Identification of transferrin in Atlantic cod Gadus morhua epidermal mucus

The previously unreported presence of transferrin in Atlantic cod Gadus morhua epidermal mucus is described. A less destructive sampling method, which may result in decreased epidermal tissue damage, is discussed.     

Conserving marine biodiversity: insights from life-history trait candidate genes in Atlantic cod (Gadus morhua)

Recent technological developments have facilitated an increased focus on identifying genomic regions underlying adaptive trait variation in natural populations, and it has been advocated that this information should be important for designating population units for conservation. In marine fishes, phenotypic studies have suggested adaptation through divergence of life-history traits among natural populations, but the distribution of adaptive genetic variation in these species is still relatively poorly known. In this study, we extract information about the geographical distribution of genetic variation for 33 single nucleotide polymorphisms (SNPs) associated with life-history trait candidate genes, and compare this to variation in 70 putatively neutral SNPs in Atlantic cod (Gadus morhua). We analyse samples covering the major population complexes in the eastern Atlantic and find strong evidence for non-neutral levels and patterns of population structuring for several of the candidate gene-associated markers, including two SNPs in the growth hormone 1 gene. Thus, this study aligns with findings from phenotypic studies, providing molecular data strongly suggesting that these or closely linked genes are under selection in natural populations of Atlantic cod. Furthermore, we find that patterns of variation in outlier markers do not align with those observed at selectively neutral markers, and that outlier markers identify conservation units on finer geographical scales than those revealed when analysing only neutral markers. Accordingly, results also suggest that information about adaptive genetic variation will be useful for targeted conservation and management in this and other marine species.     

Persistent growth effects of temperature and photoperiod in Atlantic cod Gadus morhua

Short-term environmental manipulations during the early juvenile stage have a large impact on harvesting size of Atlantic cod Gadus morhua nearly 3 years later. A group of juvenile Atlantic cod (initial mass 9·5 g) were reared for 3 months under simulated natural photoperiod or continuous light, and a range of temperatures (7, 10, 13 and 16° C, and a group called T-step, i.e. with temperature reduced successively from 16 to 13 and 10° C). After termination of the laboratory trial, the fish were moved to sea pens and reared at ambient conditions for 30 months before harvesting in June 2006. Observed growth gain from the 3 month laboratory trial was still persistent following the 30 months of sea-pen ongrowing. The T-step group displayed 15, 13, 1 and 10% superior mass gain respectively than the groups initially at 7, 10, 13 and 16° C at harvest in June 2006. Similarly, rearing under continuous light during the initial 3 month period during the early juvenile stage resulted in 1–9% larger size at harvesting compared to fish reared at simulated natural photoperiod. Gonado-somatic and hepato-somatic indices were similar in all groups. Contribution to the understanding of the mechanism behind size variation in adult fish can have wide range applications for Atlantic cod fisheries and aquaculture.     

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)     

Stages of embryonic development in the Atlantic cod Gadus morhua

The early development of the Atlantic cod, Gadus morhua was studied from fertilization until first-feeding. Multiple families were reared at 7 degrees C and a developmental staging series was prepared using morphological landmarks visible with the light microscope. Stages were named rather than numbered to allow for future additions and broadly grouped into larger time intervals called periods. The most useful staging features were found to be initially cell number, and later in development, somite number. The mean cell cycle time for the first six cleavages was 135 min and the linear regression equation for development of somites(s) over time (t) was s = 0.29t - 18.14. The segmentation period began at 220 h postfertilization (hpf), and unlike some other teleosts, the addition of new somites continued throughout the majority of embryonic development, until just prior to hatching. Hatching occurred at 256 hpf, after which individuals remained motionless at the water's surface, undergoing negative phototaxis only after the first day posthatch. The first-feeding stage was reached at the end of the third day posthatch, subsequent to development of a functional jaw and hindgut. This staging series provides an essential baseline reference for future experiments involving developing cod embryos and for the aquaculture industry.     

Gut morphology of diploid and triploid Atlantic cod,Gadus morhua

Little is known about the functional consequences of triploidy in Atlantic cod. This study compared the gut morphology of diploid and triploid offspring of wild and selected broodstock. Three‐year‐old triploid offspring of wild cod (mean weight 1695 ± 346 g) had approximately 18% fewer pyloric caeca (125 ± 9 vs 172 ± 14, P < 0.001) and a 23% shorter intestine [Relative Gut Length, (RGL); 1.40 ± 0.17 vs 1.81 ± 0.17, P < 0.05] than their diploid siblings (mean weight: 1820 ± 262 g). Two‐year‐old triploid offspring of selected broodstock (mean weight: 640 ± 64 g) had 20% fewer pyloric caeca (309 ± 17 vs 387 ± 27, P < 0.001) but similar RGL to their diploid siblings (mean weight: 820 ± 69 g). The average number of mucus cells in the columnar epithelium of pyloric caeca was significantly higher in triploid than in diploid cod (54 ± 9 vs 25 ± 5, P < 0.001). There was no correlation between pyloric caeca number and RGL, or between mucus cells and pyloric caeca number, and no significant differences between sexes for any of the measured variables. Overall, the observations highlight some differences in the digestive system of these two ploidy groups that could have an influence on nutrient utilization and performance capacity in triploids compared to diploids.     

