Connecting gut microbiome changes with fish health conditions in juvenile Atlantic cod (Gadus morhua) exposed to dispersed crude oil.

Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.

Sensory Assessment of Fish and Chicken Protein Hydrolysates. Evaluation of NMR Metabolomics Profiling as a New Prediction Tool.

Nuclear magnetic resonance (NMR) metabolomics profiling was evaluated as a new tool in sensory assessment of protein hydrolysates. Hydrolysates were produced on the basis of different raw materials (cod, salmon, and chicken), enzymes (Food Pro PNL and Bromelain), and hydrolysis time (10 and 50 min). The influence of raw material and hydrolysis parameters on sensory attributes was determined by traditional descriptive sensory analysis and 1H NMR spectroscopy. The raw material had a major influence on the attribute intensity and metabolite variation, followed by enzyme and hydrolysis time. However, the formation of bitter taste was not affected by the raw material. Partial least-squares regression (PLSR) on 1H NMR and sensory data provided good models (Q2 = 0.55-0.89) for 11 of the 17 evaluated attributes, including bitterness. Significant metabolite-attribute associations were identified. The study confirms the potential prediction of the sensory properties of protein hydrolysates from cod, salmon, and chicken based on 1H NMR metabolomics profiling.

Dioxin-like polybrominated biphenyls (PBBs) and ortho-substituted PBBs in edible cod (Gadus morhua) liver oils and canned cod livers.

This study investigates the occurrence of polybrominated biphenyls (PBBs), a legacy flame retardant, in fishery products such as medicinal grade cod liver oils and canned liver products, sourced from the North Atlantic during 1972-2017. It also assesses the dietary and supplementary (the oils were commonly administered as dietary supplements to children and youth) intake of PBBs from these products. Summed ortho-PBB concentrations ranged from 770 to 1400 pg g-1 fat in the oils and from 99 to 240 pg g-1 whole weight in canned livers, with PBB-49, 52, 101 and 153 accounting for most of these levels. Among the more toxic non-ortho-PBBs, PBB-126 and PBB-169 were not detected, but PBB-77 concentrations ranged from 0.6 to 5.78 pg g-1 fat in the oils and 0.06-0.126 pg g-1 whole weight in canned livers. During 1972-1993, PBB contamination levels were similar for cod liver oils from the Baltic Sea and other North Atlantic regions, but over the timescale of the study, Baltic Sea products appear to show a decline in PBB concentrations. As PBB-77 was the only dioxin-like PBB detected in the samples, the corresponding supplementary (oils, 1972-2001) and dietary (cod liver from 2017) intakes were very low, at < 0.001 pg TEQ kg-1 bm d-1 (or < 0.01 pg TEQ kg-1 bm d-1 upper bound) for the sum of all the measured dioxin-like PBBs -four to six orders of magnitude lower than that arising from other dioxin-like contaminants that were shown to occur in these products, from earlier studies.

Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs.

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. ß-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Environmentally realistic exposure to weathered North Sea oil: Sublethal effects in Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus).

With increasing oil and gas activities and transport in the Arctic, there is a need to understand how operational or accidental releases of substances affect marine organisms from a pristine environment. The aim of the current study was to describe and compare the responses of two marine fish species, Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus), following exposure to three levels (low, medium, high) of the water-soluble fraction of a North Sea crude oil for 16 days. The exposure system simulated environmental exposure by allowing clean seawater to percolate through gravel covered in weathered oil before being introduced to aquaria. Both polycyclic aromatic hydrocarbon (PAH) metabolite bile concentrations and cytochrome P4501A (CYP1A) levels and activity increased markedly in comparison with controls in both species, but there were no significant differences between the three exposures. Turbot possessed 4-5-fold higher concentrations of two PAH bile metabolites compared to Atlantic cod by day 8. In contrast, hepatic CYP1A activity in cod was consistently 2-6-fold higher than in turbot with increasing differences over the experimental period. Baseline DNA strand breaks in lymphocytes and kidney cells were low in both species, but was elevated for all treatments by day two. There were no marked indications of the treatments affecting immune functions in either species. This investigation demonstrated that there may be significant differences in responses between species receiving identical exposures and that DNA strand breaks in lymphocytes and kidney cells are sensitive to confinement stress. Data also indicate that some species, such as turbot, may adapt to treatments within days and weeks.

Effects of amino acid supplementations on metabolic and physiological parameters in Atlantic cod (Gadus morhua) under stress.

