The genome size and the structure and content of ribosomal RNA genes in Atlantic cod (Gadus morhua L.).

1. The haploid genome size of the Atlantic cod was estimated to 3.4 x 10(8)kb by reassociation kinetics analysis of cod sperm DNA. 2. The size of the small and large subunit ribosomal RNAs is 1.85 and 4.1 kb, respectively. 3. Restriction enzyme mapping of the rRNA coding unit revealed conservation of an Eco RI site in the coding regions of 18 S and 28 S rRNA and a Bam HI site in the 28 S rRNA coding region compared to other fish species. 4. The length of the repeat unit of the cod rDNA was found to be 30 kb. 5. The rRNA genes are repeated approximately 50 times in the cod genome and constitutes 0.08% of the cod genetic material.     

Neutrophils and B-cells in Atlantic cod (Gadus morhua L.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.     

Antimicrobial activity in the tissues of Atlantic cod (Gadus morhua L.)

Antimicrobial peptides (AMP) are important components of the innate immune system in metazoans. They have been studied widely in several fishes, but little is known about these defence factors in Atlantic cod, which is thought to have a less sophisticated adaptive immune system compared to other teleosts. The aim of the present study was to screen for potential AMPs in various tissues of Atlantic cod and to examine their spectra of activity. Acidic crude extracts were prepared from thirteen tissues (i.e. mucus, gills, skin, intestine, rectum, head kidney, spleen, blood, gall bladder, liver, ovary, muscle and peritoneal wall). Following partial purification by solid-phase extraction, 78 fractions were obtained and these were assayed for antimicrobial activity using a two-layer radial diffusion assay. Some of the fractions prepared from several tissues examined had potent activity against the test bacteria. In general, acetonitrile rich fractions displayed higher antibacterial activity than the aqueous ones. The most potent fractions were obtained from the gall bladder and they exhibited potent antimicrobial activity against 8 of the 9 test bacteria, including the cod pathogen Vibrio anguillarum. Antibacterial activity was completely eliminated or reduced upon treatment with proteinase K in most fractions. Protein profiles obtained by SDS-PAGE and two-dimensional gel electrophoresis showed that antimicrobial activity of the partially purified tissue extracts might be due to cationic, low molecular weight peptides.     

Induction of meiotic gynogenesis in Atlantic cod,Gadus morhua (L.)

Gynogenesis is one of several chromosome‐manipulating techniques used in fish. In gynogenesis the male does not contribute to the genetic material of the offspring, and the sperm cells act only as stimulators in order for the egg to start development. This technique has several applications, both in aquaculture and in biological research: gynogenetic fish may be used as a step in the production of all‐female populations, the production of isogenetic‐ and inbred lines, revealing of the sex determination mechanism, construction of genetic maps, and testing of environmental vs genetic control of different traits. The aim of this study was to develop a simple protocol for production of gynogenetic cod (Gadus morhua L.) for further use in aquaculture research. Various milt dilutions and UV‐irradiation doses were tested, in order to inactivate the sperm without destroying its ability to induce egg development. This was followed by pressure treatment of the eggs shortly after ‘fertilization’ to suppress the completion of meiosis II, and thereby restoring diploidy. A dose of 9000 erg mm^?2, followed by a 5‐min pressure treatment (58.6 MPa) 180 min‐degrees after fertilization gave 100% gynogenetic larvae. Histologically, sexual differentiated fish were all females, possibly confirming female homogamety in Atlantic cod. No particular signs of reduced growth, survival or enhanced deformity rates were observed after the fish had reached the juvenile phase. Mortality was, however, high during the egg and larval stages. This protocol has made capable the production of gynogenetic cod juveniles in significant amounts using relatively simple means; the next step will be to elaborate on the technique in order to produce mitotic gynogenetic (double haploid) individuals, which are 100% homozygous.     

Immunological differences in intestine and rectum of Atlantic cod (Gadus morhua L.)