New haemoglobin genotypes in Atlantic cod, Gadus morhua: possible relation with growth

In a preliminary study, 121 individually tagged juvenile Atlantic cod (Gadus morhua) were classified according to their haemoglobin genotypes into four groups, i.e., two main haemoglobin genotypes [Hb-I(1/2), Hb-I(2/2)] and two sub-types [Hb-I(1/2b), Hb-I(2/2b)], and reared for 3 months at 10 degrees C, 13 degrees C and T-step (fish reared at 16 degrees C and then subsequently moved to 13 and later to 10 degrees C). Overall growth rates across temperatures were 10% and 19% higher in the Hb-I(2/2b), Hb-I(1/2b) sub-types compared to corresponding Hb-I(2/2) and Hb-I(1/2) main types, respectively. Individual growth rate trajectories varied between the genotypes at all temperatures studied. Our study indicates that under certain environmental conditions higher growth in the two sub-types compared to the main genotypes could be expected. This may indicate difference in other physiological characters not studied here, but seen in previous studies, i.e., oxygen affinity and competitive performance.     

Conformation and stability of elastase from Atlantic cod, Gadus morhua

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.     

Genomic signatures of local directional selection in a high gene flow marine organism; the Atlantic cod (Gadus morhua)

Background  

Marine fishes have been shown to display low levels of genetic structuring and associated high levels of gene flow, suggesting shallow evolutionary trajectories and, possibly, limited or lacking adaptive divergence among local populations. We investigated variation in 98 gene-associated single nucleotide polymorphisms (SNPs) for evidence of selection in local populations of Atlantic cod (Gadus morhua L.) across the species distribution.     

Excess post‐hypoxic oxygen consumption in Atlantic cod Gadus morhua

Atlantic cod Gadus morhua experienced oxygen deficit () when exposed to oxygen levels below their critical level (c. 73% of p_crit) and subsequent excess post‐hypoxic oxygen consumption (C_EPHO) upon return to normoxic conditions, indicative of an oxygen debt. The mean ± s.e . C_EPHO: was 6·9 ± 1·5, suggesting that resorting to anaerobic energy production in severe hypoxia is energetically expensive.     

Evaluation of candidate reference genes in Q-PCR studies of Atlantic cod (Gadus morhua) ontogeny, with emphasis on the gastrointestinal tract

To obtain reliable relative qPCR data in developing fish larvae, stable reference genes have to be found. This study is focused on finding good candidates for normalization of qPCR data for ontogenetic studies of Atlantic cod. Ten commonly used reference genes; Acidic ribosomal protein, Actin-related protein 2, beta-actin, Elongation factor 1 A, Glyceraldehyde-3-phosphate dehydrogenase, Ribosomal protein 37, Ribosomal protein 4, Ribosomal protein S9, beta 2-Tubulin and Ubiquitin were analyzed in developing larvae from 3 to 97 day post hatch (DPH). Two different tools were used to evaluate the stabilities of these genes; the geNorm software ranks the most stable genes based on a pair-wise analysis whereas NormFinder uses a model-based approach. The same genes were also analyzed in GI tract homogenates and compared to whole larvae homogenates. During Atlantic cod larval development there are several strong candidates with Ubiquitin as the most stable. The ribosomal proteins RPL4 and RPS9 are also strong candidates. RPL37 may be used but only when normalizing qRT-PCR results from one type of tissue. We also suggest the use of multiple genes for normalization of qRT-PCR. Our study suggests that whole-larvae samples can be used to study relative expression of genes that are expressed only in certain tissues.     

Organization of the mitochondrial genome of Atlantic cod, Gadus morhua.

