Search

Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the effects of dietary AFB 1 on growth performance, health, intestinal microbiota communities and AFB 1 tissue residues of turbot and evaluate the mitigation efficacy of yeast cell wall extract, Mycosorb ® (YCWE) toward AFB 1 contaminated dietary treatments. Nine experimental diets were formulated: Diet 1 (control): AFB 1 free; Diets 2-5 or Diets 6-9: 20 μg AFB 1 /kg diet or 500 μg AFB 1 /kg diet + 0%, 0.1%, 0.2%, or 0.4% YCWE, respectively). The results showed that Diet 6 significantly decreased the concentrations of TP, GLB, C3, C4, T-CHO, TG but increased the activities of AST, ALT in serum, decreased the expressions of CAT, SOD, GPx, CYP1A but increased the expressions of CYP3A, GST- ζ 1 , p53 in liver. Diet 6 increased the AFB 1 residues in serum and muscle, altered the intestinal microbiota composition, decreased the bacterial community diversity and the abundance of some potential probiotics. However, Diet 8 and Diet 9 restored the immune response, relieved adverse effects in liver, lowered the AFB 1 residues in turbot tissues, promoted intestinal microbiota diversity and lowered the abundance of potentially pathogens. In conclusion, YCWE supplementation decreased the health effects of AFB 1 on turbot, restoring biomarkers closer to the mycotoxin-free control diet.

To understand the role of 14-3-3 beta/alpha in hypoosmotic regulation of turbot (Scophthalmus maximus), we characterized the 14-3-3 beta/alpha gene and analyzed the tissue distribution and its gene transcriptional patterns in the main expressed tissues under low salt stress. The 14-3-3 beta/alpha cDNA is 892 bp in length, incorporating an ORF of 774 bp with a putative primary structure of 257 residues. The deduced amino acid sequences shared highly conserved structures with other eukaryotes. Quantitative real-time PCR results showed that the 14-3-3 beta/alpha transcripts were widely expressed in various tissues of turbot, with most abundant in the gill (P < .05), to a lesser extent in the kidney, intestine, brain and spleen, and at low levels in the pituitary and other tissues examined. And the expression of turbot 14-3-3 beta/alpha exhibited a trend of increasing first and then decreasing with the time of stress under low salt stress, and the highest value appeared in 12 h (P < .05). After injecting different concentrations of dsRNA, the mRNA expression of 14-3-3 gene decreased significantly during the monitoring period, and the best interference effect was achieved 12 h after injecting 4 μg/g dsRNA. For the first time, the gene was silenced in fish by intramuscular injection of dsRNA. It also provides a new and effective way to study gene function at the individual level. Moreover, the mRNA interference of 14-3-3 beta/alpha would cause changes in the expression of several ion channel proteins, for example, the decrease of Na + -K + -ATPase and Na + -H + -exchanger and the increase of CFTR. As a result, 14-3-3 beta/alpha appears to be an important molecular regulator for osmosensory signal transduction in gill of turbot., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest exist., (Copyright © 2020. Published by Elsevier Inc.)

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 ( vkorc1 ) and vkorc1 like 1 ( vkorc1l1 ) were determined during the larval ontogeny of Senegalese sole ( Solea senegalensis ), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1 , and ovary and brain for ssvkorc1l1 . Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions.

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.

Nucleotide degradation in fish is an important biochemical change after death, which is closely related to freshness and sensory quality. However, except ATP-relative nucleotides, it remains unclear about changes in other nucleotide metabolites during postmortem stage. In this study, a strategy for the simultaneous quantification of 28 nucleobases, nucleosides, and nucleotides using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) with positive/negative ion switching was developed. This method showed good linearity, precision, repeatability, and recovery. Furthermore, it was successfully applied to monitor the postmortem nucleotide degradation of turbot mince during chill (4 °C) and partial freezing (-3 °C) storage for 168 h. It was noted that the patterns of the changes in nucleotide metabolites differed considerably depending on the storage temperature. Meanwhile, the different pathway and speed of nucleotide catabolism in turbot mince was summarized based on the quantification data., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The induction of the myo-inositol biosynthesis (MIB) pathway in euryhaline fishes is an important component of the cellular response to osmotic challenge. The MIPS and IMPA1 genes were sequenced in turbot and found to be highly conserved in phylogenetic evolution, especially within the fish species tested. Under salinity stress in turbot, both MIPS and IMPA1 showed adaptive expression, a turning point in the level of expression occurred at 12 h in all tissues tested. We performed an RNAi assay mediated by long fragment dsRNA prepared by transcription in vitro. The findings demonstrated that knockdown of the MIB pathway weakened the function of gill osmotic regulation, and may induce a genetic compensation response in the kidney and gill to maintain physiological function. Even though the gill and kidney conducted stress reactions or compensatory responses to salinity stress, this inadequately addressed the consequences of MIB knockdown. Therefore, the survival time of turbot under salinity stress after knockdown was obviously less than that under seawater, especially under low salt stress. Pearson's correlation analysis between gene expression and dietary myo-inositol concentration indicated that the MIB pathway had a remarkable negative feedback control, and the dynamic equilibrium mediated by negative feedback on the MIB pathway played a crucial role in osmoregulation in turbot. An RNAi assay with c-Myc in vivo and the use of a c-Myc inhibitor (10058-F4) in vitro demonstrated that c-Myc was likely to positively regulate the MIB pathway in turbot., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019. Published by Elsevier Inc.)

