The ontogeny of lymphoid organs and IgM+ B-cells in ballan wrasse (Labrus bergylta) reveals a potential site for extrarenal B-cell lymphopoiesis: The pancreas.

Vaccination of farmed fish is the most effective prophylactic measure against contagious diseases but requires specific knowledge on when the adaptive immune system is fully developed. The present work describes kidney and spleen morphogenesis as well as B-cell development in the ballan wrasse (Labrus bergylta). The kidney was present at hatching (0 days pot hatching, dph) but was not lymphoid before larvae was 50-60 dph (stage 5), containing abundant Igµ+ cells. The spleen anlage was first observed in larvae at 20-30 dph and was later populated with B-cells. Unexpectedly, we found strong RAG1 signal together with abundant Igµ+ and IgM + cells in the exocrine pancreas of larvae from when the kidney was lymphoid and onwards, suggesting that B-cell lymphopoiesis occurs not only in the head kidney (HK) but also in pancreatic tissue. In this agastric fish, the pancreas is diffused along the intestine and the early presence of IgM+ B-cells in pancreatic tissue might have a role in maintain immune homeostasis in the peritoneal cavity, making a substantial contribution to early protection. IgM-secreting cells in HK indicate the presence of systemic IgM at stage 5, before the first IgM+ cells were identified in mucosal sites. This work together with our previous study on T-cell development in this species indicates that although T- and B-cells start to develop around the same time, B-cells migrate to mucosal tissues ahead of T-cells. This early migration likely involves the production of natural antibodies, contributing significantly to early protection. Moreover, a diet composed of barnacle nauplii did not result in an earlier onset of B-cell lymphopoiesis, as seen in the previous study analysing T-cell development. Nevertheless, components for adaptive immunity indicating putative immunocompetence is likely achieved in early juveniles (>100 dph). Additionally, maternal transfer of IgM to the offspring is also described. These findings provide important insights into the development of the immune system in ballan wrasse and lay the foundation for optimizing prophylactic strategies in the future. Furthermore, this work adds valuable information to broaden the knowledge on the immune system in lower vertebrates.

The thymus and T-cell ontogeny in ballan wrasse (Labrus bergylta) is nutritionally modelled.

Marine fish larvae often experience high mortality unrelated to predation during early life stages, and farmed ballan wrasse (Labrus bergylta) is no exception. Knowing when the adaptive immune system is developed and fully functional, and how nutrition may modulate these processes is therefore of importance to establish effective prophylactic measures and will also extend the relatively limited knowledge on the immune system in lower vertebrates. The thymus anlage of ballan wrasse was found to be histologically visible for the first time at larval stage 3 (20-30 days post hatch, dph) and becomes lymphoid at stage 5 (50-60 dph) correlating with an increase of T-cell marker transcripts. At this stage, a clear zonation into a RAG1+ cortex and a RAG1- CD3ϵ+ medulla was distinguished, indicating that T-cell maturation processes in ballan wrasse are similar to other teleosts. The higher abundance of CD4-1+ compared to CD8ß+ cells in the thymus together with the apparent lack of CD8ß+ cells in gill, gut, and pharynx, where CD4-1+ cells were identified, indicates that helper T-cells have a more prominent role during larval development compared to cytotoxic T-cells. As ballan wrasse lacks a stomach but has an exceptionally high IgM expression in the hindgut, we hypothesize that helper T-cells are crucial for activation and recruitment of IgM+ B-cells and possibly other leukocytes to the gut during early development. Nutritional factors such as DHA/EPA, Zn and Se may lead to an earlier expression of certain T-cell markers as well as a larger size of the thymus, indicating an earlier onset of adaptive immunity. Including live feeds that supplies the larva with higher amounts of these nutrients can therefore be beneficial for ballan wrasse farming.

Soya saponins and prebiotics alter intestinal functions in Ballan wrasse (Labrus bergylta).

