A teleost interleukin-16 is implicated in peripheral blood leukocytes recruitment and anti-bacterial immunity.

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

Analysis of lncRNA and mRNA expression profiles in peripheral blood leukocytes of the half-smooth tongue sole (Cynoglossus semilaevis) treated with chitosan oligosaccharide.

Long noncoding RNAs (lncRNAs) play a multifaceted role in transcriptional regulation and are important regulators of immune function. Scarce information is available regarding lncRNAs in fish. Peripheral blood mononuclear cells participate in the immune response of fish and aid resistance to infection with pathogenic microorganisms. Chitosan oligosaccharide can improve cellular and humoral immunity to enhance disease resistance in fish. In this study, we obtained peripheral blood leukocytes from half-smooth tongue sole and studied the effect of chitosan oligosaccharide on the lncRNA-mRNA expression profile of these cells using high-throughput sequencing and bioinformatics techniques. A total of 609 differentially expressed mRNAs and 50 differentially expressed lncRNAs were identified. The GO term enrichment analysis of the differentially expressed genes was annotated by 220 GO terms, 137 biological processes, 18 cellular components, and 65 molecular functions. Sixteen KEGG pathways, including immune signaling pathways, metabolism, and genetic information processing, were significantly enriched in differentially expressed genes. Thirty-six differentially expressed lncRNAs and 32 differentially expressed mRNAs produced a coexpression network containing 90 relationship pairs. The prediction of lncRNA target genes revealed 244 lncRNAs that potentially cis-regulated 294 differentially expressed mRNAs. qPCR verified that the expression levels of 17 differentially expressed lncRNAs and 15 differentially expressed mRNAs were consistent with the RNA-Seq results. Among them, 6 lncRNAs and 7 mRNAs were differentially expressed genes obtained from the prediction and analysis of lncRNA target genes, and 8 lncRNAs and 4 mRNAs were differentially expressed genes that participated in the construction of the coexpression network. In peripheral blood leukocytes after chitosan oligosaccharide treatment, as well as in peripheral blood and spleen after Vibrio anguillarum stimulation, lncRNAs and mRNAs showed significant differential expression. The results indicated that they may be related to the immune response, providing novel reference information for further research on the role of lncRNAs in immune regulation in half-smooth tongue sole.

Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases.

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

Kisspeptin Influences the Reproductive Axis and Circulating Levels of microRNAs in Senegalese Sole.

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.

Oral administration of microencapsulated egg yolk immunoglobulin (IgY) in turbot (Scophthalmus maximus) to combat against Edwardsiella tarda 2CDM001 infections.

Edwardsiellosis, an extremely harmful disease can be caused by Edwardsiella tarda, severely restricts the development of turbot (Scophthalmus maximus) farming worldwide, especially in China. This study aimed to establish an effective and feasible prophylaxis by feeding chitosan-alginate coated egg yolk immunoglobulin (IgY) against E. tarda 2CDM001 infections in the process of turbot farming. Enzyme-linked immunosorbent assays proved that the obtained specific IgY could specifically target E. tarda 2CDM001 and five other E. tarda isolates (1a5p, Hz-s, 1a1s, fs-a1 and 58p8). In-vitro, the bacteriostatic effects of specific IgY showed dose dependencies at concentrations ranging from 1 to 10 mg/mL. Moreover, E. tarda 2CDM001 incubated with 10 mg/mL specific IgY could induce the destruction of cell wall structures and significantly decrease the bacterial surface hydrophobicity (p < 0.05). In this study, turbots were challenged with 107 CFU E. tarda 2CDM001 after seven days of continuous feeding with basal diets containing microencapsulated IgYs. Survival rates of the 5%, 3% and 1% microencapsulated specific IgY groups were 63.3%, 56.7% and 20% on the tenth day post infection, respectively, while the turbots in the positive control and non-specific IgY groups all died within ten days. Oral administration of basal diets containing 5% microencapsulated specific IgY significantly reduced IL-1ß, IL-8, TNF-α and C3 transcript levels in the head kidney and spleen of turbots compared with the positive and non-specific IgY groups at 24 h after E. tarda 2CDM001 challenging (p < 0.05). Pathological increase of leukocytes in the specific IgY group was significantly lower than that in the positive control and non-specific IgY groups (p < 0.05), decreasing slowly after 24 h of infection and showing a recovery trend. Erythrocyte counts and hemoglobin concentrations of turbots in positive and non-specific IgY groups showed a marked decrease compared with the negative and specific groups at 96 h after E. tarda 2CDM001 infection (p < 0.05). These results suggest that passive immunity via feeding microencapsulated specific IgY could be used as a valuable preventative in turbot against E. tarda 2CDM001 infections.

