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This field study investigates the morphological indices (condition index, hepatosomatic index) and biochemical (catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), metallothionein (MT), lipid peroxidation) parameters in liver, gills and kidney of common sole (Solea solea) originating from different sites of the Tunisian coast area impacted by different anthropogenic activities. Differences among sites and tissues for AChE, GST, CAT, MT and TBARS were found and possibly related to known sources of domestic and industrial discharges in the studied sites. Liver, gills and kidney CAT, liver and kidney MT and brain AChE were key biomarkers to discriminate fish of different sites. So, we suggest using these biomarkers in future biomonitoring.

Abnormal pigmentation of Senegalese sole has been described as one problem facing the full exploitation of its commercial production. To improve our understanding of flatfish pigmentation of this commercially important species we have evaluated eleven genes related to two different processes of pigmentation: melanophore differentiation, and melanin production. The temporal distribution of gene expression peaks corresponds well with changes in pigmentation patterns and the intensity of skin melanization. Several gene ratios were also examined to put in perspective possible genetic markers for the different stages of normal pigmentation development. Further, the phenotypic changes that occur during morphogenesis correspond well with the main transitions in gene expression that occur. Given the dramatic phenotypic alterations which flatfish undergo, including the asymmetric coloration that occurs between the ocular and the blind side, and the synchrony of the two processes of morphogenesis and pigmentation ontogenesis, these species constitute an interesting model for the study of pigmentation. In this study we present a first approximation towards explaining the genetic mechanisms for regulating pigmentation ontogeny in Senegalese sole, Solea senegalensis.

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The common sole, Solea solea (Linneus, 1758), and the Senegalese sole, Solea senegalensis (Kaup, 1858), are two important commercial species that coexist in the NW Mediterranean. In order to assess the species' ability to respond to chemical insults, a comparison of activities on enzymes involved in xenobiotic metabolism was carried out. Juveniles of both species were sampled in winter 2011 from the Ebro Delta region, and activities of selected enzymes such as acetylcholinesterase (AChE), carboxylesterase (CbE), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) were determined in several tissues. Lipid peroxidation (LP) levels in plasma were measured as a sign of oxidative stress. In vitro exposures to selected pesticides were contrasted, analysing AChE and CbE activities in several tissue homogenates. Overall, enzymatic activities were higher in S. solea except for gill GST and CbE and kidney GST, while plasmatic LP levels were similar. In vitro contrasts revealed lower IC50 values for CbE activities in S. solea, suggesting a greater buffer capacity of this enzyme to potentially reduce pesticide toxicity over AChE.

We characterized the expression patterns of pepsinogen A (tPGA) and pepsinogen C (tPGC) in turbot (Scophthalmus maximus). Quantitative expression analysis showed that tPGC was preferentially expressed in early developmental stages, and that the tPGA mRNA expression level was higher in adult fish. Full-length cDNA constructs of tPGA and tPGC were 1307 bp (from which 377 amino acids were deduced); and 1430 bp (from which 385 amino acids were deduced), respectively. The deduced proteins of tPGA and tPGC possessed signal peptides of 17 amino acids and 20 amino acids respectively. The initial transcripts of tPGA and tPGC were detected at 22 days post hatching (dph), well after the formation of gastric glands (16 dph). This suggested that the morphologic development of gastric glands was not synchronous with their functional development. In addition, tPGA and tPGC mRNAs were also expressed in muscle and ovary at much lower levels than in stomach and esophagus. The distribution of tPGA and tPGC in the turbot was investigated using in-situ hybridization, and tPGA and tPGC were first detected in the esophagus and cardiac region of the stomach, and then throughout the stomach., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Previous experiments with Senegalese sole (Solea senegalensis) have demonstrated that dietary lipid levels above 8% impaired growth and did not promote protein retention. We hypothesised that this low ability to use high-lipid diets may depend on the dietary protein level. In the present study, a 2 × 2 factorial design was applied where two dietary lipid (4-17% DM) and two dietary protein (below and above the requirement levels, 48 and 54% DM) levels were tested in juveniles for 114 d. Growth performance was not improved by the increase in dietary fat, irrespectively of the dietary protein levels. Protein retention was similar among the diets, although fish fed the diets with high lipid content resulted in significantly lower protein gain. Among the enzymes involved in amino acid catabolism, only aspartate aminotransferase activity in the liver was affected by the dietary lipid levels, being stimulated in fish fed high-lipid diets. Moreover, phosphofructokinase 1 activity was significantly elevated in the muscle of Senegalese sole fed 4% lipid diets, suggesting enhanced glycolysis in the muscle when the dietary lipid supply was limited and dietary starch increased. The results confirmed that high-lipid diets do not enhance growth, and data from the selected enzymes support the assumption that lipids are not efficiently used for energy production and protein sparing, even when dietary protein is below the protein requirement of the species. Furthermore, data suggest a significant role of glucose as the energy source in Senegalese sole.

