Fertility of gynogenetic Atlantic cod (Gadus morhua L.)

In order to investigate whether meiotic gynogenetic Atlantic cod is fertile and able to produce viable offspring, meiotic gynogenetic females were produced in spring 2010 by activating cod eggs using irradiated sperm. The extrusion of the second polar body was prevented by the application of hydrostatic pressure (56.6 MPa) 36 min after fertilization. In February 2012, their mean round weight was 972 g, and 2580 g in March 2013. In 2012, when the fish were 2 years old, about 52% were mature, 33% were immature, and 13% had undifferentiated gonads. One year later, 77% were mature, 11% were immature, and 11% had undifferentiated gonads. Several of the mature females had malformed gonads, with only one developed ovary lobe or with the two lobes fused. The mean gonadosomic index (GSI) of the 2‐year‐old mature females was 5.2%, with an estimated relative fecundity of 581 000 eggs kg ovary‐free wet weight^?1. Females were stripped for eggs when 2 and 3 years old (2012 and 2013), and fertilized with sperm from normal males. Offspring were obtained from 12 of 17 and 12 of 15 egg batches incubated in 2012 and 2013, respectively, proving that the gynogenetic females are fertile. Furthermore, larvae in all but one of the hatched groups from 2013 had commenced feeding 2 h after being startfed using rotifers 4 days after hatch, indicating viable offspring.     

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.     

Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

Complement component C3 was isolated from the plasma of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.). Fast protein liquid chromatography (FPLC) techniques, involving ion exchange and gel filtration columns, were used. The purified proteins were analysed by SDS-PAGE which showed a two-chain structure, alpha- and beta-chains, as seen in higher vertebrates. Both proteins had intra-chain thioesters located within their alpha-chains and N-terminal amino acid sequencing confirmed their identity with reference to known C3 amino acid sequences from other species. Specific antibodies were prepared against cod and halibut C3 and tested in Western blotting on sera and purified C3. The proteolytic fragmentation of C3 was tested with trypsin, pepsin, papain and the extracellular product (ECP) from the bacterium Aeromonas salmonicida ssp. achromogenes (Asa). Both trypsin and papain were successful in cleaving C3 whereas pepsin and ECP had no effect. Carbohydrate moieties were detected in the alpha- and beta-chains of cod and halibut C3 and N-linked oligosaccharides were removed from the C3 with PNGase treatment, revealing a difference in C3 glycosylation between the two species.     

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.     

Isolation of two C-reactive protein homologues from cod (Gadus morhua L.) serum

Pentraxins are important molecules in innate defence and play a role in the acute phase response of both mammals and fish. Isolation of cod pentraxins by affinity chromatography using phosphorylcholine agarose revealed two pentraxin-like proteins, referred to as PI and PII proteins. These varied in their overall charge, pentameric and subunit molecular size, glycosylation and N-terminal amino acid sequences. The PI protein was homologous with the CRP-like pentraxin previously described in cod whereas the PII protein was a new CRP homologue, which was characterized by substantial individual heterogeneity with regard to subunit size and relative density. The results indicate considerable genetic variations in the cod pentraxins.     

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.     

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.     

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.     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

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.     

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.     

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.     

Immune parameters of immunised cod (Gadus morhua L.)

The immune response of cod (Gadus morhua L.) is unusual in that specific antibody response is limited or absent. In the present study cod was immunised with haptenated and non-haptenated protein antigen at two different temperatures and the antibody response monitored over a period of 18 months. Other humoral parameters of immunological importance were also analysed, namely total immunoglobulin concentration, anti-protease and spontaneous haemolytic activity. No specific antibody response was detected but increased activity of non-specific anti-TNP antibodies was observed 10-12 weeks after immunisation, irrespective of the antigen used. This antibody activity was attributed to the adjuvant used (FCA) and did not cross react with other antigens tested. Other parameters were probably not influenced by the immunisation but seasonal fluctuations were indicated. The immunoglobulin level appeared to peak in August-September and the anti-protease activity and the haemolytic activity in October-January.     

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.     

Site fidelity of Atlantic cod Gadus morhua L. as deduced from telemetry and stable isotope studies

A study was performed to test the hypothesis that Atlantic cod Gadus morhua captured within fjords do not migrate across the sill into outer fjord segments or coastal waters. An 18 month long telemetry experiment, corroborated by recapture patterns, showed that 40% of the tagged fish crossed the sill. The majority of the fish that crossed the sill, however, returned after short excursions of only a few days. Many fish remained stationary at or near the release location, and migrations up the fjord into brackish water appeared more frequent than migration out of the fjord. The stable isotope analyses showed significant differences between the inner and the outer fjord segments, and this was seen for both adults and juveniles. This suggests that the Atlantic cod in the two areas have different diets and probably utilize different foraging ranges. Thus, both the isotope studies and the telemetry experiment suggest limited cross-sill migration and rather high site fidelity. Marketing restrictions only apply to fish caught in the fjord proper, not the comparatively clean outer fjord system beyond the sill. Based on this study of migratory tendencies, it is concluded that contamination levels in Atlantic cod in both the Frierfjord proper and the outer fjords probably result from local exposure and appear little affected by inter-fjord migration.     

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.     

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.