Effects on hematological parameters, antioxidant and immune responses, AChE, and stress indicators of olive flounders, Paralichthys olivaceus, raised in bio-floc and seawater challenged by Edwardsiella tarda.

Studies on the resistance of fish raised in bio-floc systems against bacterial infection are limited. We aimed to evaluate the changes in hematological parameters, antioxidant and immune responses, stress indicators, and acetylcholinesterase (AChE) in olive flounder, Paralichthys olivaceus, raised in bio-floc and seawater for 10 months and, then, infected with Edwardsiella tarda at concentrations of 0 (control), 6.61 × 104, 6.61 × 105, 6.61 × 106, and 6.61 × 107 CFU/g fish for 7 days. The lethal concentration 50% was 4.32 × 107 in bio-floc and 3.11 × 106 in seawater. Hematological parameters were significantly decreased by E. tarda challenge, and plasma components were significantly changed. The superoxide dismutase, catalase, and glutathione-S-transferase activities, as antioxidant responses, were significantly increased after infection, whereas the reduced glutathione level was significantly decreased. The lysozyme activity was significantly increased and the AChE level was significantly decreased after infection. Cortisol and HSP 70, as stress indicators, were also significantly increased. The results indicate that E. tarda infection affected various physiological factors in P. olivaceus. Additionally, P. olivaceus raised in seawater were more susceptible to E. tarda infection than those raised in bio-floc.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures.

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

Expression profile and localization of vitellogenin mRNA and protein during ovarian development in turbot (Scophthalmus maximus).

Egg yolk generation is a common physiological process in oviparous animals. To understand oogenesis and reproductive capacity, it is necessary to characterize vitellogenins (Vtgs), which are the precursors of major egg yolk proteins (Yps). Therefore, to improve our understanding of the entire process of egg yolk generation in female turbot (Scophthalmus maximus), we obtained full-length cDNAs of vtg genes, examined gene expression in the female liver and ovary, and analyzed Vtg synthesis in the ovary. Three distinct complete polypeptide sequences were identified and designated as VtgAa, VtgAb, and VtgC, which confirmed the multiplicity of the vtg gene in turbot and showed that it follows a "three vtg model". The expression of these three vtg genes in the female liver was far higher than that in other tissues, including the ovary. The expression of all three vtg genes was extremely low before vitellogenesis, and then increased and was maintained at a high level until the degradation stage, which was in accordance with changes in the concentration of estradiol-17ß (E2) and the gonadosomatic index. Compared with the liver, the ovary had a higher E2 level and lower vtg expression, suggesting that some other factors limit high vtg expression in the ovary of turbot. Transcripts of vtgAb and the Yps derived from them were both detected in oogonia and primary oocytes, which showed that these might possess the ability to perform autosynthesis of yolk. These findings add to our understanding of the reproductive physiology of Vtg synthesis in turbot.

The anti-androgenic effect of chronic exposure to semicarbazide on male Japanese flounder (Paralichthys olivaceus) and its potential mechanisms.

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 17ß-estradiol (E2) concentrations were significantly decreased in male flounders after SMC exposure. The expression of genes involved in T and E2 synthesis, including steroidogenic acute regulatory protein, cytochrome P450 11A1, 17α-hydroxylase, 17ß-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 (fshß), luteinizing hormone beta subunit (lhß), follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr) mRNA. In addition, down-regulated transcription of fshß and lhß 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.

Influence of vegetable diets on physiological and immune responses to thermal stress in Senegalese sole (Solea senegalensis).