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

The valorization of wastes generated in the processing of farmed fish is currently an issue of extreme relevance for the industry, aiming to accomplish the objectives of circular bioeconomy. In the present report, turbot ( Scophthalmus maximus ) by-products were subjected to Alcalase hydrolysis under the optimal conditions initially defined by response surface methodology. All the fish protein hydrolysates (FPHs) showed a high yield of digestion (>83%), very remarkable degrees of hydrolysis (30-37%), high content of soluble protein (>62 g/L), an excellent profile of amino acids, and almost total in vitro digestibility (higher than 92%). Antioxidant and antihypertensive activities were analyzed in all cases, viscera hydrolysates being the most active. The range of average molecular weights (Mw) of turbot hydrolysates varied from 1200 to 1669 Da, and peptide size distribution showed that the hydrolysate of viscera had the highest content of peptides above 1000 Da and below 200 Da.

Paralichthys olivaceus (mean weight, 280.1 ± 10.5 g; mean length, 28.37 ± 2.3 cm) was reared in bio-floc and seawater for 6 months to determine the toxic effects of waterborne nitrite exposure (0, 25, 50, 100, and 200 mg/L) for 1 week, compared to those observed with bio-floc and seawater only. The effects on antioxidant activity, immune responses, and acetylcholinesterase activity were measured. Following nitrite exposure, superoxide dismutase activity in the liver and gills was significantly elevated and catalase activity was significantly increased, except for in the gills of P. olivaceus reared in bio-floc. Further, glutathione S-transferase activity was significantly elevated in the liver and gills, and glutathione was significantly lower. Meanwhile, acetylcholinesterase activity in the liver and gills was significantly inhibited and plasma lysozyme activity and immunoglobulin M were considerably elevated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Opsins play important roles in the image-forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost-specific genome duplication (TSGD). qRT-PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA-positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE., (© 2019 Wiley Periodicals, Inc.)

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.

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry., Competing Interests: APIVITA provided support in the form of salaries for authors KG, ES and SL that participated in the Algae4A&B training activities. These authors contributed to data collection as part of the training secondments but did not have any additional role in the study design, data analysis, decision to publish, or preparation of the manuscript. Commercial affiliation of some authors does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or market products to declare here.

Methylmercury (MeHg) is a highly toxic contaminant in coastal environments and poses threats to marine fish in early life stages (ELSs). However, MeHg toxicity to fish embryos and larvae is not well investigated. This study investigated the antioxidant defenses and immune responses of flounder Paralichthys olivaceus larvae exposed to waterborne MeHg (0, 0.1, 1.0 and 10.0 μg L - 1 ) for 35 days, from embryogenesis to settlement. The results revealed that metal accumulation in the larvae was positively correlated with MeHg concentration, reduced larval growth and survival. The activities of catalase and glutathione reductase were significantly increased at 10.0 μg L - 1 , while glutathione peroxidase activity and lipid peroxidation level were significantly increased at concentrations over 1.0 μg L - 1 . The corresponding antioxidant-related genes were upregulated under MeHg exposure (cat and gpx at 10.0 μg L - 1 ; gr over 1.0 μg L - 1 ). Lysozyme content was significantly increased, but immunoglobulin M content was significantly decreased at 10.0 μg L - 1 . The immune-related genes were significantly upregulated (hsp70 at 0.1 and 10.0 μg L - 1 ; lzm and il-1β over 1.0 μg L - 1 ; tnf-α and il-6 at 10.0 μg L - 1 ) or downregulated (igm, over 0.1 μg L - 1 ). Overall, MeHg exposure induced oxidative stress and caused immunotoxicity at concentrations over 1.0 μg L - 1 and 10.0 μg L - 1 , respectively. The transcription of selected genes correlated with the corresponding biochemical markers in response to MeHg toxicity. These findings improve our knowledge to better understand the mechanisms by which marine fish at ELSs cope with oxidative stress and immunotoxicity induced by MeHg., (Copyright © 2019 Elsevier Inc. All rights reserved.)

High levels of soybean meal (SBM) in aquafeed leads to detrimental inflammatory response and oxidative stress in fish. In the present study, fermentation with Aspergillus awamori was conducted to explore the potential effects on improving the nutritional quality of soybean meal and the health status of turbot. A 63-day feeding trial (initial weight 8.53 ± 0.11 g) was carried out to evaluate the utilization of fermented soybean meal (FSM) by juvenile turbot. 0% (FM, control), 30% (S30, F30), 45% (S45, F45), and 60% (S60, F60) of fish meal were replaced with SBM or FSM, respectively. As the results showed, fermentation significantly reduced the contents of anti-nutritional factors in SBM, including raffinose (-98.8%), glycinin (-98.5%), β-conglycinin (-97.4%), trypsin inhibitors (-80%) and stachyose (-80%). A depression of fish growth performance and activities of superoxide dismutase and lysozyme were observed in S45 and S60 groups, while these inferiorities were only observed in F60 group. Meanwhile, fermentation also improved the heights of enterocytes and microvillus significantly in the F45 and F60 groups compared with those in SBM. An induced expression of anti-inflammatory cytokine transforming growth factor-β and depression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β in the distal intestine were observed in the F45 and F60 groups. Taken together, this study indicated that fermentation with Aspergillus awamori could improve the replacement level with soybean meal from 30% to 45% in turbot., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 10 8 -10 9  CFU kg -1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Primordial germ cells (PGCs) as the precursors of germ cells are responsible for transmitting genetic information to the next generation. Visualization of teleost PGCs in vivo is essential to research the origination and development of germ cells and facilitate further manipulation on PGCs isolation, cryopreservation, and surrogate breeding. In this study, artificially synthesized mRNAs constructed by fusing fluorescent protein coding region to the 3' untranslated region (3'UTR) of nanos3 or vasa (mCherry-Smnanos3 3'UTR or mCherry-Smvasa 3'UTR mRNA) were injected into turbot (Scophthalmus maximus) fertilized eggs for tracing PGCs. The results demonstrated that the fluorescent PGCs differentiated from somatic cells and aligned on both sides of the trunk at the early segmentation period, then migrated and located at the dorsal part of the gut where the gonad would form. In the same way, we also found that the zebrafish (Danio rerio) vasa 3'UTR could trace turbot PGCs, while the vasa 3'UTR s of marine medaka (Oryzias melastigma) and red seabream (Pagrus major) failed, although they could label the marine medaka PGCs. In addition, through comparative analysis, we discovered that some potential sequence elements in the3 'UTRs of nanos3 and vasa, such as GCACs, 62-bp U-rich regions and nucleotide 187-218 regions might be involved in PGCs stabilization. The results of this study provided an efficient, rapid, and specific non-transgenic approach for visualizing PGCs of economical marine fish in vivo.