Dietary long chain polyunsaturated fatty acids (FA) have been recognized of crucial importance in early development of vertebrates, contributing to the impressive morphological and physiological changes both as building blocks and to energy production. The importance of lipids along development depends on ontogenetic, phylogenetic and environmental parameters. The expression patterns of FA metabolism genes have not been characterized in developing fish embryos nor compared to lipid class profiles. Full lipid metabolism only occurred after hatching, as revealed by alterations in lipid profiles and FA gene expression. Nonetheless, transcriptional changes of some FA genes were already present in embryos at notochord formation. Many genes displayed an expression profile opposed to the decrease of lipids along the development, while others responded solely to starvation. Transcription of most genes involved in FA metabolism had a strong correlation to PPARs' mRNA levels (α1, α2, β, γ). The comparison of mRNA expression of the genes with the lipid profiles produced new insights into the FA metabolism and regulation during the development of turbot larvae, providing the basis for future studies including comparative approaches with other vertebrate species., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Myogenin (myog) encodes a highly conserved myogenic regulatory factor that is involved in terminal muscle differentiation. It has been shown in mammals that methylation of cytosines within the myog promoter plays a major role in regulating its transcription. In the present study, the Senegalese sole (Solea senegalensis) myog putative proximal promoter was identified and found to be highly conserved among teleosts. Therefore, it is plausible that it plays a similar role in controlling myog expression. Cytosine methylation of the myog promoter in skeletal muscle of Senegalese sole larvae undergoing metamorphosis was influenced by rearing temperature. A lower temperature (15 °C) significantly increased myog promoter methylation in skeletal muscle, particularly at specific CpG sites, relatively to higher rearing temperatures (18 and 21 °C). Myog transcription was downregulated at 15 °C, whereas expression of dnmt1 and dnmt3b was upregulated, consistently with the higher myog methylation observed at this temperature. Rearing temperature also affected growth and fast muscle cellularity, producing larger fibers at 21°C. Taken together, our data provide the first evidence of an epigenetic mechanism that may be underlying the temperature-induced phenotypic plasticity of muscle growth in teleosts.

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.

To study the effects and possible mechanisms of suspected endocrine disrupting compounds (EDCs), a wide variety of assays have been developed. In this work, we generated engineered Escherichia coli biosensor strains that incorporate the ligand-binding domains (LBDs) of the β-subtype estrogen receptors (ERβ) from Solea solea (sole), and Sus scrofa (pig). These strains indicate the presence of ligands for these receptors by changes in growth phenotype, and can differentiate agonist from antagonist and give a rough indication of binding affinity via dose-response curves. The resulting strains were compared with our previously reported Homo sapiens ERβ biosensor strain. In initial tests, all three of the strains correctly identified estrogenic test compounds with a high degree of certainly (Z' typically greater than 0.5), including the weakly binding test compound bisphenol A (BPA) (Z' ≈ 0.1-0.3). The modular design of the sensing element in this strain allows quick development of new species-based biosensors by simple LBD swapping, suggesting its use in initial comparative analysis of EDC impacts across multiple species. Interestingly, the growth phenotypes of the biosensor strains indicate similar binding for highly estrogenic control compounds, but suggest differences in ligand binding for more weakly binding EDCs., (Copyright © 2011 Wiley Periodicals, Inc.)

Rationale: The oxygen isotope ratio (δ(18)O value) of aragonite fish otoliths is dependent on the temperature and the δ(18)O value of the ambient water and can thus reflect the environmental history of a fish. Secondary ion mass spectrometry (SIMS) offers a spatial-resolution advantage over conventional acid-digestion techniques for stable isotope analysis of otoliths, especially given their compact nature., Methods: High-precision otolith δ(18)O analysis was conducted with an IMS-1280 ion microprobe to investigate the life history of a yellowfin sole (Limanda aspera), a Bering Sea species known to migrate ontogenetically. The otolith was cut transversely through its core and one half was roasted to eliminate organic contaminants. Values of δ(18)O were measured in 10-µm spots along three transects (two in the roasted half, one in the unroasted half) from the core toward the edge. Otolith annual growth zones were dated using the dendrochronology technique of crossdating., Results: Measured values of δ(18)O ranged from 29.0 to 34.1‰ (relative to Vienna Standard Mean Ocean Water). Ontogenetic migration from shallow to deeper waters was reflected in generally increasing δ(18)O values from age-0 to approximately age-7 and subsequent stabilization after the expected onset of maturity at age-7. Cyclical variations of δ(18)O values within juvenile otolith growth zones, up to 3.9‰ in magnitude, were caused by a combination of seasonal changes in the temperature and the δ(18)O value of the ambient water., Conclusions: The ion microprobe produced a high-precision and high-resolution record of the relative environmental conditions experienced by a yellowfin sole that was consistent with population-level studies of ontogeny. Furthermore, this study represents the first time that crossdating has been used to ensure the dating accuracy of δ(18)O measurements in otoliths., (Copyright © 2013 John Wiley & Sons, Ltd.)

