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.     

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)     

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.     

Diclidophora merlangi (Monogenea: Diclidophoridae) on Atlantic cod, Gadus morhua

Diclidophora merlangi (Kuhn, in Nordmann, 1832), as species specific to whiting, Merlangius merlangus (Linnaeus, 1758), is reported and described for the first time on the gills of Atlantic cod Gadus morhua (Linnaeus, 1758). Of the about 1,200 cod examined in this article, only 2 specimens of D. merlangi occurred on 2 fish, suggesting that they represented accidental infections. The 2 specimens showed specific traits of D. merlangi, but their size was considerably smaller than that of D. merlangi on whiting. Principal-component analyses showed that the morphology of the specimens on cod was more similar to that of D. merlangi than those of other congeneric species from the North Atlantic, additionally supporting their assignment to D. merlangi. Quantitative data documenting the potential reproductive consequences of D. merlangi occurring on cod is also provided. The testes and germaria of the specimens on cod were noticeably smaller than those of D. merlangi on whiting, but the number of testes was similar and spermatozoa were observed in the sperm duct. By contrast, the aspect and smaller size of the oocytes suggested that D. merlangi on cod could not produce viable ova, although 1 specimen exhibited an egg with normal sized and shaped capsule.     

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.     

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.     

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.     

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.     

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.     

Effect of turbidity on habitat preference of juvenile Atlantic cod, Gadus morhua

We examined the effects of turbidity on habitat preference of juvenile Atlantic cod in the laboratory, using a shuttle box where fish could select between two different habitats. In the first experiment, we compared three turbidity levels of kaolin (3, 8 and 21 beam attenuation m⁻¹). In the second experiment, we looked at the effect of turbidity media (kaolin versus algae), after controlling for spectral differences between turbidity media. Although cod preferred an intermediate turbidity of kaolin over low turbidity water, comparisons between low and high turbidity, and intermediate and high turbidity did not significantly influence habitat preference. Algae did not influence habitat preference by cod. Although other studies have found that turbidity affects both foraging and antipredator behaviour of juvenile cod, this study has shown that gradients in turbidity per se do not have a strong effect on their habitat preference.     

Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhua L.) on larval survival and development

Aims: To assess the effects of bacterial treatment at the earliest stages of cod rearing on the microbial load, larval development and performance, testing three bacterial strains (Carnobacterium divergens V41, Arthrobacter sp. and Enterococcus sp.) in vivo that were previously shown to have inhibitory potential towards fish pathogens in vitro. Methods and Results: A bacterial mixture was added eight times to the rearing water from the prehatch to the mid‐larval stage (a 38‐day period). Microbiological analysis of ova, larvae and rearing water was performed regularly. Larval performance and development were evaluated by survival rate, hypersalinity tolerance and physiological measurements. Different larval survival rates were observed within and between treatments, and possibly explained by variations in larval microflora and established probionts. Larvae from one silo, which had been bathed in the bacterial suspension, showed the highest survival rate (42·1%), lowest Vibrio levels, and were significantly heavier (19·3%) and more stress tolerant than control larvae (P < 0·01). This coincided with the intestinal establishment of two of the tested bacteria. Conclusions: Arthrobacter and Enterococcus strains added regularly to the rearing water from the postfertilized egg stage can become established in larval gastrointestinal tract. The Enterococcus strain was associated with increased larval growth, performance and microflora control, indicating its probiotic nature. Significance and Impact of the Study: Regular application of autochthonous probionts may promote larval welfare, development and stress tolerance at early stages, hence increasing production yield in intensive cod larviculture.     

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.     

Susceptibility of juvenile Atlantic cod Gadus morhua to viral haemorrhagic septicaemia virus isolated from wild-caught Atlantic cod

A European strain of viral haemorrhagic septicaemia virus (VHSV) isolated from wild-caught cod Gadus morhua (H16/7/95) was shown to cause clinical disease and mortality in excess of 80% in juvenile Atlantic cod when administered by the intra-peritoneal (i.p.) route. No virus was recovered from cod cohabiting with experimentally infected fish at a ratio of 1:1, and no VHSV-associated mortality was demonstrated following immersion infection. External signs of disease in cod were the presence of exophthalmia and ascites. Virus was identified as VHSV by enzyme-linked immunosorbent assay (ELISA) and was recovered from both brain and organ pools (kidney, liver and spleen) of 100% of i.p. infected cod mortalities. Virus was also detected using an indirect immunofluorescence test on tissue imprints of kidney, liver, spleen and brain taken from moribund fish. The fact that cod were not susceptible to VHSV following waterborne exposure raises important questions surrounding the propagation, maintenance and impact of a naturally occurring reservoir of virus in the marine environment.     

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.     

Nematode infestation of fillets from Atlantic cod, Gadus morhua, off eastern Canada

The prevalence and intensity of larval nematodes in fillets of Atlantic cod, Gadus morhua, were examined and compared with similar data from a previous survey conducted about 30 yr ago. Anisakis simplex occurred more often in the nape of the fillet, whereas Pseudoterranova decipiens was the predominant species in napeless fillets. The results suggest an increase in both the prevalence and intensity of P. decipiens in fillets of cod, especially those originating from the Gulf of St. Lawrence and coastal Nova Scotia.     

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.     

Predicting variability in catch-per-effort in Atlantic cod, Gadus morhua, trap and gillnet fisheries

The relationships between catch rates and fish abundance, hydrographic conditions and fishing effort, for Atlantic cod caught by trapnets (fixed gear) and gillnets (non-fixed gear) in the northern Gulf of St Lawrence have been quantified. Daily changes in trap catch rates were accounted for by changes in salinity, currents and mean local cod densities in 1985 ( R ²= 0.78), and predicted 1986 trap catch rate changes (by 1985 model) were correlated significantly with those observed ( r = 0.60, P < 0.05). In contrast, the daily changes in 1985 gillnet catch rates were determined by currents, maximum (not mean) local cod densities, and fishing effort (negative) ( R ²= 0.68), while predicted 1986 gillnet catch rates (by 1985 model) were significantly correlated with those observed ( r = 0.35, P < 0.05). Seasonal catchability coefficients of the traps were similar in 1985 and 1986, but for gillnets this index was an order of magnitude higher in 1986 than in 1985.