Pseudo-albinism is a pigmentation disorder observed in flatfish aquaculture with a complex, multi-factor aetiology. We tested the hypothesis that pigmentation abnormalities are an overt signal of more generalised modifications in tissue structure and function, using as a model the Senegalese sole and two important innate immune barriers, the skin and intestine, and their microbiomes. Stereological analyses in pseudo-albino sole revealed a significantly increased mucous cell number in skin (P < 0.001) and a significantly thicker muscle layer and lamina propria in gut (P < 0.001). RNA-seq transcriptome analysis of the skin and gut identified 573 differentially expressed transcripts (DETs, FDR < 0.05) between pseudo-albino and pigmented soles (one pool/tissue from 4 individuals/phenotype). DETs were mainly linked to pigment production, skin structure and regeneration and smooth muscle contraction. The microbiome (16 S rRNA analysis) was highly diverse in pigmented and pseudo-albino skin but in gut had low complexity and diverged between the two pigmentation phenotypes. Quantitative PCR revealed significantly lower loads of Mycoplasma (P < 0.05) and Vibrio bacteria (P < 0.01) in pseudo-albino compared to the control. The study revealed that pseudo-albinism in addition to pigmentation changes was associated with generalised changes in the skin and gut structure and a modification in the gut microbiome.

Pyroptosis is a newly defined gasdermin (GSDM)-dependent inflammatory type of programmed cell death. Different from mammals, which have a panel of pyroptotic GSDM members (e.g., GSDMA-E), teleosts possess only GSDME. The pyroptotic activity and regulation mechanism of teleost GSDME remain to be elucidated. In this work, we investigated the activity of the teleost Cynoglossus semilaevis (tongue sole) GSDME (CsGSDME) in association with different caspases (CASPs). We found that CsGSDME exerted pyroptotic and bactericidal activities through its N-terminal domain. Unlike human GSDME, which is exclusively cleaved by CASP3, CsGSDME was cleaved by C. semilaevis CASP (CsCASP) 1 with high efficiency and by CsCASP3 and 7 with comparatively low efficiencies, and all cleavages occurred at the 243 FEVD 246 site in the interdomain linker region of CsGSDME. Mutation of Phe 243 to Asp/Ala and Asp 246 to Ala in 243 FEVD 246 altered the cleavage preference of CsCASP1, 3, and 7. Treatment with loss-of-function CsCASP mutants or inhibition of CsCASP activity resulted in failure of CsGSDME cleavage. CsCASP1-cleaved CsGSDME induced pyroptosis, whereas CsCASP3/7-cleaved CsGSDME and F243 mutants induced switching of cell death from apoptosis to pyroptosis. Analysis of 54 teleost GSDME sequences revealed a conserved tetrapeptide motif that fits well to the inherent cleavage site of CASP1. Taken together, the results of our study demonstrate a hitherto, to our knowledge, unrecognized GSDME cleavage mode in teleosts that is clearly different from that in mammals, thus providing an important insight into the activation mechanism of CASP-mediated, GSDM-executed pyroptosis in teleosts., (Copyright © 2019 by The American Association of Immunologists, Inc.)

