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.

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.

A cohort study of gill infections, gill pathology and gill-related mortality in sea-farmed Atlantic salmon (Salmo salar L.): A descriptive analysis.

Gill disease is an important cause of economic losses, fish mortality and reduced animal welfare in salmonid farming. We performed a prospective cohort study, following groups of Atlantic salmon in Western Norway with repeated sampling and data collection from the hatchery phase and throughout the 1st year at sea. The objective was to determine if variation in pathogen prevalence and load, and zoo- and phytoplankton levels had an impact on gill health. Further to describe the temporal development of pathogen prevalence and load, and gill pathology, and how these relate to each other. Neoparamoeba perurans appeared to be the most important cause of gill pathology. No consistent covariation and no or weak associations between the extent of gill pathology and prevalence and load of SGPV, Ca. B. cysticola and D. lepeophtherii were observed. At sea, D. lepeophtherii and Ca. B. cysticola persistently infected all fish groups. Fish groups negative for SGPV at sea transfer were infected at sea and fish groups tested negative before again testing positive. This is suggestive of horizontal transmission of infection at sea and may indicate that previous SGPV infection does not protect against reinfection. Coinfections with three or more putative gill pathogens were found in all fish groups and appear to be the norm in sea-farmed Atlantic salmon in Western Norway.

Dietary Selenomethionine Reduce Mercury Tissue Levels and Modulate Methylmercury Induced Proteomic and Transcriptomic Alterations in Hippocampi of Adolescent BALB/c Mice.

Methylmercury (MeHg) is a well-known environmental contaminant, particularly harmful to the developing brain. The main human dietary exposure to MeHg occurs through seafood consumption. However, seafood also contains several nutrients, including selenium, which has been shown to interact with MeHg and potentially ameliorate its toxicity. The aim of this study was to investigate the combined effects of selenium (as selenomethionine; SeMet) and MeHg on mercury accumulation in tissues and the effects concomitant dietary exposure of these compounds exert on the hippocampal proteome and transcriptome in mice. Adolescent male BALB/c mice were exposed to SeMet and two different doses of MeHg through their diet for 11 weeks. Organs, including the brain, were sampled for mercury analyses. Hippocampi were collected and analyzed using proteomics and transcriptomics followed by multi-omics bioinformatics data analysis. The dietary presence of SeMet reduced the amount of mercury in several organs, including the brain. Proteomic and RNA-seq analyses showed that both protein and RNA expression patterns were inversely regulated in mice receiving SeMet together with MeHg compared to MeHg alone. Several pathways, proteins and RNA transcripts involved in conditions such as immune responses and inflammation, oxidative stress, cell plasticity and Alzheimer's disease were affected inversely by SeMet and MeHg, indicating that SeMet can ameliorate several toxic effects of MeHg in mice.

Analysis of immunoglobulin and T cell receptor gene expression in ballan wrasse (Labrus bergylta) revealed an extraordinarily high IgM expression in the gut.

The serum IgM concentration of ballan wrasse is relatively high, estimated to approximately 13 mg/ml in adult wild fish of 800 g. The present study revealed an unusual high abundance of IgM mRNA in the gut of ballan wrasse. Initially, transcripts encoding IgM, IgT, IgD, TCRα, TCRδ and CD3ε were quantified by RT-qPCR in several tissues of wild caught fish (approx. 800 g), indicating an elevated immune activity in hindgut and an extraordinarily high expression of IgM. Subsequently, a new RT-qPCR analysis was performed on the entire intestine, cut into four different segments, of reared fish (32-100 g). The analysis indicated immune activity along the entire intestine, but not as strong as in the hindgut. Furthermore, similar to the larger fish, the relative abundance of IgM transcripts was higher in the hindgut than in kidney and spleen, although the absolute level of IgM was in general higher in the larger fish. The secreted form of IgM was completely dominant in comparison to the membrane bound form of IgM and the other analysed genes. IgM was purified from gut mucus and external mucosal surfaces by magnetic beads coated with protein A. Mucus IgM reacted with rabbit antisera raised against serum IgM and contained subunits of the same size. Regarding the elevated immune activity in the intestine it is tempting to speculate on a possible compensatory strategy in this lineage of stomach-less fish, and that natural antibodies have an important role in the first line defence.

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.

Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae.

UNLABELLED: Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures. IMPORTANCE: Aquaculture is an increasingly important global source of high-quality food. To sustain the growth in aquaculture, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wildlife is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases that they cause. There is a possibility that viruses with enhanced virulence may spread to new species, as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.

Characterization of IgM in Norwegian cleaner fish (lumpfish and wrasses).

