Stage-specific mortality of Baltic cod (Gadus morhua L.) eggs

A study on cod egg mortality was carried out in the Bornholm Basin (southern central Baltic Sea) toward the end of July 1996. An initial egg aggregation marked by a satellite‐tracked drifter buoy was sampled repeatedly over an 11‐day period; profiles of temperature, salinity and dissolved oxygen were concurrently recorded. Three replicate estimates of mortality were obtained for each pair of subsequent developmental stages from newly spawned eggs to early larvae. A consistent pattern of stage‐specific mortality coincided well with previous experimental observations. Average daily mortality rates were 7.2% (eggs IA/IB), 38.7% (eggs (IB/II), 25.6% (eggs II/III), 40.0% (eggs III/IV), and 42.3% (eggs IV/early larvae). The cumulative mortality until hatch amounted to 99.9%. Results from hydrodynamic modelling, however, indicated that the drifter's trajectory was influenced by wind stress. Hence, the mortality rates might be biased despite the short sampling intervals; a modification of the sampling design is recommended for future studies.     

First evidence of spawning of eastern Baltic cod (Gadus morhua callarias) in the Belt Sea,the main spawning area of western Baltic cod (Gadus morhua L.)

Two cod stocks (western Baltic cod, WBC, and eastern Baltic cod, EBC) are managed in the Baltic Sea which is characterized by different main spawning areas and different main spawning periods. In this study we analyse the spatial and temporal occurrence of spawning individuals of both cod stocks in the main spawning grounds of the Baltic Sea based on eight microsatellite loci. Our results suggest that EBC (Gadus morhua callarias) has formed currently temporally stable, substantially homogeneous population not only in the Bornholm Sea (ICES SD: 25) but also in the Arkona Sea (ICES SD: 24). The presented analyses proved that EBC (G. m. callarias) can temporarily also spawn in the Belt Sea.     

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.     

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.     

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.     

Long-term stability and effective population size in North Sea and Baltic Sea cod (Gadus morhua)

DNA from archived otoliths was used to explore the temporal stability of the genetic composition of two cod populations, the Moray Firth (North Sea) sampled in 1965 and 2002, and the Bornholm Basin (Baltic Sea) sampled in 1928 and 1997. We found no significant changes in the allele frequencies for the Moray Firth population, while subtle but significant genetic changes over time were detected for the Bornholm Basin population. Estimates of the effective population size ( N _e ) generally exceeded 500 for both populations when employing a number of varieties of the temporal genetic method. However, confidence intervals were very wide and N _e 's most likely range in the thousands. There was no apparent loss of genetic variability and no evidence of a genetic bottleneck for either of the populations. Calculations of the expected levels of genetic variability under different scenarios of N _e showed that the number of alleles commonly reported at microsatellite loci in Atlantic cod is best explained by N _e 's exceeding thousand. Recent fishery-induced bottlenecks can, however, not be ruled out as an explanation for the apparent discrepancy between high levels of variability and recently reported estimates of N _e  << 1000. From life history traits and estimates of survival rates in the wild, we evaluate the compatibility of the species' biology and extremely low N _e / N ratios. Our data suggest that very small N _e 's are not likely to be of general concern for cod populations and, accordingly, most populations do not face any severe threat of losing evolutionary potential due to genetic drift.     

Growth and otolith microstructure of cod (Gadus morhua L.) larvae

Cod larvae from Irish Sea stocks were reared under differentgrowth conditions, and the otolith growth and increment formationexamined in sagittae and lapilli. Otolith increments were firstdeposited around the time of hatching and increment counts,on average, reflected larval age. The growth rate of fish larvaereared in different sized tanks was significantly different(P < 0.001), but there was no detectable effect on incrementformation. Otolith size was independent of larval size for individuals<5 mm in length. In larvae >6 mm, larger, faster growingindividuals had larger and faster growing otoliths.     