The effects of tryptophan (Trp) and phenylalanine (Phe) diet supplementation on the stress and metabolism of the Atlantic cod have been studied. Fish were fed diet supplemented with Trp or Phe or control diet for 1 week. At the end of the feeding trial, fish were subjected to air exposure or heat shock. Following samples of blood, liver and muscle were taken from the fish and were analyzed for stress and metabolic indicators. After an air exposure, plasma cortisol levels in fish fed with Trp and Phe diets were lower compared to the fish fed the control diet. Diets containing both amino acids increased significantly the liver transaminase activities in juvenile cod. During thermal stress, high Trp contents had significant effects on fructose biphosphatase activity though Phe did not. Overall, activities of glucose 6-phosphate dehydrogenase, pyruvate kinase, and phosphofructokinase increased significantly for both amino acid diets. For the thermal stress, fish had the highest values of those activities for the 3Trp diet. Trp content in the diet had significant effects on the transaminase activity in muscle during air stress compared to fish fed control and Phe diets. Muscle alanine transaminase activity for thermal stress in fish fed any diet was not significantly different from the control. Both Trp and Phe supplementations reduced the stress markers in the cod; hence, they could be used as additives for the stress attenuation. However, they also raised the activity of key enzymes in glycolysis and gluconeogenesis, mainly the Trp diets.

Exposure of first-feeding cod larvae to dispersed crude oil results in similar transcriptional and metabolic responses as food deprivation.

Exposure of first-feeding cod larvae (Gadus morhua) to dispersed oil results in reduced feeding during an important transition period. First-feeding cod larvae were subjected to a 4-d treatment of food deprivation and sampled for microarray analyses. These microarray data were combined with data from cod larvae treated with mechanically and chemically dispersed oil in an attempt to understand to what extent starvation might explain some of the effects observed in first-feeding cod larvae during oil exposure. Transcriptional profiling of cod larvae suggested that the influence of oil exposure was almost as dramatic as being completely deprived of food. Protein and cellular degradation and loss of amino acids and glucose appear to be concomitant responses to both oil exposure and starvation. Fluorescence imaging of gut content indicated low uptake of food, and reduced growth (decrease in dry weight and in carbon and nitrogen content) was also noted in oil-exposed larvae, providing phenotypic anchoring of microarray data. The study displays the importance in combining use of high-throughput molecular tools with assessment of fitness-related endpoints in order to provide a greater understanding of toxicant-induced responses. This combined-approach investigation suggests that reduction of food uptake is an important process to be included when predicting effects of accidental oil spills. Finally, when comparing data from two oil treatments, exposure to chemically dispersed oil did not appear to result in greater toxicity than exposure to mechanically dispersed oil.

Atlantic cod (Gadus morhua) larvae can biosynthesis phospholipid de novo from 2-oleoyl-glycerol and glycerol precursors.

The dietary requirement of phospholipid (PL) of fish larvae has been suggested to originate in an inefficient ability for de novo biosynthesis of PL based on dietary triacylglycerol (TAG). The main objective of the present study was to investigate whether cod larvae could synthesis PL from sn-2-monoacylglycerol (2-MAG) and glycerol precursors. A tube feeding method was used to deliver equal molar aliquots of 2-oleoyl-[1,2,3-(3)H]glycerol and [U-(14)C] glycerol together with bovine serum albumin (BSA) bound 16:0 (palmitic acid) and 22:6n-3 (docosahexaenoic acid, DHA), with or without choline chloride to the foregut of anesthetized cod larvae and thereafter monitoring the metabolism of these components in the larvae through 4 h following injection. Our results showed that both 2-MAG and glycerol precursors contributed to the de novo synthesis of phosphatidylcholine (PC) and the 2-MAG pathway predominated over the G-3-P (glycerol-3-phosphate) pathway in the synthesis of TAG and PC. The molecular ratio of PC/TAG obtained from the 2-MAG and the G-3-P pathways was 0.44-0.74 and 1.02-2.06 within the first hour of tube feeding, suggesting they might have comparable biosynthesis ability of PC and TAG under the conditions of the present study. Furthermore, supplementation of choline chloride significantly increased PC/TAG ratio (p < 0.05) for both pathways. However, further studies are needed to quantify the enzyme activity involved in the CDP-choline (cytidine diphosphate choline) pathway, and the function of choline either in simulating PC synthesis or TAG catabolism or both needs further investigation.

Impact of dietary selenium on methylmercury toxicity in juvenile Atlantic cod: a transcriptional survey.