Fertilised eggs of Senegalese sole were incubated at 15, 18 or 21 °C, and after hatching all larvae were reared at 21 °C until 30 days post-hatch. By this point larvae from the 18 or 21 °C temperature groups had 11 and 9% more muscle fibres than those from 15 °C, respectively. Hyperplastic growth during metamorphosis was higher in larvae from 18 °C. Embryonic temperature induced gene expression changes, albeit with a variable pattern throughout development. Myf5, myod2, myHC and fst mRNA levels were significantly higher at several stages prior to hatching in embryos incubated at 21 °C, whereas hsp90AB and hsp70 transcripts were present at higher levels in the 15 °C group. Myf5, myod1, myod2, pax7, myog, fst, igf-II, igf1r, hsp90AA and hsp90AB were expressed at higher levels during early development, particularly during somitogenesis. In contrast, mrf4, myHC, mylc2, igf-I, mstn1 and hsp70 were up-regulated at later stages of larval development, namely during and after metamorphosis. This study is the first example of thermal plasticity of myogenesis with prolonged effect in a flatfish., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The objective of the present study was to evaluate the modulation of acute stress response by dietary nucleotides (NT) in sole, Solea solea. A basal diet was supplemented with levels of 0 (normal diet), or 0.4 g NT/kg dry diet for 8 weeks. At the end of feeding trial, fish fed the normal and NT-supplemented diet were subjected to a standardized protocol of disturbance and sampled over a 24h recovery after the stressor exposure. Modulatory effects of NT on acute stress response (cortisol and glucose), proopiomelanocortin (POMC) and cannabinoid receptor 1 splice variants (CB1A and CB1B) mRNA levels were studied. Both plasma cortisol and glucose levels of fish fed NT-supplemented diet were significantly lower than fish fed the control diet at 1 and 4h post-stress time-points. There are no significant effects of dietary NT on POMC and HSP70 mRNA levels. In our study, both CB1A and CB1B trascript levels were induced in fish fed the normal diet at 1 and 4h post-stress intervals. Collectively, the results obtained suggest that dietary NT modulates the CB1-like receptor mRNA expressions leading to attenuation in stressor-induced plasma cortisol level in sole., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Antimicrobial peptide plays an important role in fish immunity. The small molecular antimicrobial peptide Hepcidin in turbot was studied and reported in this paper. The Ferroportin 1 (FPN1) and Transferrin Receptor (TFR) genes, which are related to Hepcidin, were cloned in turbot. The characteristics of Hepcidin and its related genes were studied, including an analysis of the expression patterns and cloning of the Hepcidin promoter, the relationship between Hepcidin and NF-κB and the regulation of iron-metabolism. The results showed that the promoter of SmHepcidin contains the binding sites of NF-κB, and NF-κB may directly or indirectly receive feedback signals from SmHepcidin. In the liver, spleen and kidney, in which there was an increased SmHepcidin expression level, SmFPN1 dramatically decreased and SmTFR was also either decreased or exhibited no obvious change after bacterial/viral infection and an injection of exogenous Hepcidin protein. RNAi experiments in turbot kidney cells confirmed the expression changes of these gene patterns. Furthermore, the administration of exogenous Hepcidin protein, which regulates the level of chelatable iron in cells, further confirmed the function of Hepcidin in iron metabolism. It is speculated that the rapidly increased expression of SmHepcidin may induce changes in the expression of related genes, and that the in vivo chelatable iron concentration which participates in the antibacterial process was also changed when exogenous pathogens are present in turbot. It is suggested that SmHepcidin plays a defensive role against pathogenic infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sediment polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCls) and polybrominated diphenyl ethers (PBDEs) were measured during winter 2011 at seven sites on the Catalan coast (NW Mediterranean). Also at these sites, the benthic fish Solea solea and Solea senegalensis were fished for biomarker analysis. Chemical concentrations did not exceed the values considered safe by sediment quality guidelines (SQGs) for PAHs. However, levels of DDT (94.5 ng/g d.w.) and PCBs (15.1 ng/g d.w.) were significant in the Ebre Delta. Nonspecific immune parameters in S. solea showed a positive correlation between lysozyme activity in plasma and PAH content in sediment (r=0.982, p<0.01) and the opposite trend occurred with the complement test (ACH50) (r=-0.837; p<0.05). Cell counts (monocytes, lymphocytes and granulocytes) were elevated at most PAH-affected sites. A high concentration of bile FACs (fluorescent aromatic compounds) occurred at the northern station., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pentraxins are a family of evolutionarily conserved proteins that play an important part in innate immunity. C-reactive protein (CRP) is a member of the pentraxin family and in humans is known to be the major acute phase protein. In this work, we report the identification and analysis of a CRP, CsCRP, from half-smooth tongue sole (Cynoglossus semilaevis). CsCRP is composed of 228 amino acid residues and possesses a Pentraxin/CRP domain. Expression of CsCRP occurred in a wide range of tissues and was upregulated by pathogen infection in kidney, spleen, blood, and, in particular, liver. Following bacterial infection, CsCRP level in blood rose rapidly within 12 h and was approximately 3.8 fold of that of the basal level. Purified recombinant CsCRP (rCsCRP) was able to interact with Gram-negative and Gram-positive bacteria including those of pathogenic nature in a dose-dependent manner. When peripheral blood leukocytes (PBL) were infected with bacterial pathogen in the presence of rCsCRP, the respiratory burst and phagocytic capacity of the cells were increased to significant extents. Taken together, these results indicate that CsCRP is an acute phase protein that plays a role in innate immune defense against bacterial infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