Sexual maturation of cod (Gadus morhua L.) in the southern Baltic (1990–2006)

Baltic cod, like other species, is susceptible to inter‐annual fluctuations in sexual maturation, depending on the length, age, sex, extent of the habitat area, and stock abundance of the cod population. Maturity is one of the biological indicators used to detect changes in a stock that can be caused by fishing. To address these issues specifically for the eastern Baltic cod stock, long‐term data (1990–2006) from Polish research vessels in the southern Baltic were examined. To date, the ICES has used the same maturity ogives over extended periods and assumed invariant sex ratios for the assessment of eastern Baltic cod. The combined maturity ogives calculated in the present study were markedly lower, particularly for age groups 2–4 (5), in all periods, than those used in the ICES assessment. Moreover, the proportion of females increased with length and age, suggesting that annual verification of the sex ratio is needed. The present study also revealed that the total length (L_50%) and the age (mean age‐at‐maturity; MAM_50%) at which 50% achieved first sexual maturity were higher for females than for males in the study period. The long‐term mean L_50% and MAM_50% for females were 43.9 cm and 4.3 years, respectively, and for males 34.8 cm and 3.4 years. There was also a spatial difference between calculated maturity ogives, with slightly lower L_50% (range: 1.4–8.6 cm) in the Gdańsk Basin than in the Bornholm Basin. The increasing trend in fishing mortality observed in 1993–2004 (ICES data) did not translate into a temporal trend in calculated maturity ogives. However, changes in L_50% and MAM_50% reflected recruitment variations (ICES data). The significance of these findings is discussed in the context of the environment and recruits abundance.     

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.     

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.     

Reproductive potential of the Eastern Baltic cod Gadus morhua callarias L. population

Trends in interannual variation in maturation and spawning terms of various age cohorts in the Eastern Baltic cod population in 1997-2009 were studied. Specific features in the age structure of the mature population part that were established by the end of the first decade of the 21st century were clarified. The role of cod age cohorts in the current population reproduction was considered taking into account the data on cod recruitment and fecundity.     

Epidemiology of pseudobranchial tumours in Atlantic cod(Gadus morhua) from the North Sea and the Baltic Sea

Investigations during several cruises (1979–1981) showed that Atlantic cod with pseudobranchial tumours occurred in all areas investigated. Statistical analysis of data revealed that there was a marked prevalence of the condition in the centre of the German Bight, whereas a lower incidence of the condition was observed on the Dogger Bank, off the Dutch coast and in the western Baltic Sea. The higher prevalence of pseudobranchial tumours in the German Bight coincides with a dense population of cod and impacts of pollution.     

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.     

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.     

Histology of pseudobranchial tumours in Atlantic cod(Gadus morhua) from the North Sea and the Baltic Sea

Atlantic cod bearing pseudobranchial tumours were investigated from both North Sea and Baltic Sea areas. Light and electron microscopic studies revealed a tumorous structure previously described in cod from Atlantic and Pacific waters. Additionally, cysts, multinucleated cells and early stages of mitosis are described. These findings support the hypothesis that pseudobranchial tumours are instigated by a protozoan infection causing xenotumours associated with the pseudobranch organ. The cells contained in these lesions showed similarities to amoebae.     

Male reproductive competition in spawning aggregations of cod (Gadus morhua, L.)

Reproductive competition may lead to a large skew in reproductive success among individuals. Very few studies have analysed the paternity contribution of individual males in spawning aggregations of fish species with huge census population sizes. We quantified the variance in male reproductive success in spawning aggregations of cod under experimental conditions over an entire spawning season. Male reproductive success was estimated by microsatellite-based parentage analysis of offspring produced in six separate groups of spawning cod. In total, 1340 offspring and 102 spawnings distributed across a spawning season were analysed. Our results show that multiple males contributed sperm to most spawnings but that paternity frequencies were highly skewed among males, with larger males on average siring higher proportions of offspring. It was further indicated that male reproductive success was dependent on the magnitude of the size difference between a female and a male. We discuss our results in relation to the cod mating system. Finally, we suggest that the highly skewed distribution of paternity success observed in cod may be a factor contributing to the low effective population size/census population size ratios observed in many marine organisms.     

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.