A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse (Labrus bergylta) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic (Aquate™ SG, 0·4 %) expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 - IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.

Enniatin B and beauvericin affect intestinal cell function and hematological processes in Atlantic salmon (Salmo salar) after acute exposure.

Unintentional use of mold-infested plant-based feed ingredients are sources of mycotoxins in fish feeds. The presence of the emerging mycotoxins ENNB and BEA in Norwegian commercial fish feeds and plant-based feed ingredients has raised concerns regarding the health effects on farmed Atlantic salmon (Salmon salar). Atlantic salmon pre-smolts were exposed to non-lethal doses of BEA and ENNB (ctrl, 50 and 500 µg/kg feed for 12 h), after which total RNA sequencing of the intestine and liver was carried out to evaluate gut health and identify possible hepatological changes after acute dietary exposure. ENNB and BEA did not trigger acute toxicity, however ENNB caused the onset of pathways linked to acute intestinal inflammation and BEA exposures caused the onset of hepatic hematological disruption. The prevalence and concentration of ENNB found in today's commercial feed could affect the fish health if consumed over a longer time-period.

Co-exposure to UV radiation and crude oil increases acute embryotoxicity and sublethal malformations in the early life stages of Atlantic haddock (Melanogrammus aeglefinus).

Crude oil causes severe abnormalities in developing fish. Photomodification of constituents in crude oil increases its toxicity several fold. We report on the effect of crude oil, in combination with ultraviolet (UV) radiation, on Atlantic haddock (Melanogrammus aeglefinus) embryos. Accumulation of crude oil on the eggshell makes haddock embryos particularly susceptible to exposure. At high latitudes, they can be exposed to UV radiation many hours a day. Haddock embryos were exposed to crude oil (5-300 µg oil/L nominal loading concentrations) for three days in the presence and absence of UV radiation (290-400 nm). UV radiation partly degraded the eggs' outer membrane resulting in less accumulation of oil droplets in the treatment with highest oil concentration (300 µg oil/L). The co-exposure treatments resulted in acute toxicity, manifested by massive tissue necrosis and subsequent mortality, reducing LC50 at hatching stage by 60 % to 0.24 µg totPAH/L compared to 0.62 µg totPAH/L in crude oil only. In the treatment with nominal low oil concentrations (5-30 µg oil/L), only co-exposure to UV led to sublethal morphological heart defects. Including phototoxicity as a parameter in risk assessments of accidental oil spills is recommended.

Beauvericin (BEA) and enniatin B (ENNB)-induced impairment of mitochondria and lysosomes - Potential sources of intracellular reactive iron triggering ferroptosis in Atlantic salmon primary hepatocytes.

Beauvericin (BEA) and enniatin B (ENNB) are emerging mycotoxins frequently detected in plant-based fish feed. With ionophoric properties, they have shown cytotoxic potential in mammalian models. Sensitivity in fish is still largely unknown. Primary hepatocytes isolated from Atlantic salmon (Salmo salar) were used as a model and exposed to BEA and ENNB (0.05-10 µM) for 48 h. Microscopy, evaluation of cell viability, total ATP, total H2O2, total iron content, total Gpx enzyme activity, and RNA sequencing were used to characterize the toxicodynamics of BEA and ENNB. Both mycotoxins became cytotoxic at ≥ 5 µM, causing condensation of the hepatocytes followed by formation of blister-like protrusions on the cell's membrane. RNA sequencing analysis at sub-cytotoxic levels indicated BEA and ENNB exposed hepatocytes to experience increased energy expenditure, elevated oxidative stress, and iron homeostasis disturbances sensitizing the hepatocytes to ferroptosis. The present study provides valuable knowledge disclosing the toxic action of these mycotoxins in Atlantic salmon primary hepatocytes.

Photo-enhanced toxicity of crude oil on early developmental stages of Atlantic cod (Gadus morhua).