In this study, laboratory experiments were carried out in order to come to a better understanding of the fate of polycyclic aromatic hydrocarbons (PAHs) in the marine environment and especially on their bioaccumulation, biotransformation and genotoxic effects in fish. Juveniles of turbot (Scophthalmus maximus) were exposed to PAHs through different routes via (1) a mixture of dissolved PAHs, (2) a PAH-polluted sediment and (3) an oil fuel elutriate. Fish were exposed 4 days followed by a 6-day depuration period. In each experiment, PAH concentrations in the seawater of the tanks were analysed regularly by gas chromatography coupled with mass spectrometry. Muscle and liver samples were also analysed for parent PAH levels and PAH bioconcentration factors were calculated. Biotransformation was evaluated by measuring the levels of PAH metabolites in fish bile. Genotoxicity was assessed by the alkaline comet assay. Regardless of exposure route, the parent PAH concentrations in the liver and muscle showed a peak level 1 day after the beginning of the exposure, followed by a decrease up to the background level towards the end of the experiment, except for the exposure to dissolved PAHs for which levels were relatively low throughout the study. As a consequence, no bioaccumulation was observed in fish tissues at the end of the experiment. In contrast, regardless of exposure routes, a rapid production of biliary metabolites was observed throughout the whole exposure experiment. This was especially true for 1-hydroxypyrene, the major metabolite of pyrene. After 6 days of recovery in clean water, a significant decrease in the total metabolite concentrations occurred in bile. Fish exposed through either route displayed a significant increase in DNA strand breaks after 4 days of exposure, and significant correlations were observed between the level of biliary PAH metabolites and the level of DNA lesions in fish erythrocytes. Overall results indicate that exposure to either a mixture of dissolved PAHs, a PAH-contaminated sediment or a dispersed oil fuel elutriate leads to biotransformation and increase in DNA damage in fish. The quantification of PAH metabolites in bile and DNA damage in erythrocytes appear to be suitable for environmental monitoring of marine pollution either in the case of accidental oil spills or sediment contamination.