The mitochondrial DNA (mtDNA) from the Atlantic cod, Gadus morhua, was mapped using 11 different restriction enzymes and cloned into plasmid vectors. Sequence data obtained from more than 10 kilobases of cod mtDNA show that the genome organization, genetic code, and the overall codon usage have been conserved throughout the evolution of vertebrates. Comparison of the derived amino acid sequences of proteins encoded by cod mtDNA to the ones encoded by Xenopus laevis mtDNA revealed that the amino acid identity range from 46% to 93% for the different proteins. ND4L is most divergent while COI is most conserved. GUG was found as the translation initiation codon of the COI gene, indicating a dual coding function for this codon. The sequences of the 997 base pair displacement-loop (D-loop)-containing region and the origin of L-strand replication (oriL), are presented. Only few of the primary and secondary structure features found to be conserved among mammalian mitochondrial D-loops, can be identified in cod. Presence of CSB-2 in the D-loop-containing region and the conserved hairpin structure at oriL, indicates that replication of bony fish mtDNA may follow the same general scheme as described for higher vertebrates.     

Evidence of microsatellite hitch-hiking selection in Atlantic cod (Gadus morhua L.): implications for inferring population structure in nonmodel organisms

Microsatellites have gained wide application for elucidating population structure in nonmodel organisms. Since they are generally noncoding, neutrality is assumed but rarely tested. In Atlantic cod (Gadus morhua L.), microsatellite studies have revealed highly heterogeneous estimates of genetic differentiation among loci. In particular one locus, Gmo 132, has demonstrated elevated genetic differentiation. We investigated possible hitch-hiking selection at this and other microsatellite loci in Atlantic cod. We employed 11 loci for analysing samples from the Baltic Sea, North Sea, Barents Sea and Newfoundland covering a large part of the species' distributional range. The 'classical' Lewontin-Krakauer test for selection based on variance in estimates of F(ST) and (standardized genetic differentiation) revealed only one significant pairwise test (North Sea-Barents Sea), and the source of the elevated variance could not be ascribed exclusively to Gmo 132. In contrast, different variants of the recently developed ln Rtheta test for selective sweeps at microsatellite loci revealed a high number of significant outcomes of pair-wise tests for Gmo 132. Further, the presence of selection was indicated in at least one other locus. The results suggest that many previous estimates of genetic differentiation in cod based on microsatellites are inflated, and in some cases relationships among populations are obscured by one or more loci being the subject to hitch-hiking selection. Likewise, temporal estimates of effective population sizes in Atlantic cod may be flawed. We recommend, generally, to use a higher number of microsatellite loci to elucidate population structure in marine fishes and other nonmodel species to allow for identification of outlier loci that are subject to selection.     

Use of substratum ripples for flow refuging by Atlantic cod, Gadus morhua

The ability to maintain position in a current without actively swimming (station-holding) was measured on substratum ripples for Atlantic cod, Gadus morhua, a bentho-pelagic fusiform species. The current velocities tested ranged from 0–111 cm sec^-1. Ripples were sinusoidal, with twelve combinations of ripple wavelength (10, 25, 50, 125 cm) and ripple amplitude (1.0, 2.5, 5.0 cm). Ripple wavelengths were chosen to approximate 0.5, 1.0, 2.0 and 5.0 times fish total length. The potential of ripples to locally retard current and thereby provide a refuge from the flow was measured as a velocity ratio, u_trough/u_free-stream, where u_trough is the flow velocity measured at a height of 0.5 cm from the bottom of a trough and u_free-stream the flow velocity measured at a height of 10 cm above ripple crests. Cod usually swam steadily above substratum ripple crests in the free-stream flow. They used substratum ripples to hold station on only 3 of the 12 ripples tested by refuging from the flow in the ripple troughs (flow refuging). These ripples had wavelengths approaching twice the body length, with ripple amplitudes that produced velocity ratios of 0.44–0.65, thus providing at least a 35% flow reduction in the troughs. In addition, these ripples were only used at intermediate velocities starting at 49–78 cm sec^-1 and ending at 81–109 cm sec^-1 depending on the ripple morphology, suggesting there may be costs involved in flow refuging, probably in stability control. Flow refuging on substratum ripples could dramatically impact the physiology and ecology of cod in high current velocities by providing areas of retreat for energetic savings, but also offering opportunities for enhanced feeding and migration.     

The demography of a haemoglobin polymorphism in the Atlantic cod, Gadus morhua L.

Genetic stability was investigated at a polymorphic haemoglobin gene-locus in 13 124 Atlantic cod, Gadus morhua L. A total of 30 year-classes, sampled over 18 years, were variously sampled in 33 named localities. Regional populations showed distinctive allele frequencies, and each major cod fishery showed some degree of genetic imbalance, which was expressed as excessive numbers of homozygotes. This imbalance occurred seasonally in a proportion of the population samples in each of the major cod fisheries. The findings are attributable to migrations of distinctive genetic populations.     

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.