Photo-enhanced toxicity of crude oil is produced by exposure to ultraviolet (UV) radiation. Atlantic cod (Gadus morhua) embryos were exposed to crude oil with and without UV radiation (290-400 nm) from 3 days post fertilization (dpf) until 6 dpf. Embryos from the co-exposure experiment were continually exposed to UV radiation until hatching at 11 dpf. Differences in body burden levels and cyp1a expression in cod embryos were observed between the exposure regimes. High doses of crude oil produced increased mortality in cod co-exposed embryos, as well as craniofacial malformations and heart deformities in larvae from both experiments. A higher number of differentially expressed genes (DEGs) and pathways were revealed in the co-exposure experiment, indicating a photo-enhanced effect of crude oil toxicity. Our results provide mechanistic insights into crude oil and photo-enhanced crude oil toxicity, suggesting that UV radiation increases the toxicity of crude oil in early life stages of Atlantic cod.

Cardiac dysfunction affects eye development and vision by reducing supply of lipids in fish.

Developing organisms are especially vulnerable to environmental stressors. Crude oil exposure in early life stages of fish result in multiple functional and developmental defects, including cardiac dysfunction and abnormal and smaller eyes. Phenanthrene (Phe) has a reversible impact on cardiac function, and under exposure Phe reduces cardiac contractility. Exposure to a known L-type channel blocker, nicardipine hydrochloride (Nic) also disrupts cardiac function and creates eye deformities. We aimed to investigate whether cardiac dysfunction was the major underlying mechanism of crude oil-, Phe- and Nic-induced eye malformations. We exposed Atlantic haddock (Melanogrammus aeglefinus) early embryos to Nic and crude oil (Oil) and late embryos/early larvae to Phe exposure. All three exposures resulted in cardiac abnormalities and lead to severe, eye, jaw and spinal deformities at early larval stages. At 3 days post hatching, larvae from the exposures and corresponding controls were dissected. Eyes, trunk, head and yolk sac were subjected to lipid profiling, and eyes were also subjected to transcriptomic profiling. Among most enriched pathways in the eye transcriptomes were fatty acid metabolism, calcium signaling and phototransduction. Changes in lipid profiles and the transcriptome suggested that the dysfunctional and abnormal eyes in our exposures were due to both disruption of signaling pathways and insufficient supply of essential fatty acids and other nutrients form the yolk.

Dietary Lipid Modulation of Intestinal Serotonin in Ballan Wrasse (Labrus bergylta)-In Vitro Analyses.

Serotonin (5-HT) is pivotal in the complex regulation of gut motility and consequent digestion of nutrients via multiple receptors. We investigated the serotonergic system in an agastric fish species, the ballan wrasse (Labrus bergylta) as it represents a unique model for intestinal function. Here we present evidence of the presence of enterochromaffin cells (EC cells) in the gut of ballan wrasse comprising transcriptomic data on EC markers like adra2a, trpa1, adgrg4, lmxa1, spack1, serpina10, as well as the localization of 5-HT and mRNA of the rate limiting enzyme; tryptophan hydroxylase (tph1) in the gut epithelium. Second, we examined the effects of dietary marine lipids on the enteric serotonergic system in this stomach-less teleost by administrating a hydrolyzed lipid bolus in ex vivo guts in an organ bath system. Modulation of the mRNA expression from the tryptophan hydroxylase tph1 (EC cells isoform), tph2 (neural isoform), and other genes involved in the serotonergic machinery were tracked. Our results showed no evidence to confirm that the dietary lipid meal did boost the production of 5-HT within the EC cells as mRNA tph1 was weakly regulated postprandially. However, dietary lipid seemed to upregulate the post-prandial expression of tph2 found in the serotonergic neurons. 5-HT in the intestinal tissue increased 3 hours after "exposure" of lipids, as was observed in the mRNA expression of tph2. This suggest that serotonergic neurons and not EC cells are responsible for the substantial increment of 5-HT after a lipid-reach "meal" in ballan wrasse. Cells expressing tph1 were identified in the gut epithelium, characteristic for EC cells. However, Tph1 positive cells were also present in the lamina propria. Characterization of these cells together with their implications in the serotonergic system will contribute to broad the scarce knowledge of the serotonergic system across teleosts.