Information on the occurrence of persistent organic pollutants (POPs) in fish from Tunisia is scarce. In this study, thirty one persistent organic pollutants including organochlorine pesticides (OCPs) (dichlorodihenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB)), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) were determined in solea solea muscle, from Bizerte Lagoon (northern Tunisia) and from the Mediterranean Sea (reference area) (northern Mediterranean). In the Bizerte Lagoon, contaminant concentrations generally followed this order: PCBs>DDTs>PBDEs>MeO-PBDEs>HCB>HCHs; while in the Mediterranean Sea, pollutant concentration followed this order: MeO-PBDEs>PCBs>DDTs>PBDEs>HCB>HCHs. Mean levels of organochlorine compounds were 1018 and 380 ng g(-1) lipid weight (lw) in fish from Bizerte Lagoon and the Mediterranean Sea, respectively. Mean concentrations of organobromine compounds were 279 and 301 ng g(-1) lw in sole from Bizerte Lagoon and the Mediterranean Sea, respectively. Organohalogen concentrations in fish from Bizerte Lagoon were similar or slightly lower than those reported for other marine fish species from other locations around the world. PCB, HCH, HCB and PBDE levels were negatively correlated with lipid content, while no such correlation was seen for DDTs. Assessment based on several available guidelines suggested an insignificant human health risk for dietary intake of HCB, lindane and PBDEs associated with consumption of sole. However, the estimated lifetime cancer risk from dietary exposure to DDTs and PCBs is a potential concern., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Coastal habitats play a major role as nurseries for many fish species; however, they are also submitted to pollutants and oxygen fluctuations. Fry's concept of metabolic scope for activity was used to evaluate the effect of polychlorinated biphenyls (PCBs) on the aerobic metabolism in juvenile common sole (0-1 year old). Aerobic metabolic scope (AMS) in control and PCB-contaminated fish via food pathway was determined using respirometry techniques. Furthermore, the hypoxia tolerance in control and PCB-contaminated fish was evaluated by assessing their critical oxygen concentration (O(2crit)). Our results showed that while PCB-contaminated fish were able to maintain a constant AMS and O(2crit), PCBs tend to affect their aerobic metabolism by acting on maximal oxygen consumption (MO(2max)) in hypoxia and standard metabolic rate, but only at the highest PCB concentration between 30 and 60 days of exposure. In conclusion, we can hypothetise that the tested PCB-exposures may not impair the tolerance to hypoxia and the survival of common sole in their natural environment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Edwarsiella tarda is highly resistant to the action of cationic antimicrobial peptides (CAMPs). However, the mechanism underlying CAMP resistance is not clear. The enzyme UDP-glucose dehydrogenase (Ugd) that converts UDP-glucose into UDP-glucuronic acid may be important for this resistance. In this study, a ugd gene was identified in E. tarda and its functional role was analyzed using an in-frame deletion mutant Δugd and the complemented strain ugd+. The lipopolysaccharide (LPS) produced by Δugd consisted of a truncated core oligosaccharide (OS) with no O-antigen attached. The ugd mutant was extremely sensitive to CAMPs, presumably because of alterations in LPS structure. The mutant also exhibited enhanced autoaggregation and biofilm formation and reduced hemolytic activity. Using different infection models we found that Δugd was impaired in survival within macrophages and displayed significantly attenuated virulence and an impaired ability to persist within the host. The expression of ugd was induced by polymyxin B and under the control of PhoP and RcsB, two response regulators of the bacterial two-component systems that we identified previously. Moreover, vaccination of turbot (Scophthalmus maximus) with Δugd by intraperitoneal injection elicited significant protection against the wild-type E. tarda strain, suggesting that Δugd may be promising as a potential vaccine candidate against edwardsiellosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Senegalese sole, a high-value flatfish, is a good candidate for aquaculture production. Nevertheless, there are still issues regarding this species' sensitivity to stress in captivity. We aimed to characterize the hepatic proteome expression for this species in response to repeated handling and identify potential molecular markers that indicate a physiological response to chronic stress. Two groups of fish were reared in duplicate for 28 days, one of them weekly exposed to handling stress (including hypoxia) for 3 min, and the other left undisturbed. Two-dimensional electrophoresis enabled the detection of 287 spots significantly affected by repeated handling stress (Wilcoxon-Mann-Whitney U test, p < 0.05), 33 of which could be reliably identified by peptide mass spectrometry. Chronic exposure to stress seems to have affected protein synthesis, folding and turnover (40S ribosomal protein S12, cathepsin B, disulfide-isomerase A3 precursor, cell-division cycle 48, and five distinct heat shock proteins), amino acid metabolism, urea cycle and methylation/folate pathways (methionine adenosyltransferase I α, phenylalanine hydroxylase, mitochondrial agmatinase, serine hydroxymethyltransferase, 3-hydroxyanthranilate 3,4-dioxygenase, and betaine homocysteine methyltransferase), cytoskeletal (40S ribosomal protein SA, α-actin, β-actin, α-tubulin, and cytokeratin K18), aldehyde detoxification (aldehyde dehydrogenase 4A1 family and aldehyde dehydrogenase 7A1 family), carbohydrate metabolism and energy homeostasis (fatty acid-binding protein, enolase 3, enolase 1, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, aconitase 1, mitochondrial ATP synthase α-subunit, and electron-transfer flavoprotein α polypeptide), iron and selenium homeostasis (transferrin and selenium binding protein 1), steroid hormone metabolism (3-oxo-5-β-steroid 4-dehydrogenase), and purine salvage (hypoxanthine phosphoribosyltransferase). Further characterization is required to fully assess the potential of these markers for the monitoring of fish stress response to chronic stressors of aquaculture environment.

To investigate the occurrence and bioaccumulation of organic contaminants of emerging concern (CECs) near four major wastewater ocean outfalls in the Southern California Bight, more than 75 pharmaceutical and personal care products, current-use pesticides, and industrial/commercial chemicals were analyzed in sediment and liver tissues of hornyhead turbot (Pleuronichthys verticalis) using gas and liquid chromatography-mass spectrometry. Although most CECs targeted were infrequently detected or not detectable, triclosan, 4-nonylphenol (4-NP) and bis(2-ethylhexylphthalate) were detected in all sediments at median (maximum) concentrations of 5.1 (8.6), 30 (380), and 121 (470) µg/kg, respectively. In the liver, 4-NP and polybrominated diphenyl ether (PBDE) congeners 47 and 99 were detected in >90% of samples at median (maximum) concentrations of 85 (290) and 210 (480) µg/kg, respectively. The sedative diazepam was detected in all liver samples, but was infrequently detected in sediments. Sediment and liver concentrations across outfall locations ranged over several orders of magnitude and were elevated relative to a reference site. Relative to sediment, accumulation in liver of PBDEs 47 and 99 was comparable to that for legacy organochlorines, confirming their high bioaccumulation potential and suggesting their inclusion in future tissue monitoring studies. Mean tissue PBDE and diazepam concentrations were higher in livers from male versus female P. verticalis, suggesting that gender differences also be considered in designing such studies., (Copyright © 2012 SETAC.)