Selenium (Se) and its derivatives are known to have protective effects against mercury (Hg) toxicity in mammals. In this study we wanted to evaluate whether Se co-exposure affect the transcription of methylmercury (MeHg) toxicity-relevant genes in early life stages of fish. Juvenile Atlantic cod were exposed to regular feed (control), Se-spiked feed (3mg Se kg(-1)), MeHg-spiked feed (10mg Hg kg(-1)) or to Se- and MeHg-spiked feed (3mg Se kg(-1) and 10mg Hg kg(-1), respectively) for ten weeks. Liver tissue was harvested for transcriptional analysis when the fish were weighing 11.4 ± 3.2g. Accumulated levels of Hg in liver of the two groups of fish exposed to MeHg were 1.5mg Hg kg(-1) wet weight, or 44-fold higher than in the control group, while the Se concentrations differed with less than 2-fold between the fish groups. Selenium co-exposure had no effect on the accumulated levels of Hg in liver tissue; however, MeHg co-exposure reduced the accumulated level of Se. Dietary exposure to MeHg had no effect on fish growth. Interaction effects between Se and MeHg exposure were observed for the transcriptional levels of CAT, GPX1, GPX3, NFE2L2, UBA52, SEPP1 and DNMT1. Significant effects of MeHg exposure were seen for DNMT1 and PPARG, while effects of Se exposure were seen for GPX4B and SEPP1A, as well as for DNA methyltransferase activity. The transcriptional results suggest, by considering up-regulation as a proxy for negative impact and at the tested concentrations, a pro-oxidative effect of Se co-exposure with MeHg, rather than an antioxidative effect.

An investigation of appetite-related peptide transcript expression in Atlantic cod (Gadus morhua) brain following a Camelina sativa meal-supplemented feeding trial.

Camelina sativa is a hardy oilseed crop with seeds that contain high levels of ω3 polyunsaturated fatty acids and protein, which are critical components of fish feed. Camelina might thus be used as a cheaper and more sustainable supplement to fish-based products in aquaculture. Atlantic cod, Gadus morhua, is a species of interest in the aquaculture industry due to a decrease in wild populations and subsequent collapse of some cod fisheries. As cod are carnivorous fish, it is necessary to determine how this species physiologically tolerates plant-based diets. In this study, juvenile Atlantic cod were subjected to 13 weeks of either 15 or 30% camelina meal (CM)-supplemented diets or a control fish meal feed. Growth and food intake were evaluated and the mRNA expression of appetite-related hormones [pro-melanin-concentrating hormone (pmch), hypocretin (synonym: orexin, hcrt), neuropeptide Y (npy) and cocaine- and amphetamine-regulated transcript (cart)] was assessed using quantitative real-time PCR in brain regions related to food intake regulation (telencephalon/preoptic area, optic tectum/thalamus and hypothalamus). CM inclusion diets caused decreases in both growth and food intake in Atlantic cod. Optic tectum pmch transcript expression was significantly higher in fish fed the 30% CM diet compared to fish fed the 15% CM diet. In the hypothalamus, compared to fish fed the control diet, hcrt expression was significantly higher in fish fed the 30% CM diet, while npy transcript expression was significantly higher in fish fed the 15% CM diet. cart mRNA expression was not affected by diet in any brain region. Further studies are needed to determine which factors (e.g. anti-nutritional factors, palatability and nutritional deficits) contribute to reduced feed intake and growth, as well as the maximum CM inclusion level that does not negatively influence feed intake, growth rate and the transcript expression of appetite-related factors in Atlantic cod.

Aryl hydrocarbon receptor protein and Cyp1A1 gene induction by LPS and phenanthrene in Atlantic cod (Gadus morhua) head kidney cells.