The substitution of fish resources as ingredients for aquafeeds by those based on vegetable sources is needed to ensure aquaculture sustainability in the future. It is known that Senegalese sole (Solea senegalensis) accepts high dietary content of plant ingredients without altering growth or flesh quality parameters. However, scarce information is available regarding the long-term impact of vegetable diets (combining the inclusion of both vegetable protein and oils) on the stress response and immunity of this fish species. This study aims to evaluate the concomitant effect of the extended use of vegetable protein-based diets with fish oil (FO) replacement (0, 50 or 100%) by vegetable oils (VO), on the response to acute (10 min) or prolonged (4 days) stress, induced by thermal shock. Plasma levels of cortisol, glucose and lactate as well as hepatic levels of glucose, glycogen and lactate were evaluated as primary and secondary responses to stress, 6 and 18 months after feeding the experimental diets (6 and 18 MAF). The brain monoaminergic activity in telencephalon and hypothalamus, and non-specific immune parameters were also evaluated. As expected, thermal shock induced an increase in values of plasma parameters related to stress, which was more evident in acute than in prolonged stress. Stress also affected lactate levels in the liver and the values of the alternative complement pathway-ACH50 in the plasma. Dietary substitution of FO induced an effect per se on some parameters such as decreased hepatic glucose and glycogen levels and peroxidase activity in plasma as well enhanced serotonergic activity in brain of non-stressed fish. The results obtained in some parameters indicate that there is an interaction between the use of vegetable diets with the physiological response to thermal stress, as is the case of the hepatic lactate, serotonergic neurotransmission in brain, and the activity of ACH50 in plasma. These results suggest that the inclusion of VO in plant protein based diets point to a slightly inhibited stress response, more evident for an acute than a prolonged stress.

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses.

The present study examined the short and mid-term effects of a rise in temperature from 18°C to 24°C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6°C, after 1month of acclimation at 18°C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1h, 24h, 3days and 1week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration. Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1h as a short-term response, and a consecutive increase after one week, as a mid-term response. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis. Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol.

Identification of exosomes and its signature miRNAs of male and female Cynoglossus semilaevis.

Exosomes are small membrane particles which are widely found in various cell lines and physiological fluids in mammalian. MicroRNAs (miRNAs) enclosed in exosomes have been identified as proper signatures for many diseases and response to therapies. However, the composition of exosomes and enclosed miRNAs in fishes has not been investigated. Cynoglossus semilaevis is an important commercial flatfish with ambiguous distinction between males and females before sex maturation, which leads to screening difficulty in reproduction and cultivation. An effective detection method was required for sex differentiation of C. semilaevis. In this work, we successfully identified exosomes in C. semilaevis serum. The analysis of nucleotide composition showed that miRNA dominated in exosomes. Thereafter the miRNA profiles in exosomes from males and females were sequenced and compared to identify the signature miRNAs corresponding to sex differentiation. The functions of signature miRNAs were analyzed by target matching and annotation. Furthermore, 7 miRNAs with high expression in males were selected from signature miRNAs as the markers for sex identification with their expression profiles verified by real time quantitative PCR. Exosomes were first found in fish serum in this work. Investigation of marker miRNAs supplies an effective index for the filtration of male and female C. semilaevis in cultivation.

Insight into the heat resistance of fish via blood: Effects of heat stress on metabolism, oxidative stress and antioxidant response of olive flounder Paralichthys olivaceus and turbot Scophthalmus maximus.

High temperature has direct confinement on fish survival and growth, especially under the background of global warming. Selection of fish line with heat resistance is an important means to address this problem. In the present study, we analyzed the difference in heat resistance between families of olive flounder Paralichthys olivaceus and turbot Scophthalmus maximus, two flatfish species occupying slightly different thermal niches. Then the chosen families were tested to determine their differential response to heat stress (ΔT = +8 °C and +12 °C) in blood, including anaerobic metabolism (lactate), oxidative stress (lipid peroxidation and protein carbonylation) and antioxidant enzymes. Results showed a difference in heat resistance between families of the two species. Among the chosen parameters, growth traits had a significant effect on contents of lactate and malondialdehyd (MDA), and activities of catalase (CAT) and glutathione S-transferase in flounder (P < 0.05), and on MDA content and CAT activity in turbot (P < 0.05). In comparison with heat-sensitive family of each species, levels of all studied parameters were lower and more stable in heat-resistant families after heat stress. What's more, heat resistance of fish significantly influenced contents of lactate and MDA and activity of CAT in flounder (P < 0.05), as well as contents of lactate, MDA and carbonyl and activity of superoxide dismutate (SOD) in turbot (P < 0.05). These results demonstrated that such physiological phenotypes as anaerobic metabolism, oxidative stress and antioxidant enzymes are good biomarkers of fish heat-resistance, being potentially valuable in fish breeding. However, these markers should be applied with more caution when there is a growth discrepancy between fish families.

Plasma levels of follicle-stimulating and luteinizing hormones during the reproductive cycle of wild and cultured Senegalese sole (Solea senegalensis).