Physical and nutrient stimuli differentially modulate gut motility patterns, gut transit rate, and transcriptome in an agastric fish, the ballan wrasse.

The effects of nutrient and mechanical sensing on gut motility and intestinal metabolism in lower vertebrates remains largely unknown. Here we present the transcriptome response to luminal stimulation by nutrients and an inert bolus on nutrient response pathways and also the response on gut motility in a stomachless fish with a short digestive tract; the ballan wrasse (Labrus berggylta). Using an in vitro model, we differentiate how signals initiated by physical stretch (cellulose and plastic beads) and nutrients (lipid and protein) modulate the gut evacuation rate, motility patterns and the transcriptome. Intestinal stretch generated by inert cellulose initiated a faster evacuation of digesta out of the anterior intestine compared to digestible protein and lipid. Stretch on the intestine upregulated genes associated with increased muscle activity, whereas nutrients stimulated increased expression of several neuropeptides and receptors which are directly involved in gut motility regulation. Although administration of protein and lipid resulted in similar bulbous evacuation times, differences in intestinal motility, transit between the segments and gene expression between the two were observed. Lipid induced increased frequency of ripples and standing contraction in the middle section of the intestine compared to the protein group. We suggest that this difference in motility was modulated by factors [prepronociceptin (pnoca), prodynorphin (pdyn) and neuromedin U (nmu), opioid neurotransmitters and peptides] that are known to inhibit gastrointestinal motility and were upregulated by protein and not lipid. Our findings show that physical pressure in the intestine initiate contractions propelling the bolus distally, directly towards the exit, whereas the stimuli from nutrients modulates the motility to prolong the residence time of digesta in the digestive tract for optimal digestion.

Effects of Cholecystokinin (CCK) on Gut Motility in the Stomachless Fish Ballan Wrasse (Labrus bergylta).

Cholecystokinin (CCK) is well-known as a key hormone that inhibits stomach emptying and stimulates midgut motility in gastric species. However, the function of CCK related to gut motility in agastric fish, especially in fish with a short digestive tract such as ballan wrasse, remains unknown. Here we present a detailed description of the spatio-temporal quantification of intestinal motility activity in vitro comprising the complete intestinal tract in ballan wrasse. We show that CCK modulates intestinal motility, having multiple effects on motility patterns depending on location in the gut and types of contractions. CCK reduced propagating contractions in the foregut, but it increased both non-propagating and propagating contractions in the hindgut. CCK also altered the direction of propagating contractions, as it reduced anterograde ripples and slow propagating contractions. The velocity of propagating contractions was slowed down by CCK. CCK also reduced the amplitude of standing contractions and ripples, but it did not alter the amplitude of slow propagating contractions. The presence of CCKA receptor antagonist modulated the motility responses of ballan wrasse intestines when exposed to CCK. We also showed that CCK reduced the intestinal length and stimulated motility to empty the gallbladder. Based on our findings we hypothesize that CCK, mainly through the CCKA receptor, modulates non-propagating and propagating contractions to optimize digestion and absorption and regulate the intestinal evacuation in ballan wrasse. We also found evidence that the modulation of intestinal motility by CCK is different in agastric fish from that in gastric vertebrates. We suggest that this is an evolutionary adaptation to optimize digestion without a stomach.