The objective of this study was to evaluate interactions between environmental toxicants and cod immune cells during inflammation. Phenanthrene is abundant in plant oils (rapeseed, palm, and soya oil) as compared to fish oils, and consequently constitute an undesirable element in plant replacement diets in aquaculture. Phenanthrene was added to head kidney cell cultures, alone or together with LPS (lipopolysaccharide) or poly I: C (polyinosinic acid: polycytidylic acid), and the responses were evaluated in terms of protein and gene expression. The results showed that LPS, poly I: C or phenanthrene, added to the cultures separately, induced aryl hydrocarbon receptor (AhR) protein expression. Phenanthrene treatment in combination with LPS induced AhR protein expression and Cyp1A1 gene transcription, which not was observed combining poly I: C and phenanthrene. Phenanthrene exposure up regulated the transcription of common stress and detoxification enzymes like catalase, caspase 3 and glutathione S-transferase alfa 3 subunit B (GSTAB3), while LPS exposure alone or combined with phenanthrene down regulated GSTAB3 and catalase in cod leukocytes. It seems clear that immune regulation and phenanthrene induced signaling pathways interact; transcriptional down regulation of detoxification and antioxidant enzymes by LPS could indicate that combating bacterial infections is the number one priority in these cells, and that AhR and Cyp1A1 is somehow involved in this signaling cascade. LPS seems to affect the mitogen activated protein kinases (MAPKs) pathways (P-p38 and ERK1/2) thus modulating the AhR protein and Cyp1A1 gene transcription, while phenanthrene possibly activates AhR by ligand binding.

Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua).

For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod.

Substitution of fish oil with camelina oil and inclusion of camelina meal in diets fed to Atlantic cod (Gadus morhua) and their effects on growth, tissue lipid classes, and fatty acids.

Developing a commercially relevant Atlantic cod aquaculture industry will require improvements in feed sustainability. Camelina oil and meal are potential replacements of fish oil and fish meal in aquaculture feeds. Camelina oil is high in 18:3ω3 (30%), with an ω3/ω6 ratio > 1. Camelina meal has a considerable crude protein level (38%), which includes significant amounts of methionine and phenylalanine. Four diets were tested; each diet was fed to triplicate tanks (3 tanks per diet) of Atlantic cod (14.4 g/fish; 70 fish per tank) for 13 wk. The diets included a fish oil/fish meal control (FO) and three diets which replaced 100% of fish oil with camelina oil: one diet contained fish meal (100CO), another solvent extracted fish meal (100COSEFM), and another had fish meal partially reduced by 15% inclusion of camelina meal (100CO15CM). Growth was measured (length and weight) and tissue samples were collected for lipid analysis (muscle, liver, brain, gut, spleen, skin, and carcass) at wk 0 (before feeding the experimental diet) and at wk 13. Cod fed camelina oil had a lower (P < 0.001) final weight than cod fed the FO diet (50.8 ± 10.3 g/fish). Cod fed 100CO15CM had a lower (P < 0.001) final weight (35.0 ± 8.0 g) than those fed 100CO (43.6 ± 8.9 g) and 100COSEFM (46.7 ± 10.7 g). Cod tissues in the 100COSEFM treatment were most impacted by dietary fatty acid profile. Multivariate statistics revealed that FO and 100COSEFM tissue fatty acid profiles were 21 to 31% different, depending on tissue type. The full replacement of fish oil with camelina oil, plus solvent extracted fish meal had an overarching effect on the entire fatty acid profile of the whole animal. Fatty acid mass balance calculations indicated that cod fed 100COSEFM elongated 13% of 18:3ω3 to 20:3ω3 and oxidized the remaining 87%, whereas cod fed fish oil showed a much lower (P < 0.001) elongation of 18:3ω3 of 1.6%. These results suggest that excess 18:3ω3 from camelina oil caused some fatty acid elongation, but little desaturation. Energy budget estimates indicated that cod fed 100COSEFM deposited the most energy throughout the trial (60 kJ/fish; P = 0.019), mostly in the liver (90%). Excess camelina lipids were not necessarily utilized for energy, which likely impacted growth. Feeding 100% camelina oil to Atlantic cod impacted growth and lipid and fatty acid composition; however, additional removal of fish oil from fish meal caused the greatest change in cod lipid composition and utilization.

Acetylcholinesterase activity in the host-parasite system of the cod Gadus morhua and acanthocephalan Echinorhynchus gadi from the southern Baltic Sea.

Acetylcholinesterase (AChE) activity measurement is widely used as a specific biomarker of neurotoxic effects. The aim of this study was to evaluate AChE activity in a host fish (the cod) and its acanthocephalan parasite Echinorhynchus gadi from the southern Baltic. AChE activity in hosts and parasites was inversely related: the highest cod AChE activity corresponded to the lowest E. gadi enzymatic activity and vice versa ("mirror effect"). This is the first report on the simultaneous application of this biomarker in cod and its acanthocephalan parasites. Results obtained for the host-parasite system are complementary and provide comprehensive information about the response of this biomarker. Analysis of the system allows for detection of a greater number of factors influencing AChE activity in the marine environment than separate analysis of the host and parasites. Thus, AChE activity measurement in a host-parasite system may be considered to be a promising tool for biomonitoring.