4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic-pituitary-interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17β-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC) mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7 day, to either 10(-6) or 10(-8) M 4-NP, or 10(-8) M E2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Effects of 55 and 45% dietary protein levels (55P and 45P diets, respectively) and temperature (12 and 18 °C) on hepatic activity of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase and lipid peroxidation (LPO) levels of Solea senegalensis juveniles were studied. Further, effects of acute thermal shocks provoked by a drop (18 °C to 12 °C) or a rise (12 °C to 18 °C) of water temperature on sole oxidative state was also evaluated. Dietary protein reduction increased LPO levels though no major alterations were found on antioxidant enzyme activities between dietary treatments. At 12 °C GR activity was higher and SOD activity was lower than 18 °C but LPO levels were not affected. In both thermal shock cases, LPO levels increased in 55P group, probably due to insufficient antioxidant enzyme activation. In contrast, fish of 45P group under acute exposition to warmer and colder temperature exhibited no substantial changes and a significant decrease on LPO levels, respectively, along with no major changes in antioxidant enzymes. Overall, results suggest that independently of rearing temperatures 45P group was more susceptible to oxidative stress than 55P group. Thermal shock either due to rise or drop of temperature seemed to induce oxidative stress in 55P group., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Production of all-female populations in turbot can increase farmer's benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3-6 months earlier than males. Puberty in males occurs earlier than in females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production. A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21. In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b). We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species. Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish. Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming.

For several decades, perfluorooctane sulfonate (PFOS) has widely been used as a fluorinated surfactant in aqueous film forming foams used as hydrocarbon fuel fire extinguishers. Due to concerns regarding its environmental persistence and toxicological effects, PFOS has recently been replaced by novel fluorinated surfactants such as Forafac®1157, developed by the DuPont company. The major component of Forafac®1157 is a 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB), and a link between the trade name and the exact chemical structure is presented here to the scientific community for the first time. In the present work, the structure of the 6:2 FTAB was elucidated by (1)H, (13)C and (19)F nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Moreover, its major metabolites from blue mussel (Mytilus edulis) and turbot (Scophthalmus maximus) and its photolytic transformation products were identified. Contrary to what has earlier been observed for PFOS, the 6:2 FTAB was extensively metabolized by blue mussel and turbot exposed to Forafac®1157. The major metabolite was a deacetylated betaine species, from which mono- and di-demethylated metabolites also were formed. Another abundant metabolite was the 6:2 fluorotelomer sulfonamide. In another experiment, Forafac®1157 was subjected to UV-light induced photolysis. The experimental conditions aimed to simulate Arctic conditions and the deacetylated species was again the primary transformation product of 6:2 FTAB. A 6:2 fluorotelomer sulfonamide was also formed along with a non-identified transformation product. The environmental presence of most of the metabolites and transformation products was qualitatively demonstrated by analysis of soil samples taken in close proximity to an airport fire training facility., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mercury concentrations in three flatfish species - flounder (Platichtys flesus), plaice (Pleuronectes platessa), and Baltic turbot (Scophthalmus maximus), netted in the southern Baltic Sea were assessed and compared to concentrations of this metal in sediments, sea water, and flatfish food - bivalve Macoma balthica, isopod Saduria entomon, and sprat (Sprattus sprattus). Collected simultaneously with flatfish in 2009 and 2010. Different concentrations of mercury depending on species, tissue or organ, sex, individual length, kind of food, and region were determined. The muscle tissues of turbot had the highest concentrations of the metal. The bioaccumulation (BF) and biomagnification (BMF) factors has been counted showing that the muscle tissues of turbot have maximum affinity for mercury, and thus best reflected the metal contamination of the Baltic Sea environment. The data suggest that the common Baltic turbot (S. maximus) is an important model species, suitable and cost-effective to biomonitor environmental mercury pollution for ecological research., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5-10.8% of fat) followed by fins (3.4-6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4-4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n-6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n-6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish - the muscle., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Overlapping external morphometric characters easily confound the flatfishes Solea aegyptiaca and Solea solea (Soleidae) in areas of the Mediterranean Sea where both species live in sympatry. This leads to uncertainties in the fisheries and marketing of the species, in addition to misinterpretations in biogeography and conservation studies. This paper describes a simple restriction fragment length-based diagnostic test that differentiates S. solea from S. aegyptiaca, as well as from other species of the Soleidae family. Furthermore, the two species living in sympatry in the Gulf of Kavala (North Aegean Sea, Greece) present significant qualitative differences in muscle fatty acid composition, a property that can also be used to distinguish the two cryptic species.