In this study, potential immunological and hematological effects of different concentrations (0, 1, 10, and 50 μg L -l ) of waterborne zinc pyrithione (ZnPT) were studied in the blood of the olive flounder Paralichthys olivaceus over 30 days. Reduced alternative complement activity (ACH50) and lysozyme activity were measured in fish exposed to 10 and/or 50 μg L -l of ZnPT for 20 days. Decreased levels of total Ig were also observed in response to 10 and/or 50 μg L -l ZnPT during the exposure period. Levels of cortisol, a marker of stress, were significantly increased by 10 and 50 μg L -l ZnPT from day 10, and by 1 μg L -l exposure on day 30. The levels of red blood cells (RBCs) and white blood cells (WBCs) decreased following exposure to 10 and/or 50 μg L -l ZnPT, while no significant change was observed in hemoglobin level. Concentrations of total protein and albumin were significantly reduced with 50 μg L -l ZnPT at day 20. Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities were significantly increased following exposure to 10 and/or 50 μg L -l ZnPT. Lipid peroxidation was induced by ZnPT, and higher concentrations (10 and 50 μg L -l ) significantly increased intracellular malondialdehyde levels during exposure. Regarding the subsequent antioxidant response, intracellular glutathione levels increased significantly in response to 10 and 50 μg L -l ZnPT on days 20 and 30. Similarly, catalase and superoxide dismutase activity was significantly increased in response to 10 and 50 μg L -l ZnPT after day 10. Taken together, changes in the studied parameters suggested the immunotoxicity of ZnPT, with modulations observed in hematological homeostasis and oxidative stress induction in the blood of olive flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adding immunopotentiators to plant protein based diets has been a feasible way to improve fish growth performance and healthy status. In this study, an 8-week trial was carried out to explore the effects of resveratrol, a natural polyphenolic compound, on growth performance, anti-oxidative capacity and immune responses in turbot fed soybean meal based diet. As the results showed, replacement 45% fish meal with soybean meal (SBM) significantly depressed the fish growth, feed utilization and the heights of villi and microvilli in distal intestine. The mRNA levels of hepatic antioxidant enzymes, including superoxide dismutase (sod), glutathione peroxidase (gsh-px) and peroxiredoxin 6 (prx 6), were highly inhibited in SBM group. The inflammation related genes in intestine were also responsive to soybean meal. Supplying resveratrol showed no effects on fish growth performance but significantly restored the intestinal morphology and improved the mRNA levels of hepatic antioxidant enzymes as well as the activity of SOD. Meanwhile, resveratrol significantly improved the mRNA levels of anti-inflammatory cytokine transforming growth factor-β and inhibited the expression of pro-inflammatory cytokines tumor necrosis factor-α (tnf-ɑ), interleukin-1β (il-1β) and interleukin-8 (il-8). The results indicate that resveratrol could attenuate the oxidative stress and inflammatory response induced by soybean meal in turbot. This study shows resveratrol is an effective immunopotentiator to carnivorous fishes fed plant protein sources., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Phytochemicals are widely present in the aquatic environment and they are derived from many anthropogenic activities. The isoflavone daidzein is a natural compound that is found in the soya products used as habitual constituents of aquafeeds. Nevertheless, this isoflavone possesses oestrogenic and apoptotic properties. The present study determined the effects of daidzein (at 20 mg/L) during the first month and a half of life (from 7 to 44 days post-hatching -dph-) of the flatfish Senegalese sole, Solea senegalensis, focusing at the metamorphosis. We have analysed different gene expression levels and immunohistochemical protein patterns implicated in some oestrogenic, apoptosis and enzymatic pathways. In general, the oestrogen receptor (ERβ) and stimulating apoptosis death receptor factor (Fas) transcript levels showed similar baseline patterns and transcriptional responses induced by daidzein. Both ERβ and Fas were up-regulated by this isoflavone at the pre-metamorphosis and metamorphosis, and they were down-regulated in post-metamorphosed stages. The expression pattern of the apoptotic effector caspase (Casp6) was exclusively up-regulated at the pre-metamorphic phase. The Birc5 transcripts (i.e. anti-apoptosis, Survivin) were down-regulated by daidzein during certain metamorphic and post-metamorphosed stages. Besides, daidzein showed an up-regulating effect on both enzymatic complexes, the haemoprotein CYP1A and the acetylcholinesterase (AChE), except for a temporary AChE down-regulation in some post-metamorphosed stages. Immunostaining analysis only showed increased CYP1A signals in the liver of daidzein exposed fish. Overall, a majority of the transcriptional oestrogenic and apoptotic imbalances could be gradually and/or temporarily stabilised. Most controls and exposed larvae (70-80%) developed and grew following normal ontogenetic developmental patterns.

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., (Copyright © 2019. Published by Elsevier Ltd.)

Accumulating evidence suggests that the growth hormone (GH)/insulin-like growth factor (IGF) system participates in fish reproduction. To understand the physiological functions of the GH/IGF system, the mRNA expression profiles of all known members of the GH/IGF system, including hepatic and ovarian gh, GH receptor (ghr), IGFs (igf-i, igf-ii), IGF-I receptor (igf-ir) and IGF binding protein (igfbp1, igfbp2), pituitary gh, and hepatic vitellogenin (vtg) were investigated during ovarian development in turbot Scophthalmus maximus. Results showed that ghr, igf-i, igf-ii, igf-ir, and igfbp2 were expressed in the liver and ovary, whereas igfbp1 and gh were undetected. The hepatosomatic index (HSI) and gonadosomatic index (GSI) gradually increased and peaked during the late vitellogenesis (Latvtg) and migratory nucleus (Mig-nucl) stages, respectively. The mRNA expression profiles of ovarian ghr, igf-ii, hepatic igf-ir, vtg, and pituitary gh were similar to the HSI; ovarian igf-i and igf-ir expression was close to the GSI. However, the hepatic mRNA levels of ghr, igf-i, and igf-ii peaked at the early vitellogenesis (Evtg) stage, and then drastically declined during ovarian development. The mRNA expression of hepatic igfbp2 decreased and reached the lowest at the atresia (Atre) stage, whereas that of ovarian igfbp2 increased and peaked at Latvtg stage. Furthermore, significant correlations between pituitary gh, ovarian ghr, igf-i, and igf-ii, and hepatic ghr, igf-i, igf-ir, and igf-ii were observed, respectively. These results suggest that GH/IGF members appear to play distinct roles in the regulation of ovarian development in turbot and will be valuable for fish reproduction and broodstock management of aqua-cultured fish species.

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3β signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3β signaling pathway via suppressing the IRS2a and IRS2b genes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present work, growth and digestive enzyme activities of total acid and alkaline proteases, pepsin, trypsin, lipase, and α-amylase, as well as partial characterization of enzyme activity, were studied in diploid and triploid turbot. Growth was similar between both groups. Acid protease activity increased consistently during the experiment, for both diploid (2n) and triploid (3n) fish. The alkaline protease activity was always higher for triploids throughout the experiment. Proteolytic acid activity (pH 2) was generally higher for diploids, at all temperatures tested. Higher activity was at pH 2 and 3 for 2n and 3n fish, respectively. Regarding temperature, acid and alkaline protease activity was higher at 37 °C and 60 °C, respectively, for both groups. The general increase in pancreatic enzymes (trypsin and amylase) before 35 days after hatching (DAH) and posterior decrease until 60 DAH. There was a marked effect on enzyme activity when changing from live prey to pellets (35 DAH), especially on triploids.

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot., (Copyright © 2019. Published by Elsevier Ltd.)