Effect of replacement of fish oil with camelina (Camelina sativa) oil on growth, lipid class and fatty acid composition of farmed juvenile Atlantic cod (Gadus morhua).

Camelina (Camelina sativa) oil was tested as a replacement for fish oil in diets for farmed Atlantic cod (Gadus morhua). Camelina differs from other plant oilseeds previously used in aquaculture with high lipid (40 %), α-linolenic acid (40 %), antioxidants and low proportions of saturated fats. Dietary treatments were fed to cod (19 g fish⁻¹ initial weight) for 9 weeks and included a fish oil control (FO), 40 % (CO40) and 80 % (CO80) replacement of fish oil with camelina oil. There was no effect of replacing fish oil with camelina oil included at levels up to 80 % on the growth performance. Cod fed CO80 stored more lipid in the liver (p < 0.01), including more neutral lipid (p < 0.05) and triacylglycerol (p < 0.05). Cod fed CO80 decreased in total polyunsaturated fatty acids (PUFAs) in muscle compared to CO40 and FO (p < 0.05), increased in monounsaturated fatty acids (p < 0.01), decreased in total ω3 fatty acids (FO > CO40 > CO80; p < 0.01) and increased in total ω6 fatty acids (FO < CO40 < CO80; p < 0.01). In the liver, long-chain (LC) PUFA such as 20:4ω6, 20:5ω3, 22:5ω3 and 22:6ω3 decreased when fish oil was removed from the diet (p < 0.05), and increased in 18-carbon fatty acids (p < 0.01). Camelina oil can reduce the amount of fish oil needed to meet lipid requirements, although replacing 80 % of fish oil reduced LC PUFAs in both tissues. A comparison of BF3 and H2SO4 as catalysts to transmethylate cod liver and muscle lipids revealed small but significant differences in some fatty acid proportions.

Cytokine gene expression and prostaglandin production in head kidney leukocytes isolated from Atlantic cod (Gadus morhua) added different levels of arachidonic acid and eicosapentaenoic acid.

Primary head kidney leukocytes from Atlantic cod were isolated to evaluate the use of arachidonic acid and eicosapentaenoic acid by cyclooxygenases and the production of prostaglandins E2 and E3. The expression of cyclooxygenase genes and selected interleukin genes like Interleukin 1ß, Interleukin 6, interleukin 8 and interleukin 10 were monitored. Increasing concentrations of eicosapentaenoic acid and arachidonic acid in equal amounts increased cyclooxygenase2 transcription as well as cell secretion of prostaglandin E2. Even though the ratio of the two fatty acids was 1:1, the ratio between prostaglandin E2 and E3 was 50:1. The addition of arachidonic acid alone increased prostaglandin E2 secretion but did not induce cyclooxygenase2 transcription. However, when the concentration of eicosapentaenoic acid was increased, maintaining arachidonic acid constant, both prostaglandin E3 and prostaglandin E2 production was induced and the prostaglandin E2 production was higher than in cell cultures only added arachidonic acid. An up-regulation of cyclooxygenase2 transcription was also observed. The addition of the two fatty acids also affected the immune response by alteration of leukocytic cytokines gene expression. According to our results the Cyclooxygenase in cod seem to prefer arachidonic acid as substrate. Therefore, we suggest that the shift from marine oils (rich in n-3 fatty acids) to plant oils (higher in n-6 fatty acids) in the diet of commercially reared Atlantic cod could have negative effects on the whole organism through the increase in the production of prostaglandins belonging to those derived from n-6 fatty acids.

Changes in regional brain monoaminergic activity and temporary down-regulation in stress response from dietary supplementation with l-tryptophan in Atlantic cod (Gadus morhua).

The brain monoamines serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA) both play an integrative role in behavioural and neuroendocrine responses to challenges, and comparative models suggest common mechanisms for dietary modulation of transmission by these signal substances in vertebrates. Previous studies in teleosts demonstrate that 7 d of dietary administration with L-tryptophan (Trp), the direct precursor of 5-HT, suppresses the endocrine stress response. The present study investigated how long the suppressive effects of a Trp-enriched feed regimen, at doses corresponding to two, three or four times the Trp levels in commercial feed, last in juvenile Atlantic cod (Gadus morhua) when the fish are reintroduced to a diet with standard amino acid composition. We also wanted to determine whether Trp supplementation induced changes in brain monoaminergic neurochemistry in those forebrain structures innervated by DA and 5-HTergic neurons, by measuring regional activity of DA and 5-HT in the lateral pallial regions (Dl) of the telencephalon and nucleus lateralis tuberis (NLT) of the hypothalamus. Dietary Trp resulted in a dose-dependent suppression in plasma cortisol among fish exposed to confinement stress on the first day following experimental diet; however, such an effect was not observed at 2 or 6 d after Trp treatment. Feeding the fish with moderate Trp doses also evoked a general increase in DA and 5-HT-ergic activity, suggesting that these neural circuits within the NLT and Dl may be indirectly involved in regulating the acute stress response.