Loss of stomach, loss of appetite? Sequencing of the ballan wrasse (Labrus bergylta) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

BACKGROUND: The ballan wrasse (Labrus bergylta) belongs to a large teleost family containing more than 600 species showing several unique evolutionary traits such as lack of stomach and hermaphroditism. Agastric fish are found throughout the teleost phylogeny, in quite diverse and unrelated lineages, indicating stomach loss has occurred independently multiple times in the course of evolution. By assembling the ballan wrasse genome and transcriptome we aimed to determine the genetic basis for its digestive system function and appetite regulation. Among other, this knowledge will aid the formulation of aquaculture diets that meet the nutritional needs of agastric species. RESULTS: Long and short read sequencing technologies were combined to generate a ballan wrasse genome of 805 Mbp. Analysis of the genome and transcriptome assemblies confirmed the absence of genes that code for proteins involved in gastric function. The gene coding for the appetite stimulating protein ghrelin was also absent in wrasse. Gene synteny mapping identified several appetite-controlling genes and their paralogs previously undescribed in fish. Transcriptome profiling along the length of the intestine found a declining expression gradient from the anterior to the posterior, and a distinct expression profile in the hind gut. CONCLUSIONS: We showed gene loss has occurred for all known genes related to stomach function in the ballan wrasse, while the remaining functions of the digestive tract appear intact. The results also show appetite control in ballan wrasse has undergone substantial changes. The loss of ghrelin suggests that other genes, such as motilin, may play a ghrelin like role. The wrasse genome offers novel insight in to the evolutionary traits of this large family. As the stomach plays a major role in protein digestion, the lack of genes related to stomach digestion in wrasse suggests it requires formulated diets with higher levels of readily digestible protein than those for gastric species.

High dietary arachidonic acid levels induce changes in complex lipids and immune-related eicosanoids and increase levels of oxidised metabolites in zebrafish (Danio rerio).

This study explores the effect of high dietary arachidonic acid (ARA) levels (high ARA) compared with low dietary ARA levels (control) on the general metabolism using zebrafish as the model organism. The fatty acid composition of today's 'modern diet' tends towards higher n-6 PUFA levels in relation to n-3 PUFA. Low dietary n-3:n-6 PUFA ratio is a health concern, as n-6 PUFA give rise to eicosanoids and PG, which are traditionally considered pro-inflammatory, especially when derived from ARA. Juvenile zebrafish fed a high-ARA diet for 17 d had a lower whole-body n-3:n-6 PUFA ratio compared with zebrafish fed a low-ARA (control) diet (0·6 in the control group v. 0·2 in the high-ARA group). Metabolic profiling revealed altered levels of eicosanoids, PUFA, dicarboxylic acids and complex lipids such as glycerophospholipids and lysophospholipids as the most significant differences compared with the control group. ARA-derived hydroxylated eicosanoids, such as hydroxy-eicosatetraenoic acids, were elevated in response to high-ARA feed. In addition, increased levels of oxidised lipids and amino acids indicated an oxidised environment due to n-6 PUFA excess in the fish. To conclude, our results indicate that an ARA-enriched diet induces changes in complex lipids and immune-related eicosanoids and increases levels of oxidised lipids and amino acids, suggesting oxidative stress and lipid peroxidation.

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.

Transcriptional effects of metal-rich acid drainage water from the abandoned Løkken Mine on Atlantic salmon (Salmo salar) smolt.

Runoff of metals represents one of the major environmental challenges related to historic and ongoing mining activity. In this study, transcriptomics (direct RNA sequencing [RNA-seq] and reverse-transcription quantitative polymerase chain reaction [RT-qPCR]) was used to predict toxicity of metal-rich acid mine drainage (AMD) water collected in the abandoned copper (Cu) mine called Løkken Mine on Atlantic salmon liver and kidney, the main target organs of Cu-induced toxicity in fish. Smolts were exposed to control and diluted AMD water, which contains a mixture of metals but is especially enriched with Cu, at 4 concentrations in freshwater (FW) for 96 h, and then were transferred to and kept in seawater (SW) for another 24 h. Significant accumulation of Cu was observed in the gills, but not liver and kidney tissues, after 96 h of exposure. Short-term exposure to metal-rich ADM (high exposure group) significantly upregulated 3201 transcripts and downregulated 3782 transcripts in liver. The strongest effect attributed to exposure was observed on the KEGG pathway "protein processing in endoplasmic reticulum," followed by "steroid biosynthesis." Gene ontology (GO) analysis suggested that exposure predominantly affected "protein folding," possibly by disrupting disulfide bonds as a result of endoplasmic-reticulum-generated stress, and "sterol biosynthetic processes." Transfer to uncontaminated SW for 24 h amended the transcription of several genes, suggesting a transient effect of treatment on some mechanisms. In conclusion, the data show that trace metals in AMD from abandoned pyrite mines might disturb molecular mechanisms linked to protein folding in Atlantic salmon smolt endoplasmic reticulum.