Subcellular cadmium (Cd) partitioning was investigated in the liver of two marine fish species, the European sea bass Dicentrarchus labrax and the Senegalese sole Solea senegalensis, dietary exposed to an environmentally realistic Cd dose for two months followed by a two-month depuration. The two species displayed different handling strategies during the depuration period. Cd was largely bound to detoxifying fractions such as heat stable proteins (HSP) including metallothioneins (MT) in sea bass, while Cd was more linked to sensitive fractions such as organelles in sole. Whole liver concentrations and subcellular partitioning were also determined for essential elements. The greatest impairment of essential metal homeostasis due to Cd exposure was found in sole. These elements followed the Cd partitioning pattern, suggesting that they are involved in antioxidant responses against Cd toxicity. Cd consumption diminished sole growth in terms of body weight, probably due to lipid storage impairment. The contrasting partitioning patterns showed by the two species might imply different pathways for Cd elimination from the liver. In sea bass, MT-bound Cd would be excreted through bile or released into blood, crossing the cell membrane via a protein transporter. In sole, MRG-bound Cd would be sequestered by organelles before being released into the blood via vesicular exocytosis. These distinct strategies in cellular Cd handling in the liver might account for differential sensitivity to Cd toxicity and differential Cd excretion pathways between the two marine fish species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Concentrations of iodine-129 ( 129 I) and atomic ratios of 129 I/ 127 I in livestock (grass and milk), agricultural (cabbage, Japanese radish, and rice), and fishery (flatfish and brown alga) products collected from locations around the first Japanese commercial spent nuclear fuel reprocessing plant in Rokkasho were measured from 2006 to 2016. The actual spent nuclear fuel rods were cut and processed to test the functioning of the plant that discharged controlled amounts of 129 I to the atmosphere and coastal seawater during the period from 2006 to 2008 (the "cutting period"). Statistically significant increases in 129 I concentration and 129 I/ 127 I ratio were observed during the cutting period in livestock products and flatfish. On the other hand, these parameters were statistically comparable during and after the cutting period in the other products. The radiation dose through the ingestion of the maximum 129 I concentrations, measured in the different products, was estimated to be in the nanoSievert per year level. This value is much smaller than 1 mSv yr -1 , which is the permissible authentic radiation dose for the general public. The 129 I levels in the samples, especially in milk and flatfish, are discussed in context of the 129 I discharge history from the plant.

This study extends our knowledge of the bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) in marine organisms and investigates its possible determinants. PAH levels were measured in Solea solea tissue and in marine sediments collected from three areas of the northern Adriatic Sea characterized by different anthropic impacts (Venetian Lagoon, Po Delta, and fishing grounds off Chioggia). The possibility of differential PAH bioaccumulation in different tissues (muscle, liver and gills) was investigated by seeking relationships between mean individual and total PAH concentrations in tissue and sediment samples, the physicochemical properties of PAHs (rings and K ow ), and some key biological variables (lipid content of tissues, body size, habitat). The present study demonstrated that the lipid content might not be the only determinant of PAH bioaccumulation in common sole tissues. The habitat characteristics, the tissue types and some physicochemical properties of compounds were closely related to PAH bioaccumulation., (Copyright © 2018. Published by Elsevier Ltd.)

Wastewater treatment plant effluent introduces a mixture of pollutants into marine environments; however, the impacts of chronic sublethal exposures on populations are often unclear. Presence of estrogenic agents in sediments and uptake of these compounds by demersal flatfishes has been reported at the Orange County Sanitation District (OCSD) wastewater outfall. Furthermore, estrogenic activity has been identified in male flatfish in the area, potentially contributing to observed population declines in the OCSD region. Rising ocean temperatures may further contribute to flatfish declines as relationships between temperature and abundance have been reported in the Southern California Bight. To investigate declines, sex ratios, condition factor, organ health indices, hormones, and vitellogenin were quantified in flatfish collected at OCSD outfall and reference sites. Additionally, historical temperature data was examined for trends with population abundances. Rather than being linked to estrogenic activity, results indicated that population declines were more correlated to increases in ocean temperature., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The expanding aquaculture industry plays an important role in feeding the growing human population and with the expansion, sustainable bacterial disease control, such as probiotics, becomes increasingly important. Tropodithietic acid (TDA)-producing Phaeobacter spp. can protect live feed, for example rotifers and Artemia as well as larvae of turbot and cod against pathogenic vibrios. Here, we show that the emerging live feed, copepods, is unaffected by colonization of the fish pathogen Vibrio anguillarum, making them potential infection vectors. However, TDA-producing Phaeobacter inhibens was able to significantly inhibit V. anguillarum in non-axenic cultures of copepod Acartia tonsa and the copepod feed Rhodomonas salina. Vibrio grew to 10 6  CFU ml -1 and 10 7  CFU ml -1 in copepod and R. salina cultures, respectively. However, vibrio counts remained at the inoculum level (10 4  CFU ml -1 ) when P. inhibens was also added. We further developed a semi-strain-specific qPCR for V. anguillarum to detect and quantify the pathogen in non-axenic systems. In conclusion, P. inhibens efficiently inhibits the fish larval pathogen V. anguillarum in the emerging live feed, copepods, supporting its use as a probiotic in aquaculture. Furthermore, qPCR provides an effective method for detecting vibrio pathogens in complex non-axenic live feed systems., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Bacillus sp. SJ-10 (BSJ-10) was identified from traditional Korean fermented fish, the previously recognized prebiotic β-glucooligosaccharides (BGO), and their combination as a synbiotic were prepared to evaluate their individual and synergistic effects in olive flounder (Paralichthys olivaceus). Four diets (one control and three treatments) were formulated containing neither BSJ-10 nor BGO (control), 1 × 10 8  CFU g -1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 10 8  CFU g -1 BSJ-10 + 0.1% BGO (BSJ-10 + BGO). Triplicates of 15 fish (weight 10 ± 0.25 g) were randomly allocated to the four diet groups and fed one of the diets for 8 weeks. At the end of the experiment, fish weight gain (WG), specific growth rate (SGR), feed conversion ratio, and protein efficiency ratio in BSJ-10, BGO and BSJ-10 + BGO diets were positively modulated (P < 0.05) compared with control. Specially, WG and SGR were significantly (P < 0.05) higher in BSJ-10 + BGO than that of BSJ-10 and BGO (individual component). The innate immune parameters such as respiratory burst, superoxide dismutase, and lysozyme activity (LSZ) of fish fed BSJ-10 and BSJ-10 + BGO (both groups) were significantly (P < 0.05) higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10 + BGO were significantly higher compared with individual component. Compared with control, intestinal BSJ-10 content, expression of interleukin (IL)-1β in liver and kidney, and tumor necrosis factor (TNF)-α in liver were higher in both groups, but microvillus length was increased (P < 0.05) only in BSJ-10 + BGO. During in vivo challenge experiment with Streptococcus iniae (1 × 10 8  CFU ml -1 ), survival rate of fish was significantly higher in all treatment groups versus control. Moreover, in BSJ-10 + BGO, protection against S. iniae infection and transcription of TNF-α and IL-6 in gill were significantly (P < 0.05) higher than the individual component. Collectively, an improved WG, SGR, MPO, LSZ, transcription of IL-6 and TNF-α, and cumulative survival rate against streptococcosis clearly demonstrates a synergistic outcome of diet BSJ-10 + BGO as synbiotic in olive flounder., (Copyright © 2018. Published by Elsevier Ltd.)