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

Kisspeptin is thought to have a major role in the control of the onset of puberty in vertebrates. However, our current understanding of its function in fish and how it integrates with other hormones is incomplete due to the high diversity of this group of animals and a still limited amount of available data. This study examined the temporal and spatial changes in expression of kisspeptin, gonadotropins and their respective receptors in the Senegalese sole during a full reproductive cycle. Kiss2 and kiss2r expression was determined by qRT-PCR in the forebrain and midbrain while expression of fshβ and lhβ was determined in the pituitary and fshr and lhr in the gonads. Plasma levels of testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β were measured by ELISA and gonadal maturation was assessed histologically. In males, kiss2 and kiss2r expression in the brain areas examined was highest towards the end of winter, just before the spawning season, which took place the following spring. This coincided with maximum levels of pituitary fshβ and lhβ, plasma T and 11-KT and the highest number of maturing fish. However, these associations were not evident in females, since the highest expression of kiss2, kiss2r and gonadotropins were observed in the fall, winter or spring, depending upon the variable and tissue considered. Taken together, these data show not only temporal and spatial, but also sex-specific differences in the expression of kisspeptin and its receptor. Thus, while expression of kiss2 in Senegalese sole males agrees with what one would expect according to its proposed role as a major regulator of the onset of reproduction, in females the situation was not so clear, since kiss2 and kiss2r expression was highest either before or during the spawning season., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In vertebrates, stress and thyroid systems interact closely, most likely because of the involvement of both systems in energy metabolism. However, studies on these interactions, especially during larval development, are scarce. Recently, cDNAs coding for corticotropin-releasing hormone (CRH) and CRH-binding protein (CRH-BP), two key players in the regulation of the neuroendocrine stress response, were characterized for the flatfish Senegalese sole (Solea senegalensis). To investigate the involvement of stress and thyroid systems in this species, the effects of food deprivation during early development of S. senegalensis were assessed. Growth was arrested in food-deprived post-larvae, which was also reflected by decreased carbon and nitrogen contents, indicating increased catabolism. Food deprivation induces chronic stress, as illustrated by enhanced whole-body cortisol levels, as well as an up regulation of crh and a decrease of crh-bp expression levels. Furthermore, whole-body total T3 concentrations of food-deprived post-larvae were reduced, although tshβ subunit expression levels remained unaffected. Our results show that food deprivation is a chronic stressor that induces energy-releasing catabolic processes that compensate for the reduced energy intake, and inhibits anabolic processes via the peripheral thyroid system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH-sialidase (K-s)-WGA, GSA I-B(4) in the late vitellogenic stage, and in wild fish also SNA and K-s-PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I-B(4) only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B(4) in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA(120) during the late vitellogenic stage, also SNA, PNA, K-s-PNA, GSA I-B(4) in cultured fish, DBA, and K-s-WGA in wild ones which stained with SBA, HPA, and GSA I-B(4) in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K-s-PNA, SBA, HPA, K-s-WGA, GSA I-B(4), UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I-B(4), GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I-B(4) reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture-rearing conditions., (Copyright © 2012 Wiley Periodicals, Inc.)

Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26°C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18°C were acclimated to either cold (12°C) or warm (26°C) environmental temperatures for 21 days. Fish maintained at 18°C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26°C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12°C. When sole is acclimated to 26°C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation.

An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional-translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in fish. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal flatfish Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per-Arnt-Sim or basic helix-loop-helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression profiles were inverted in retina and optic tectum compared to liver. Such differences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their different phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity.

To test how iodine and both iodine and selenium supplementation affected the thyroid status as well as growth and survival in Senegalese sole, larvae were reared in a recirculation system from 15 to 34 DAH. Sets of three tanks were assigned to each of the following three diets: control (C), iodine (I) and iodine and selenium (I + Se). Samples were collected at 15, 27 and 34 DAH to determine dry weight, iodine and selenium levels, GPx and ORD activities, thyroid hormone levels and thyroid follicles histology. At 34 DAH, fish from the control (C) treatment suffered from hyperplasia of the thyroid follicles (goitre), whereas iodine-treated larvae did not (I and I + Se). Lower survival rates in the C groups were probably a consequence of the hyperplasia. Moreover, there was an improvement in thyroid hormone status in I- and I + Se-treated larvae, showing that further supplementation of live feed with iodine can be crucial for fish at early life stages, as it seems to sustain normal larval development, when reared in a recirculation system. Selenium did not affect the results. Together with previous results, this indicates selenium supplement is more important at younger life stages.

Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.