Transcriptional responses to temperature and low oxygen stress in Atlantic salmon studied with next-generation sequencing technology.

BACKGROUND: Warmer seawater as a result of climate change may impose environmental challenges for Atlantic salmon aquaculture in its southernmost geographic range. Seawater temperatures above optimal level for growth may be reached in the warmest summer weeks. Caged fish can experience temperature and low oxygen saturation stress during such episodes, raising fish welfare and productivity concerns. In this work we compare the transcriptional responses in Atlantic salmon exposed to chronic high temperature (19°C) and low oxygen saturation (4-5 mg/L) stress. RESULTS: We used next-generation sequencing and RT-qPCR to screen for effects, and focused on growth regulation and oxidative stress in fish exposed to sub-optimal conditions. Both prolonged temperature (45 days) and low oxygen (120 days) stress had a significant negative effect on growth. The main effect of heat stress appears to be a general reduced transcriptional rate in salmon liver, while mechanisms typically associated with responses induced by chemical drugs were stimulated. Heat stress significantly down-regulated several transcripts encoding proteins involved in the protection against oxidative stress, including CuZn SOD, Mn SOD, GPx1 and GR, as well as additional stress markers HIF1A, CYP1A, MTOR and PSMC2 (RT-qPCR data). In salmon held at low oxygen concentration for four months protein ubiquitination (protein catabolism) was the most strongly affected pathway. According to the RT-qPCR data, low oxygen stress significantly up-regulated the transcriptional levels of IGFBP1B and down-regulated the levels of GR. Pathway analysis suggests that high temperature and low oxygen saturation stress affects many similar mechanisms in Atlantic salmon. Based on the gene lists, six out of the top ten predicted upstream transcriptional regulators, 1,2-dithiol-3-thione sirolimus, CD437, 5-fluorouracil, HNF4A and NFE2L2, were similar between the two treatments. CONCLUSIONS: In conclusion, temperature and low oxygen saturation stress affect many identical mechanisms in liver cells resulting in a metabolic depression, but these effects are not necessarily mediated through altered transcription of the same genes.

Is chemically dispersed oil more toxic to Atlantic cod (Gadus morhua) larvae than mechanically dispersed oil? A transcriptional evaluation.

BACKGROUND: The use of dispersants can be an effective way to deal with acute oil spills to limit environmental damage, however very little is known about whether chemically dispersed oil have the same toxic effect on marine organisms as mechanically dispersed oil. We exposed Atlantic cod larvae to chemically and mechanically dispersed oil for four days during the first-feeding stage of development, and collected larvae at 14 days post hatch for transcriptional analysis. A genome-wide microarray was used to screen for effects and to assess whether molecular responses to chemically and mechanically dispersed oil were similar, given the same exposure to oil (droplet distribution and concentration) with and without the addition of a chemical dispersant (Dasic NS). RESULTS: Mechanically dispersed oil induced expression changes in almost three times as many transcripts compared to chemically dispersed oil (fold change >+/-1.5). Functional analyses suggest that chemically dispersed oil affects partly different pathways than mechanically dispersed oil. By comparing the alteration in gene transcription in cod larvae exposed to the highest concentrations of either chemically or mechanically dispersed oil directly, the chemically dispersed oil affected transcription of genes involved nucleosome regulation, i.e. genes encoding proteins participating in DNA replication and chromatin formation and regulation of cell proliferation, whereas the mechanically dispersed oil most strongly affected genes encoding proteins involved in proteasome-mediated protein degradation. Cyp1a was the transcript that was most strongly affected in both exposure groups, with a 60-fold induction in the two high-exposure groups according to the RT-qPCR data, but no significant difference in transcriptional levels was observed between the two treatments. CONCLUSIONS: In summary, dispersants do not appear to add to the magnitude of transcriptional responses of oil compounds but rather appear to lower or modify the transcriptional effect on cod larvae.