Spermatid perinuclear RNA-binding protein (Strbp) is a kind of double-stranded (ds) RNA specific binding protein that plays important roles in mammalian spermatogenesis. In this study, we have isolated and characterized the strbp gene of Chinese tongue sole, Cynoglossus semilaevis, termed as CS-strbp. The CS-strbp genomic sequence contained fifteen exons and fourteen introns. Its cDNA was 2655 bp in length, encoding a 666-amino-acid protein with two conserved ds binding motifs. Using quantitative PCR, we found that CS-strbp mRNA exhibited sex-biased and tissue-specific distribution, predominantly expressed in the fertile male testis, though the expression levels varied throughout different developmental stages. Comparison of methylation profile in different sexual genotypes demonstrated the low methylation level of CS-strbp promoter in male and pseudo-male, which is consistent with the high expression levels in those genotypes. In situ hybridization revealed that CS-strbp mRNA mainly localized in male germ cells, especially in spermatids and spermatozoa. Given these findings, we postulate that CS-strbp might function in spermatogenesis of Chinese tongue sole., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In order to better understand the influence of changing salinity conditions on the trophic transfer of 137 Cs in marine fish that live in dynamic coastal environments, its depuration kinetics was investigated in controlled aquaria. The juvenile turbot Scophthalmus maximus was acclimated to three distinct salinity conditions (10, 25 and 38) and then single-fed with compounded pellets that were radiolabelled with 137 Cs. At the end of a 21-d depuration period, assimilation efficiencies (i.e. AEs = proportion of 137 Cs ingested that is actually assimilated by turbots) were determined from observational data acquired over the three weeks. Our results showed that AEs of 137 Cs in the turbots acclimated to the highest salinity condition were significantly lower than for the other conditions (p < 0.05). Osmoregulation likely explains the decreasing AE observed at the highest salinity condition. Indeed, observations indicate that fish depurate ingested 137 Cs at a higher rate when they increase ion excretion, needed to counterbalance the elevated salinity. Such data confirm that ambient salinity plays an important role in trophic transfer of 137 Cs in some fish species. Implications for such findings extend to seafood safety and climate change impact studies, where the salinity of coastal waters may shift in future years in response to changing weather patterns., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

It is well known that the marine organisms are used as biological indicators for environmental pollution studies. Among these studies, the research on oxidative stress has been increasing in recent years. In this study, mussels (Mytilus galloprovincialis) and fish (Solea solea) samples were collected seasonally from İnciraltı, İzmir, Turkey. This station was in an area where fishing is carried out for human consumption. The relationship between 210 Po and oxidative stress markers (lipid peroxidation (LPO), H 2 O 2 and proline) was investigated in the mussel tissue (digestive gland, gills) and fish tissue (liver, gills) samples. The present study indicated that H 2 O 2 accumulated with increasing 210 Po concentration in mussel samples. Statistically significant correlation were found between H 2 O 2 and 210 Po and LPO and proline in mussel samples. This correlation between LPO and proline can be attributed to common environmental parameters (other than 210 Po) affecting expression of both LPO and proline levels. There was not a significant correlation between 210 Po and LPO levels. Similarly, a significant correlation was not found between 210 Po and proline., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Larval turbot (Scophthalmus maximus) undergo metamorphosis, a late post-embryonic developmental event that precedes juvenile transition. Insulin-like growth factors (IGFs) are important endocrine/autocrine/paracrine factors that provide essential signals to control of the embryonic and postnatal development of vertebrate species, including fish. Accumulating evidence suggests that IGFs are involved in regulating the metamorphic development of flatfish. This mini review focus on the functions of all known IGFs (IGF-I and IGF-II) during the metamorphic development of turbot. Information about IGFs and insulin-like growth factors binding proteins (IGFBPs) from other teleosts is also included in this review to provide an overview of IGFs functions in the metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGFs system in controlling of flatfish metamorphosis and contributing to the improvement of broodstock management strategies for larval turbot., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.

