (titanium dioxide nanoparticles) UV filter, individually and in a binary mixture, in the turbot (Scophthalmus maximus). Fish were intraperitoneally injected and a multi-level assessment was carried out 3 and 7 days later. Oxybenzone and titanium dioxide nanoparticles induced mild effects on turbot, both isolated and in mixture. Neither oxidative stress (intestine, liver and kidney) nor neurotoxicity (brain) was found. However, liver metabolic function was altered after 7 days, suggesting the impairment of the aerobic metabolism. An increased motility rate in oxybenzone treatment was the only behavioural alteration (day 7). The intestine and liver were preferentially targeted, while kidney and brain were unaffected. Both infra- and supra-additive interactions were perceived, with a toxicodynamic nature, resulting either in favourable or unfavourable toxicological outcomes, which were markedly dependent on the organ, parameter and post-injection time. The combined exposure to the UV filters did not show a consistent increment in toxicity in comparison with the isolated exposures, which is an ecologically relevant finding providing key information towards the formulation of environmentally safe sunscreen products.

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.

Nitrate (NO3−) is one of the common inorganic nitrogen compound pollutants in natural ecosystems, which may have serious risks for aquatic organisms. However, its toxicological mechanism remains unclear. In the current study, juvenile turbot (Scophthalmus maximus) were exposed to different concentrations of NO3− (CK− 3.57 ± 0.16, LN − 60.80 ± 1.21, MN − 203.13 ± 10.97 and HN − 414.16 ± 15.22 mg/L NO3–N) for 60 d. The blood biochemical assays results revealed that elevated NO3− exposure significantly increased the concentrations of plasma NO3−, NO2−, MetHb, K+, cortisol, glucose, triglyceride, lactate, while significantly decreased the concentrations of plasma Hb, Na+ and Cl−, which meant that NO3− caused hypoxic stress and further affected the osmoregulation and metabolism in fish. Besides, exposure to MN and HN induced a significant decrease in the level of antioxidants, including SOD (Point: 60th day, MN, HN v.s. CK: 258.36, 203.73 v.s. 326.95 U/mL), CAT (1.97, 1.17 v.s. 2.37 U/mL), GSH (25.38, 20.74 v.s. 37.00 μmol/L), and GPx (85.32, 71.46 v.s. 129.36 U/mL), and a significant increase of MDA (7.54, 9.73 v.s. 5.27 nmol/L), suggesting that NO3− exposure leading to a disruption of the redox status in fish. Also, further research revealed that NO3− exposure altered the mRNA levels of p53 (HN: up to 4.28 folds) and p53-regulated downstream genes such as Bcl-2 (inferior to 0.44 folds), caspase-3 (up to 2.90 folds) and caspase-7 (up to 3.49 folds), indicating that NO3− exposure induced abnormal apoptosis in the fish gills. Moreover, IBRv2 analysis showed that the toxicity of NO3− exposure to turbot was dose-dependent, and the toxicity peaked on the 15th day. In short, NO3− is an environmental toxicological factor that cannot be ignored, because its toxic effects are long-term and could cause irreversible damage to fish. These results would be beneficial to improve our understanding of the toxicity mechanism of NO3− to fish, which provides baseline evidence for the risk assessment of environmental NO3− in aquatic ecosystems.

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. This study was financially supported by grant AGL2017-83577-R awarded by the Ministerio de Economía y Competitividad (Spain) and the European Regional Development Fund (ERDF, European Union), by grant ED431C2017/31 from the Xunta de Galicia (Spain) and by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634429. FF-I was contracted by a grant from the Xunta de Galicia (Plan I2C) SI

Metals are considered to be one of the most hazardous substances due to their potential for accumulation, magnification, persistence, and wide distribution in water, sediments, and aquatic organisms. Demersal fish species, such as turbot (Psetta maxima maeotica), are accepted by the scientific communities as suitable bioindicators of heavy metal pollution in the aquatic environment. The present study uses a machine learning approach, which is based on multiple linear and non-linear models, in order to effectively estimate the concentrations of heavy metals in both turbot muscle and liver tissues. For multiple linear regression (MLR) models, the stepwise method was used, while non-linear models were developed by applying random forest (RF) algorithm. The models were based on data that were provided from scientific literature, attributed to 11 heavy metals (As, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Ni, Zn) from both muscle and liver tissues of turbot exemplars. Significant MLR models were recorded for Ca, Fe, Mg, and Na in muscle tissue and K, Cu, Zn, and Na in turbot liver tissue. The non-linear tree-based RF prediction models (over 70% prediction accuracy) were identified for As, Cd, Cu, K, Mg, and Zn in muscle tissue and As, Ca, Cd, Mg, and Fe in turbot liver tissue. Both machine learning MLR and non-linear tree-based RF prediction models were identified to be suitable for predicting the heavy metal concentration from both turbot muscle and liver tissues. The models can be used for improving the knowledge and economic efficiency of linked heavy metals food safety and environment pollution studies.

To gain insights into the influence of heat stress on lipid metabolism in juvenile turbot (Scophthalmus maximus), we analyzed the correlations between data obtained by transcriptome sequencing and metabolome sequencing of the kidney under different high temperature stimuli (20 °C, 25 °C and 28 °C) and control conditions (14 °C). We identified the differentially expressed genes and metabolites, which were found to be enriched in seven pathways (steroid hormone biosynthesis, primary bile acid biosynthesis, glycerophospholipid metabolism, linoleic acid metabolism, sphingolipid metabolism, glycerolipid metabolism and biosynthesis of unsaturated fatty acids) associated with lipid metabolism, according to KEGG pathway analysis. After correlation analysis of these differentially expressed genes, the most representative genes (lpcat2, Etnk1, TAZ, SCP2, ch25hl and gpd1l) and metabolites (citicoline, UPD-6-sulfoquinovose, dihydroxyacetone, taurine and o-phosphocholine) were selected according to their correlation coefficients. These genes and metabolites were found to be the key points to regulate lipid deposition and maintain lipid homeostasis through varying degrees of up-regulation or down-regulation under heat stress, so as to relieve the disorder of lipid metabolism caused by heat stress, which is of great significance for breeding new heat-resistant varieties of turbot and provides a reliable theoretical basis for optimizing actual production. These results provide new clues for understanding the roles of lipid metabolism in fish under heat stress.

Since the DeepWater Horizon oil spill and the use at 1450 m depth of dispersant as a technical response, the need of relevant ecotoxicological data on deep-sea ecosystems becomes crucial. In this context, this study focused on the effect of high hydrostatic pressure (10.1 MPa) on turbot hepatocytes isolated from fish exposed either to chemically dispersed oil, mechanically dispersed oil or dispersant alone. Potential combined effects of oil/dispersant and hydrostatic pressure, were assessed on cell mortality (total cell death, necrosis and apoptosis), cell viability and on hepatocyte oxygen consumption (MO2). No change in cell mortality was observed in any of the experimental conditions, whereas, the results of cell viability showed a strong and significant increase in the two oil groups independently of the pressure exposure. Finally, oil exposure and hydrostatic pressure have additive effects on oxygen consumption at a cellular level. Presence of dispersant prevent any MO2 increase in our experimental conditions. These mechanistic effects leading to this increased energetic demand and its eventual inhibition by dispersant must be investigated in further experiments.