During the development of an embryo into a juvenile, the physiology and behavior of a fish change greatly, affecting exposure to and uptake of environmental pollutants. Based on experimental data with sole (Solea solea), an existing bioaccumulation model was adapted and validated to calculate the development of concentrations of persistent organic pollutants in the tissue of developing fish. Simulation revealed that toxic tissue concentrations of pollutants with log octanol-water partition ratio (K(OW)) > 5 peak at the moment when the larvae become free-feeding, when the lipid reserves are depleted. This may explain the delayed effects observed in fish early-life-stage experiments with exposed eggs. In the field, eggs can be exposed through maternal transfer to adult pollutant tissue concentrations, which will increase in the larva to peak tissue concentrations, exceeding those of the adult fish. The results demonstrate the risk of underestimating the effects of lipophilic persistent organic pollutants with log K(OW) > 5 in short-term, early-life-stage fish tests and underscore the importance of maternal transfer as an exposure route in the field situation., (Copyright © 2012 SETAC.)

Prostaglandin levels in different tissues and cyclooxygenase (COX-2) gene expression were compared between wild and cultured Senegalese sole (Solea senegalensis) broodstock in which a significantly different fatty acid profile, particularly lower tissue levels of arachidonic acid (ARA, 20:4n-6) and higher levels of eicosapentaenoic acid (EPA, 20:5n-3) in the cultured fish compared to wild had already been described. This is the first report of the COX-2 mRNA expression in Senegalese sole. Cyclooxygenase (COX-2) mRNA expression and prostaglandin (2- and 3-series) levels were determined in tissues from 32 broodstock fish, 16 (8 males and 8 females) from each origin wild and cultured (G1). Transcripts of COX-2 were highly expressed in gills, sperm-duct (s-duct), testis, oviduct and spleen compared to liver, kidney and muscle. Differences in COX-2 transcripts expression were found in response to the origin of the fish and expression was significantly higher in s-duct and gills from wild fish compared to cultured. Wild fish showed significantly higher levels of total 2-series PGs and lower levels of 3-series compared to cultured fish. The significance of the lower COX-2 expression and lower PG 2-series production in some of the tissues of cultured fish was discussed in relation to the previously described differences in fatty acid profile (lower tissue levels of ARA and higher levels of EPA and EPA/ARA ratio in cultured fish) and the reproductive failure to spawn viable eggs from G1 cultured Senegalese sole compared to successful spawning from captive wild broodstock., (Copyright © 2012 Elsevier Inc. All rights reserved.)

T lymphocyte activation requires a combination of signals, one of which is provided by interaction between CD28 and its ligands on antigen presenting cells. Although CD28-like sequences have been identified in a few teleosts, the function of fish CD28 is virtually unknown. In this study, we cloned and analyzed a CD28 gene, CsCD28, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsCD28 contains 229 residues and shares 20.2%-40.3% overall sequence identities with known fish CD28 sequences. CsCD28 possesses structural features conserved in mammalian and teleost CD28, which include the MYPPPY motif in the extracellular immunoglobulin-like domain and the YXN motif in the cytoplasmic domain. The CsCD28 gene is 2746 bp and composed of four exons and three introns, which in organization resemble those of mammalian and trout CD28. Quantitative real time RT-PCR analysis showed that CsCD28 expression occurred predominately in kidney, spleen, gut, and gill. CsCD28 is localized on the surface of head kidney lymphocytes, and antibody ligation of CsCD28 induced significant levels of cellular proliferation. Taken together, these results indicate that CsCD28 is similar to mammalian CD28 in genetic and protein structures and possibly plays a role in T cell activation.

Chemokines are small cytokines that, based on their structural differences, are classified into four groups, one of which is called CXC chemokines. In this study, we identified a CXC chemokine, CsCXCe1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its function. The deduced amino acid sequence of CsCXCe1 contains 115 residues and is phylogenetically distinct from known CXC chemokines. CsCXCe1 possesses the conserved RCXC motif in the form of RCWC but lacks the ELR sequence that is found in some CXC chemokines. Expression of CsCXCe1 as determined by quantitative real time RT-PCR occurred abundantly in immune organs and was upregulated by bacterial and viral infection in time dependent manners. Purified recombinant CsCXCe1 (rCsCXCe1) exhibited comparable chemotactic activities against tongue sole and turbot (Scophthalmus maximus) peripheral blood leukocytes (PBL). Microscopic analysis identified lymphocytes as the major cellular population in PBL that responds to rCsCXCe1. Mutational study showed that when the two cysteine residues in the RCWC motif of CsCXCe1 were substituted by serine, the chemoattractive activity of CsCXCe1 was completely lost. Further study showed that treatment of PBL with rCsCXCe1 (i) stimulated cellular proliferation and respiratory burst activity, (ii) upregulated the expression of a wide spectrum of immune relevant genes, and (iii) enhanced cellular resistance against bacterial infection. Taken together, these results indicate that CsCXCe1 is likely a new type of CXC chemokine that exerts chemotactic and immunostimulatory effects on PBL., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5'-terminal untranslated region (UTR), a 452 bp 3'-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5'-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h post-infection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)