Chemical pollution of the marine environment from anthropogenic sources is a global concern due to the potential for long term effects on the ecosystem. Chemical monitoring schemes can detect harmful substances in water, sediment or biota. However, the presence of specific determinants gives no information on how individual species or the ecosystem are impacted. Biological effects techniques are therefore used in environmental monitoring schemes to provide a clearer picture of whether the chemicals present are causing deleterious effects on the species sampled, or whether organisms have been exposed to certain classes of organic contaminants that they may be able to metabolise. Using these techniques, we can provide an assessment of the health of our marine environment (Thain et al., 2008)., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Semicarbazide (SMC), a new marine pollutant, has anti-estrogenic effects on female Japanese flounder (Paralichthys olivaceus). However, whether SMC also affects the reproductive endocrine system of male marine organisms is currently unclear. In this study, Japanese flounder embryos were exposed to 1, 10, and 100 μg/L SMC for 130 days. Plasma testosterone (T) and 17β-estradiol (E 2 ) concentrations were significantly decreased in male flounders after SMC exposure. The expression of genes involved in T and E 2 synthesis, including steroidogenic acute regulatory protein, cytochrome P450 11A1, 17α-hydroxylase, 17β-hydroxysteroid dehydrogenase and cytochrome P450 19A, was down-regulated in the gonads, which may explain the decrease in plasma sex hormones levels. Moreover, SMC-mediated changes in the transcription of these steroidogenic genes were associated with reduced levels of follicle-stimulating hormone beta subunit (fshβ), luteinizing hormone beta subunit (lhβ), follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr) mRNA. In addition, down-regulated transcription of fshβ and lhβ in the SMC exposure groups was affected by reduced mRNA levels of seabream gonadotropin-releasing hormone (sbgnrh), g-protein-coupled receptor 54 (gpr54) in the kisspeptin/gpr54 system, as well as the gamma-aminobutyric acid (GABA) synthesis enzyme glutamic acid decarboxylase (gad). Overall, our results showed that environmentally relevant concentrations of SMC exerted anti-androgenic effects in male flounders via impacting HPG axis, kiss/gpr54 system and GABA synthesis, providing theoretical support for investigating reproductive toxicity of environmental pollutants that interfere with the neuroendocrine system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Seafood represent an important food source for human, and seafood quality is associated with marine environment quality. PAHs are one of the main organic environmental contaminants and they can be introduced into the body through different way (ingestion, inhalation, dermal absorption). We present data on bioaccumulation of the sixteen PAHs, defined priority by the U.S.- EPA, in Sardina pilchardus, Solea solea and Donax trunculus, three species caught in the Catania Gulf and highly consumed by the local population. The risk to develop chronic systemic and carcinogenic effects due to the consumption of these target species was evaluated through the EDI, THQ and CR. EDI derived from D. trunculus ingestion falls within the range calculated by the EFSA. The THQ is less than 1, and the CR calculated for the Benzo(a)Pyrene is at the limit of the ARL (1✕10 -5 ). EDI derived from S. pilchardus and S. solea ingestion are below the range calculated by the EFSA. The THQ is less than 1, and the CR is below the acceptable risk level. The contamination level found in local seafood determines a low risk to develop chronic systemic effects, but the cancer risk could be of health concern especially for high-frequency molluscs consumers., (Copyright © 2018. Published by Elsevier Ltd.)

Impacted marine environments lead to metal accumulation in edible marine fish, ultimately impairing human health. Nevertheless, metal accumulation is highly variable among marine fish species. In addition to ecological features, differences in bioaccumulation can be attributed to species-related physiological processes, which were investigated in two marine fish present in the Canary Current Large Marine Ecosystem (CCLME), where natural and anthropogenic metal exposure occurs. The European sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis were exposed for two months to two environmentally realistic dietary cadmium (Cd) doses before a depuration period. Organotropism (i.e., Cd repartition between organs) was studied in two storage compartments (the liver and muscle) and in an excretion vector (bile). To better understand the importance of physiological factors, the significance of hepatic metallothionein (MT) concentrations in accumulation and elimination kinetics in the two species was explored. Accumulation was faster in the sea bass muscle and liver, as inferred by earlier Cd increase and a higher accumulation rate. The elimination efficiency was also higher in the sea bass liver compared to sole, as highlighted by greater biliary excretion. In the liver, no induction of MT synthesis was attributed to metal exposure, challenging the relevance of using MT concentration as a biomarker of metal contamination. However, the basal MT pools were always greater in the liver of sea bass than in sole. This species-specific characteristic might have enhanced Cd biliary elimination and relocation to other organs such as muscle through the formation of more Cd/MT complexes. Thus, MT basal concentrations seem to play a key role in the variability observed in terms of metal concentrations in marine fish species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Essential nutrients are critical for physiological processes of organisms. In fish, they are obtained primarily from the diet, and their transfer and accumulation are known to be impacted by environmental variables such as water temperature, pH and salinity, as well as by diet composition and matrices. Yet, prey items consumed by fish may also contain toxic compounds such as marine toxins associated with harmful algae. These biotoxins have the potential to affect essential trace element assimilation in fish through chemical interactions such as the formation of trace element-toxin complexes or by affecting general fish physiology as in the modification of ion-specific transport pathways. We assessed the influence of dietary exposure to brevetoxins (PbTxs), ichthyotoxic neurotoxins produced by the dinoflagellate Karenia brevis, on trophic transfer of two essential trace elements, Mn and Zn, in a fish model. Using ecologically relevant concentrations of PbTxs and trace elements in controlled laboratory conditions, juvenile turbots Scophthalmus maximus were given food containing PbTxs before or at the same time as a feeding with radiotracers of the chosen essential elements ( 54 Mn and 65 Zn). Treatments included simultaneous exposure (PbTxs +  54 Mn +  65 Zn) in a single-feeding, 3-week daily pre-exposure to dietary PbTx followed by a single feeding with 54 Mn and 65 Zn, and a control ( 54 Mn and 65 Zn only). After a 21-day depuration period, turbot tissue brevetoxin levels were quantified and assimilation efficiencies of 54 Mn and 65 Zn were assessed. PbTxs were found in turbot tissues in each exposure treatment, demonstrating dietary trophic transfer of these toxins; yet, no differences in assimilation efficiencies of Mn or Zn were found between treatments or the control (p > 0.05). These results indicate that, in our experimental conditions, PbTx exposure does not significantly affect the trophic transfer of Mn and Zn in fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Studying dietary metal transfer kinetics is essential to gain a better understanding in global metal accumulation rates and its impacts in marine fish. While there exists a solid understanding on the influence of various biotic factors on this transfer, metal assimilation in fish might be also affected by abiotic factors, as has been observed in marine invertebrates. The present study therefore aims to understand the potential effects of two climate-related master variables, temperature and pH, on the assimilation efficiency (AE) of essential (Co and Zn) and non-essential (Ag) metals in the turbot Scophthalmus maximus using radiotracer tools. Juvenile turbots were acclimated for 8 weeks at two temperatures (17 and 20 °C) and pH (7.5 and 8.0) regimes, under controlled laboratory conditions, and then fed with radiolabelled shrimp ( 57 Co, 65 Zn and 110m Ag). Assimilation efficiencies of Co and Ag in juvenile turbot, determined after a 21-day depuration period, were not affected by pre-exposition to the different environmental conditions. In contrast, temperature did significantly influence Zn AE (p < 0.05), while pH variations did not affect the assimilation of any of the metals studied. In fact, temperature is known to affect gut physiology, specifically the membrane properties of anterior intestine cells where Zn is adsorbed and assimilated from the ingested food. These results are relevant to accurately assess the influence of abiotic factors in AEs of metals in fish as they are highly element-dependent and also modulated by metabolic processes.