The intensive culture of the Senegalese sole (Solea senegalensis) is hampered by the low or null fertilization rates exhibited by the first generation (F1) of reared males. To investigate the regulation of the reproductive processes in this species by the pituitary gonadotropins follicle-stimulating and luteinizing hormones (Fsh and Lh, respectively), we developed a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for Lh measurements. Quantification of the Fsh and Lh plasma levels in cultured sole using the Lh ELISA developed here, and a previously developed ELISA for Fsh, indicated that in both males and females circulating Fsh steadily increased during autumn and winter and prior to the major spawning in spring, whereas an Lh surge occurred specifically during spawning. The increase in Fsh was associated with a rise of plasma levels of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17ß (E2), but that of Lh was concomitant with a strong decline of the levels of E2 in females and of 11-KT in males, possibly reflecting a rapid steroidogenic shift promoting the final maturation of gametes. Comparison of the plasma levels of gonadotropins and steroids between wild and F1 fish during autumn and spring revealed that F1 males showed significantly lower plasma Lh titres compared to wild males, whereas the levels of T and 11-KT were similar or more elevated in the F1 fish. These data suggest that an impaired Lh secretion during spawning, and perhaps altered Lh-mediated mechanisms in the testis, may be underlying causes for the low reproductive performance of Senegalese sole F1 males.

Wnt/ß-catenin signaling participates in the regulation of lipogenesis in the liver of juvenile turbot (Scophthalmus maximus L.).

In this study, the mechanism that Wnt/ß-catenin signaling inhibits lipogenesis was investigated in the liver of juvenile turbot (Scophthalmus maximus L.) by LiCl or XAV939 treatment. Wnt/ß-catenin signaling was activated by LiCl treatment or inhibited by XAV939 treatment through regulating the expression of glycogen synthase kinase-3ß (GSK-3ß) and Wnt10b. In addition, the expression of lipoprotein lipase (LPL), fatty acid synthetase (FAS), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα) was inhibited by LiCl treatment, but induced by XAV939 treatment. In the plasma of juvenile turbot, the level of nonesterified fatty acid (NEFA), glycerol, triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) was decreased by LiCl treatment, which was related to the decrease of the activity of LPL and FAS. Thus the inhibitory effect of Wnt/ß-catenin signaling on lipogenesis was associated with the expression of key enzymes and transcriptional factors. Wnt/ß-catenin signaling may participate in inhibiting lipogenesis by inhibiting the expression of PPARγ and C/EBPα in the liver of juvenile turbot.

Effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus).

Nitrite (NO2(-)) is commonly present as contaminant in aquatic environment and toxic to aquatic organisms. In the present study, we investigated the effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Fish were exposed to various concentrations of nitrite (0, 0.02, 0.08, 0.4 and 0.8mM) for 96 h. Fish blood and gills were collected to assay haematological parameters, oxidative stress and expression of genes after 0, 24, 48 and 96 h of exposure. In blood, the data showed that the levels of methemoglobin (MetHb), triglyceride (TG), potassium (K(+)), cortisol, heat shock protein 70 (HSP70) and glucose significantly increased in treatments with higher concentrations of nitrite (0.4 and/or 0.8mM) after 48 and 96 h, while the levels of haemoglobin (Hb) and sodium (Na(+)) significantly decreased in these treatments. In gills, nitrite (0.4 and/or 0.8mM) apparently reduced the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH), increased the formation of malondialdehyde (MDA), up-regulated the mRNA levels of c-jun amino-terminal kinase (JUK1), p53, caspase-3, caspase-7 and caspase-9 after 48 and 96 h of exposure. The results suggested caspase-dependent and JUK signaling pathways played important roles in nitrite-induced apoptosis in fish. Further, this study provides new insights into how nitrite affects the physiological responses and apoptosis in a marine fish.

ORF75 of megalocytivirus RBIV-C1: A global transcription regulator and an effective vaccine candidate.

Megalocytivirus, a DNA virus belonging to the Iridoviridae family, is a severe pathogen to a wide range of marine and freshwater fish. In this study, using turbot (Scophthalmus maximus) as a host model, we examined the immunoprotective property of one megalocytivirus gene, ORF75, in the form of DNA vaccine (named pORF75). Immunofluorescence microscopy and RT-PCR analysis showed that P444, the protein encoded by ORF75, was naturally produced in the tissues of turbot during megalocytivirus infection, and that the vaccine gene in pORF75 was expressed in fish cells transfected with pORF75 and in the tissues of turbot immunized with pORF75. Following vaccination of turbot with pORF75, a high level of survival (73%) was observed against a lethal megalocytivirus challenge. Consistently, viral replication in the vaccinated fish was significantly inhibited. Immune response analysis showed that pORF75-vaccinated fish (i) exhibited upregulated expression of the genes involved in innate and adaptive immunity, (ii) possessed specific memory immune cells that showed significant response to secondary antigen stimulation, and (iii) produced specific serum antibodies which, when co-introduced into turbot with megalocytivirus, blocked viral replication. Furthermore, whole-genome transcriptome analysis revealed that ORF75 knockdown altered the transcription of 43 viral genes. Taken together, these results indicate that ORF75 encoded a highly protective immunogen that is also a global transcription regulator of megalocytivirus.