Transcriptional evidence for low contribution of oil droplets to acute toxicity from dispersed oil in first feeding Atlantic cod (Gadus morhua) larvae.

We evaluated the potential contribution of oil droplets to the toxicity of dispersed oil to first feeding fish larvae. Atlantic cod larvae were exposed to five concentrations of either artificially weathered (200°C residue) dispersed oil (D1-D5) containing oil droplets [medium size 11-13 µm based on volume] and water-soluble fraction [WSF] or the filtered dispersions containing only the corresponding equilibrium WSFs only (W1-W5). The larvae were exposed for 4 days and harvested for transcriptional analysis at 13 days post hatching. The most significant differently expressed genes were observed in cod larvae exposed to the highest concentration of the dispersed oil (containing 10.41 ± 0.46 µg ∑PAH/L), with CYP1A showing the strongest response. Functional analysis further showed that the top scored network as analyzed with Ingenuity Pathway Analysis was "Drug Metabolism, Endocrine System Development and Function, Lipid Metabolism". Oil exposure also increased the expression of genes involved in bone resorption and decreased the expression of genes related to bone formation. In conclusion, oil exposure affects drug metabolism, endocrine regulation, cell differentiation and proliferation, apoptosis, fatty acid biosynthesis and tissue development in Atlantic cod larvae. The altered gene transcription was dominated by the WSF and the corresponding oil droplet fraction only had a moderate contribution to the observed changes.

Transcriptional responses in juvenile Atlantic cod (Gadus morhua) after exposure to mercury-contaminated sediments obtained near the wreck of the German WW2 submarine U-864, and from Bergen Harbor, Western Norway.

The main aim of the present work was to investigate the effects of mercury (Hg)-enriched sediments on fish. Sediments near the sunken German WW2 submarine U-864, which according to historical documents included 67 tons of metallic Hg in its cargo, are enriched of Hg leaking from the wreckage. Juvenile Atlantic cod (Gadus morhua) were exposed to two field-collected polluted sediments (U-864: inorganic Hg and Bergen Harbor (Vågen): inorganic Hg, PCB and PAH) or two comparable reference sediments for 5 weeks in the laboratory, and transcriptional responses evaluated in gills and liver. Gills of fish exposed to the Hg-enriched sunken WW2 submarine U-864 sediment contained four fold higher Hg levels compared to the control fish. An increase in Hg content in liver in the U-864 fish was also observed. The transcriptional results showed that calreticulin, HSP70 and heme oxygenase mRNA were significantly up-regulated in gills in fish exposed to the Hg-enriched sediments, whereas calreticulin, heme oxygenase, transferrin and WAP65 were significantly up-regulated and glutathione peroxidase 4B and zona pellucida 3 were significantly down-regulated in liver tissue. In gills and liver of cod exposed to the mixed-contaminated Vågen sediment, CYP1A showed the highest induction. In conclusion, the experiment shows that sediment-bound Hg is available to the fish and affects the transcription of oxidative stress responsive enzymes, suggesting that the Hg-enriched sediments may negatively affect the local wildlife. Furthermore, the mixed contaminated sediments of Vågen affected similar responses in addition to Ah-receptor mediated responses reflecting exposure to PAHs and PCBs.