The turbot, Scophthalmus maximus, belongs to the flatfishes (order Pleuronectiformes), which display substantial asymmetry of the olfactory organs and forebrain. Sox genes code for SRY-related HMG domain-bearing transcription factors involved in various developmental processes. Group B1 Sox genes as Sox2 and Sox19 appear to play major roles in neural development. Here, we characterized by in situ hybridization the developmental expression of Sox2 and Sox19 genes in metamorphic and postmetamorphic specimens and young adults of both sexes. Expression of S. maximus Sox2 (Sm-Sox2) and Sm-Sox19 mRNAs was detected in ependymal cells of different regions of the telencephalon, preoptic region, hypothalamus, and thalamus at all stages investigated. Sm-Sox2 expression but not Sm-Sox19 occurred in neurons located in particular regions such as the dorsal nucleus of the ventral telencephalon, the medial zone of the dorsal telencephalon, preoptic area and hypothalamus. Although Sm-Sox2 and Sm-Sox19 are expressed differentially in gonads, no sex differences in their expression were observed between male and female forebrains. We also investigated the topographical relation between Sox expression and cell proliferation using series double immunostained for a radial glial marker (BLBP) and cell proliferation marker (PCNA). Sm-Sox2 and Sm-Sox19 were strongly expressed in ependymal cells located in neurogenic niches revealed by the BLBP and PCNA immunostaining. Comparison with other teleosts indicates similar expression of Sox2 and Sox19 in the telencephalon, supporting conserved roles for both genes in teleost brains., (© 2017 Wiley Periodicals, Inc.)

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

R-spondin 1 (Rspo1) is a potential female-determining gene in mammals that could regulate the Wnt/β-catenin signaling pathway. The deletion of Rspo1 causes sex reversal in females. To investigate sexual determination and differentiation, we cloned and analyzed the Rspo1 gene in Cynoglossus semilaevis. Phylogenetic and gene structure analyses revealed that Rspo1 gene exhibited high sequence conservation and contained an N-terminal signal peptide, two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat, and a C-terminal region enriched with basic charged amino acids. qRT-PCR revealed that Rspo1 expressed sexual dimorphism in gonad, with higher expression levels in the ovary than in the testis, thus, suggesting the involvement of Rspo1 in gonad differentiation. In situ hybridization results demonstrated that Rspo1 was expressed in premature germ cells, including spermatogonia and spermatocytes in the testis and stage II and stage III oocytes in the ovary. The methylation levels in two CpG sites of Rspo1 promoter significantly differed among females, males, and pseudomales. After 30days of exposure to high temperature, the expression of Rspo1 significantly decreased in female individuals, some of which were prone to males. However, no difference of Rspo1 gene expression was observed between the control group and high-temperature group in males. These preliminary findings suggested that Rspo1 played a crucial role in sex determination and development. This study laid the groundwork for further sex control breeding techniques in C. semilaevis., (Copyright © 2017. Published by Elsevier B.V.)

Recently, gonadotropin-inhibitory hormone (GnIH) has emerged as an important regulator of reproduction in birds and mammals. This RFamide neuropeptide has neuromodulatory functions and controls the synthesis and/or release of gonadotropin-releasing hormone (GnRH) and gonadotropins. Although teleosts represent about half of all living vertebrates, scientific and technological advances on the Gnih system in fish are scarce, contradictory, and inconclusive. Research on the fish Gnih system appears necessary to better clarify its role in the neuroendocrine and environmental control of vertebrate reproduction. In this study, we cloned a full-length sequence for the Gnih precursor of a flatfish, the Senegalese sole, coding for three putative Gnih peptides (ssGnih). We also generated specific antibodies against these ssGnih peptides, and used them to localize Gnih cells and their projections in the brain and pituitary. The expression of gnih was particularly evident in the diencephalon, but also in the olfactory bulbs/cerebral hemispheres, optic tectum/tegmentum, retina, and pituitary. The three antibodies used provided consistent results and showed that ssGnih-immunoreactive perikarya were present in the olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum and rostral rhombencephalon, and their fibers innervated the brain and pituitary profusely. Intramuscular injection of ssGnih-3 provoked a significant reduction in gnrh-3 and lh expression, whereas ssGnih-2 treatment did not affect transcript levels of the main reproductive genes. Our results reveal the existence of a functional Gnih system in the sole brain, profusely innervating different brain areas and the pituitary gland, which could represent an important factor in the neuroendocrine control of flatfish reproduction., (© 2017 Wiley Periodicals, Inc.)