The defence system of the distal gut (hindgut and rectum) of Atlantic cod, (Gadus morhua L.) was studied using (immuno)histochemical, electron microscopical and real-time quantitative PCR techniques. The uptake and transport of macromolecules in the intestinal epithelium was also investigated.In this study we observed that cod has many and large goblet cells in its intestinal epithelium and that IgM⁺ cells are present in the lamina propria and their number is considerably higher in the rectum than in the intestine. Myeloperoxidase staining revealed low numbers of granulocytes in and under the epithelium of the distal intestine, whereas high numbers were found clustered in the submucosa of the rectum. Electron microscopy not only confirmed these observations, but also revealed the presence of lymphoid cells and macrophages within the intestinal epithelium. Acid phosphatase staining demonstrated more positive macrophage-like cells in the rectum than in the distal intestine. Antigen uptake studies showed a diffused absorption of horse radish peroxidase (HRP) and LTB-GFP, whereas ferritin uptake could not be detected.Basal gene expression of cytokines (IL-1β, IL-8 and IL-10) and immune relevant molecules (hepcidin and BPI/LPB) were compared in both the intestine and rectum and revealed approximately 2–9 times higher expression in the rectum, of which IL-1β expression showed the most prominent difference.The present results clearly indicate that intestinal immunity is very prominent in the rectum of cod.     

Development of ten new EST-derived microsatellites in Atlantic cod (Gadus morhua L.)

Ten polymorphic microsatellite markers were developed from approximately 1,300 expressed sequence tags (ESTs) of Atlantic cod (Gadus morhua L.). Thirty two primer pairs were designed for EST sequences containing perfect di- tri- tetra- and pentanucleotide motifs and characterised in 96 unrelated fish. Ten markers were successfully amplified with number of alleles from 2 to 13 per locus and observed and expected heterozygosity ranging from 0.03 to 0.69 and 0.03 to 0.74, respectively. Loci Gmo-C131, C132 and C136 deviated from Hardy-Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C131 and Gmo-C132 and C128 and Gmo-C133. The gene identity was determined at five of the loci, confirming the associated microsatellites as Type I markers. The new microsatellites reported in this work can be used for conservation and enhancement of wild stocks for commercial harvesting. Jon-Ivar Westgaard and Tekle Tafese have contributed equally to the work.     

Development of twenty sequence-tagged microsatellites for the Atlantic cod (Gadus morhua L.)

Fifty-four primer pairs were designed for expressed sequence tag (EST) sequences containing perfect di- and tri-nucleotide motifs and characterised in 96 unrelated fish. Twenty markers were successfully amplified with number of alleles from 2 to 10 per locus and observed and expected heterozygosity ranging from 0.01 to 0.56 and 0.03 to 0.70, respectively. Loci Gmo-C213, Gmo-C246 and Gmo-C247 deviated from Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C213 and Gmo-C222, Gmo-C233 and Gmo-C229, C223 and Gmo-C236 and C229 and Gmo-C236. The gene identity was determined at 10 of the loci, confirming the associated microsatellites as Type I markers. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and constructing linkage maps of Atlantic cod.     

Characterization of 18 new microsatellite loci in Atlantic cod (Gadus morhua L.)

Eighteen new microsatellite loci consisting of 10 di‐, 5 tri‐, 2 tetra‐ and 1 heptanucleotide repeats are introduced for the Atlantic cod (Gadus morhua L.). All loci were co‐amplified in two polymerase chain reactions (plus two previously published microsatellites) and all products were typed clearly. The number of alleles per locus ranged from six (PGmo130) to 45 (PGmo76) and the observed heterozygosity ranged from 0.356 (PGmo130) to 0.957 (PGmo95). All loci except one followed Hardy–Weinberg expectations. Genetic linkage disequilibrium analysis between all pairs of loci did not yield any significant values.     

New polymorphic di-nucleotide microsatellite markers for Atlantic cod (Gadus morhua L.)

Twenty three polymorphic microsatellite markers were developed from approximately 2,300 expressed sequence tags (ESTs) of Atlantic cod (Gadus morhua L.). Seventy two primer pairs were designed for EST sequences containing perfect di-nucleotide motifs and characterised in 96 unrelated fish. Twenty three markers were successfully amplified with number of alleles from 2 to 18 per locus and observed and expected heterozygosity ranging from 0.03 to 1.00 and 0.04 to 0.90, respectively. Loci Gmo-C280, Gmo-C283, Gmo-C290 and Gmo-C293 deviated from Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C267 and Gmo-C269 and Gmo-C262 and Gmo-C291. The gene identity was determined at three of the loci, confirming the associated microsatellites as Type I markers. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and constructing linkage maps of Atlantic cod.     