The use of cleaner fish in Norwegian aquaculture has to a large extent been based on wild catches, but breeding of lumpfish and ballan wrasse is currently increasing. Due to disease problems and required vaccine development, tools to study immune responses and a better understanding of the immune system in these species is demanded. The present study comprises lumpfish (Cyclopterus lumpus) and five species of wrasses: Ballan wrasse (Labrus bergylta), rock cook (Centrolabrus exoletus), cuckoo wrasse (Labrus mixtus), corkwing wrasse (Symphodus melops), and goldsinny wrasse (Ctenolabrus rupestris). We present a comparison of the IgM sequences, phylogenetic relationship to other teleosts and characteristic features of IgM in the species studied. The lumpfish IgM heavy chain sequence was assembled from high throughput cDNA sequencing whereas the wrasse sequences were determined by molecular cloning. The secreted form of the IgM heavy chain from all species consisted of four constant Ig domains. IgM was purified from lumpfish and ballan wrasse sera by gel filtration followed by anion exchange chromatography, and polyclonal sera were produced against these proteins. Antisera against ballan wrasse IgM showed cross-reactivity to all analyzed species of wrasses, some cross-reactivity to lumpfish, very low reaction to salmon, and no reaction to cod. Anti- IgM sera against lumpfish cross-reacted to the light chain of all species studied. Wrasses and lumpfish IgM showed high binding affinities for protein A. IgM concentration in adult ballan wrasse (700-800 g) was measured by single radial immunodiffusion assay and found to be 13.4 mg/ml which is about 36% of the total protein concentration. The IgM concentration in lumpfish (600-3600 g) was estimated to 1-2.6 mg/ml, which corresponds to approximately 3% of the total protein concentration.

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.

Vitamin A and arachidonic acid altered the skeletal mineralization in Atlantic cod (Gadus morhua) larvae without any interactions on the transcriptional level.

The main object of this study was to evaluate the impact of different levels of vitamin A (VA) and arachidonic acid (ARA) in relation to eicosapentaenoic acid (EPA) on mineralization and gene expression in Atlantic cod larvae (Gadus morhua). First-feeding larvae were fed enriched rotifers from start-feeding until 29 days post hatch (dph). Larvae in four tanks were fed one of the following diets: control (EPA/ARA ratio: 15.8, 0.9µg VA g(-1)), control+VA (EPA/ARA ratio: 15.8, 7.8µg VA g(-1)), High ARA (EPA/ARA ratio: 0.9, 1.5µg VA g(-1)) or High ARA+VA (EPA/ARA ratio: 0.9, 12.0µg VA g(-1)). Larvae fed High ARA+VA were shorter at 29dph compared to the other groups and had significantly less mineralized bones when comparing larvae of similar size, showing interaction effects between VA and ARA. Although transcriptomic analysis did not reveal any interaction effects, a higher number of genes were differentially expressed in the high ARA fed larvae compared to control+VA fed larvae. Furthermore, bglap1, bglap2 and col10a1 were all down-regulated in larvae fed High ARA-diets and to a greater extent than larvae fed VA supplemented diet, indicating an additive effect on mineralization. In conclusion, this study showed that the dietary increase in ARA and VA altered the skeletal metabolism during larval development, most likely through signaling pathways specific for each nutrient rather than an interaction. The present study also demonstrates that VA could affect the larval response to ARA, even within the accepted non-toxic/non-deficient range.

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.

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.

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.

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.

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.

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.

Pathogen recognition and mechanisms in Atlantic cod (Gadus morhua) head kidney cells: bacteria (LPS) and virus (poly I:C) signals through different pathways and affect distinct genes.

Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (polyinosinic acid:polycytidylic acid), bacterial (lipopolysaccharide) and polyclonal activator (phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of interleukin 8; interleukin-1ß; cyclooxygenase 2; leukocyte derived chemotaxin 2; carboxyl-esterase 2 and environmental biomarker cytochrome P450 1A. Mitogen activated protein kinase p38 and cathepsin F were down regulated by LPS. The antiviral responses induced by double stranded RNA clearly increased transcription of Toll like receptor 3 and interferon stimulating gene 15. The phytohaemoagglutinin response seemed to be more non-specific. Special for the phytohaemoagglutinin induction was the increase in major histocompatibility complex class I. CC chemokine type 2 mRNA expression was increased by phytohaemoagglutinin, lipopolysaccharide and polyinosinic acid:polycytidylic acid, while mitogen activated protein kinase p38 and leukocyte derived chemotaxin 2 were down regulated by phytohaemoagglutinin. Oxidative stress related genes like catalase and glutaredoxin and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Eicosanoids like prostaglandin 2, leukotriene B4 and B5 were constitutively produced by cod head kidney cells in vitro. The most remarkable feature of eicosanoid secretion is the higher production of leukotrienes against prostaglandins, indicating that the lipooxygenase pathway is preferred over the cyclooxygenase pathway. Although there were no significant differences in eicosanoid secretion between the groups, polyinosinic acid:polycytidylic acid showed a clear tendency to increase the levels of leukotriene B4 and B5. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that cytochrome P450 1A was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor activation in Atlantic cod.