Transcriptional regulation of cytokines in the intestine of Atlantic cod fed yeast derived mannan oligosaccharide or ß-glucan and challenged with Vibrio anguillarum.

Immunomodulatory feed additives are expected to exert their primary influence at the intestinal level through the expression of cytokines, which in turn affect the immune responses in fish. In two separate experiments a yeast-derived mannan oligosaccharide product (YM) or a purified ß-glucan (BG) product were fed to Atlantic cod (Gadus morhua L.) for 5 weeks, after which they were bath-challenged with a bacterial pathogen--Vibrio anguillarum. The transcription of selected cytokines (proinflammatory--il1b, il8, ifng; anti-inflammatory--il10) in different intestinal segments was analysed using qPCR. In the case of YM study, the effect of the compound was observed in both the posterior intestine and rectum of Atlantic cod, upon challenge with the pathogen. iIl1b expression in the posterior intestine and rectum of post-challenge fish was significantly higher than that of pre-challenge fish. In the case of il8 the difference was confined to rectum. The expression of ifng was altered only in the anterior intestine upon YM feeding. In the BG trial, the additive had a differential effect on the expression of the cytokine genes. In anterior intestine and rectum, the purified ß-glucan additive significantly elevated the expression of il1b when challenged with V. anguillarum. An effect of BG on the anti-inflammatory cytokine il10 was visible in the rectum after the pathogen challenge. The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggest that both mannan oligosaccharides and ß-glucans impact the ability of Atlantic cod to respond to the pathogen.

Biomarker responses in Atlantic cod (Gadus morhua) exposed to produced water from a North Sea oil field: laboratory and field assessments.

Biological markers of produced water (PW) exposure were studied in Atlantic cod (Gadus morhua) in both laboratory and field experiments, using authentic PW from a North Sea oil field. In the laboratory study, the PW exposure yielded significantly elevated levels of metabolites of polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs) in bile even at the lowest exposure dose (0.125% PW). Other biomarkers (hepatic CYP1A induction and DNA adduct formation) responded at 0.25% and 0.5% PW concentrations. In the field study, bile metabolite markers and hepatic CYP1A were clearly increased in fish caged close to the PW outfall. Induction of plasma vitellogenin was not found in laboratory or field exposures, suggesting that the levels of oestrogen agonists (such as APs) might not have been sufficient to elicit induction, under the present conditions. The applicability of the biomarkers for use in water column biomonitoring programs is discussed.

An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS.

Compound-specific protein expression signatures (PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity, however, there are concerns regarding the reproducibility of this method. The aim of this study was to define an estrogen-responsive PES in plasma of Atlantic cod (Gadus morhua) using the SELDI-TOF MS technique. Protein expression analysis of male cod exposed to 17ß-estradiol (E2) showed that 27 plasma peaks were differentially expressed following exposure. The reproducibility of this result was evaluated by reanalyzing the samples six months later, and a significant change in expression was confirmed for 13 of the 27 peaks detected in the first analysis. The performance of the reproducible E2-responsive PES, constituting these 13 peaks, was then tested on samples from juvenile cod exposed to 4-nonylphenol, North Sea oil, or North Sea oil spiked with alkylphenols. Principal component analysis revealed that nonylphenol-exposed cod could be separated from unexposed cod based on the E2-responsive PES, indicating that the PES can be used to assess estrogenic exposure of both juvenile and adult specimens of cod. A targeted antibody-assisted SELDI-TOF MS approach was carried out in an attempt to identify the E2-responsive peaks. Results indicated that 2 peaks were fragments of the well-known biomarkers VTG and/or ZRP. In this study, the SELDI-TOF MS technology has shown its potential for defining compound-specific PESs in fish. Nevertheless, thorough validation of reproducibility, specificity and sensitivity of a PES is required before it can be applied in environmental monitoring.