Polymorphisms and DNA methylation level in the CpG site of the GHR1 gene associated with mRNA expression, growth traits and hormone level of half-smooth tongue sole (Cynoglossus semilaevis).

The objectives of the present study were to estimate the GHR1 gene mutations and methylation status of CpGs, and whether those mutations and methylation were involved in the regulation of GHR1 gene expression, hormone level and growth traits in half-smooth tongue sole (Cynoglossus semilaevis). Identification of single-nucleotide polymorphisms was performed on 43 male fish. Through polymerase chain reaction-single-strand conformation polymorphism and sequencing, two SNPs were found. SNP1 [c.G1357A (p.Val376Ile)] creating one CpG site located in exon 8 was named L1 locus, and SNP2 (c.G1479A) located in exon 9 was named L2 locus. Individuals were divided into three genotypes, AA, AG and GG according to L1 locus (GG genotype had one more CpG site because of the mutation), and into two genotypes, AA- and GG-based on L2 locus. The results showed that only L1 locus was significantly associated with body weight (P < 0.01), gonad weight (P ≤ 0.05), triiodothyronine (T3) level (P ≤ 0.05) and mRNA expression (P < 0.01). At L1 locus, newly created CpG site in GG genotype was highly methylated (93.3 %), while there was no difference of methylation level in the other two CpG sites among three genotypes. AA genotype and AG genotype having higher T3 level were significantly different (P ≤ 0.05) from GG genotype. There were significant differences among body weights of AA, AG and GG genotypes (P < 0.01). Gonad weights of AA genotype and AG genotype were significantly lower than GG genotype. The GHR1 mRNA expression of GG genotype was significantly lower than AA and AG genotypes (P < 0.01). These implied that mutations and methylation status of GHR1 gene might influence the hormone level, growth traits and gene expression in male half-smooth tongue sole and the L1 locus could be regarded as a potential candidate genetic and epigenetic marker in half-smooth tongue sole selection.

Effects of selected xenobiotics on hepatic and plasmatic biomarkers in juveniles of Solea senegalensis.

In recent years, Solea senegalensis has increasingly been used in pollution monitoring studies. In order to assess its response to some particular widespread pollutants, juveniles of S. senegalensis were administered an intraperitoneal injection of the model aryl hydrocarbon receptor agonist ß-naphtoflavone (ßNF) and chemicals of environmental concern, such as the fungicide ketoconazole (KETO), the lipid regulator gemfibrozil (GEM), the surfactant nonylphenol (NP) and the synthetic hormone ethinylestradiol (EE2). Two days after injection, the effect of these chemicals was followed up as alterations of hepatic microsomal activities of the cytochrome P450 (CYPs) and associated reductases, carboxylesterases (CbEs) and the conjugation enzyme uridine diphosphate glucuronyltransferase (UDPGT). In the cytosolic fraction of the liver, the effect on CbEs, glutathione S-transferase (GST) and antioxidant activities was also considered. Alterations on the endocrine reproductive system were evaluated by plasma levels of vitellogenin (VTG) and the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone (11KT) and the progestin 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P). Injection with the model compound ßNF induced the hydrolysis rate of the seven CYP substrates assayed. The xenobiotic GEM induced three CYP-related activities (e.g. ECOD) and UDPGT, but depressed antioxidant defenses. EE2 induced four CYPs, more significantly ECOD and BFCOD activities. The xenoestrogens NP and EE2 altered the activities of CbE in microsomes and catalase, and were the only treatments that induced de novo VTG synthesis. In addition, the progestin 17,20ß-P, was induced in NP-injected fish. None of the treatments caused statistically significant effects on steroid plasma levels. In conclusion, the CYP substrates assayed responded specifically to treatments and juveniles of S. senegalensis appear good candidates for assessing xenobiotics exposure.