Minisatellite DNA fingerprints of Atlantic cod (Gadus morhua)

The human minisatellite probes 33.6 and 33.15 cross–hybridized to Hae III and Hinf I digested cod DNA, revealing complex fragment patterns in both Arctic and coastal cod. The DNA fingerprints were highly polymorphic, individual specific and stable in the germline. The potential applications of multi locus probes in cod research are discussed.     

Variable region diversity of the Atlantic cod (Gadus morhua L.) immunoglobulin heavy chain

This study was undertaken to determine whether a lack of VH domain diversity could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod VH regions was studied in 113 cDNA and 2 genomic clones. A fourth VH family and a second putative JH element were identified. The expressed VH repertoire showed a clear bias in the pattern of VH family utilization, with about 80% of the clones belonging to the VH-III family. Furthermore, the VH-III family was complex and could be subdivided into several subfamilies, while little variation was seen within the other families. The VH family bias gives a somewhat reduced variability of the VH gene region of cod, but not lower than that of the rabbit IgM repertoire. The H chain CDR3 region of cod was longer than that of trout, frog and mouse, and also highly variable in sequence, probably reflecting a relative importance of this region in cod. On the other hand, the CDR3 length variability was restricted, and this may reduce the diversity of the cod VH region.     

Nodavirus in farmed Atlantic cod Gadus morhua in Norway

Viral encephalopathy and retinopathy (VER) was diagnosed in 5 to 24 g sized farmed Atlantic cod Gadus morhua kept in sea cages at Parisvatn, Hordaland county, on the west coast of Norway. Moderate mortality (10 to 15%) was observed, along with anorexia and abnormal swimming behaviour, such as looping or spiral swimming and reduced coordination. Nodavirus was detected by 2 different real-time RT-PCR assays, and this was later confirmed by immunohistochemistry. This is the first report of an outbreak of VER in farmed cod in Norway, and the first report that VER affect cod exceeding 5 g in size.     

The carbohydrate moiety of serum IgM from Atlantic cod (Gadus morhua L.)

The carbohydrate moiety of cod serum IgM was analysed using oligosaccharide sequencing techniques. The carbohydrate moiety constituted about 10% of the molecular weight of cod IgM, was associated with the constant region of the heavy chains (Fc), and was composed of N-linked complex type oligosaccharides. Considerable heterogeneity was observed. Sixteen different glycan structures were identified, over 60% were sialylated and 40% contained core fucose. The carbohydrate moiety of cod IgM was shown to provide protection against protease digestion, and partial deglycosylation abolished the antigen binding property of natural cod anti-TNP-BSA antibody.     

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.?noatunensis compared to saline formulation. The antigen epitope was a 20-25?kDa component of F.?noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.?noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.?noatunensis in cod.     

The intracellular lifestyle of Francisella noatunensis in Atlantic cod (Gadus morhua L.) leucocytes

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor^® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates egression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.     

Induction of meiotic gynogenesis in Atlantic cod (Gadus morhua L.) through pressure shock

The Atlantic cod, Gadus morhua, is one of the most important species for commercial fisheries and a promising candidate for aquaculture. Precocious sexual maturation of males is one of the major issues compromising large scale production. The potential approaches to this problem include production of all female populations. Consequently, the objective of this study was to develop an effective protocol to induce meiotic gynogenesis in the Atlantic cod by using hydrostatic pressure shock. Our first experiment tested the relevance of gamete quality on achievement of chromosome manipulation and identified the best time interval between fertilization and pressure shock. Our second experiment was designed to determine the optimal pressure value and duration of the pressure shock. Eight combinations of pressure values and durations were tested. Among them, the 34.47 MPa/6 min combination gave the best survival rate (23.6 ± 3.9%), the highest percentage of normal larvae (15.7 ± 3.6%), and the highest percentage of meiotic diploids (88.89%). In both experiments, haploid controls served as an indirect reference for paternal DNA inactivation. Chromosome counting confirmed the restoration of diploidy in gynogenetic fish. The present study optimizes a procedure for the induction of meiotic gynogenesis in the Atlantic cod, thus laying the basis for further applications towards producing monosex and defining the sex determination system.     

Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)