Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.     

The escape response of food-deprived cod larvae (Gadus morhua L.)

The escape response of Atlantic cod larvae (Gadus morhua) 25 and 47 days post hatch (dph) - either fed or deprived of food for three days - was studied. Larval escape responses were provoked by water movement from the suction of a fixed-position pipette. Escape latency, distance, speed, burst speed, and vertical and lateral escape angles were quantified using motion tracking software designed for 3-D silhouette video recordings. Escape performance, expressed as escape distance and escape speed, improved with age. The escape angles were normally distributed and highly variable, ranging from − 170° to 170° and − 40° to 105° for lateral and vertical escape angles respectively. No food deprivation-induced effects in any of the behaviours were found, suggesting that there are no condition-related behavioural effects (size-independent effects) in escape response performance after 3 d of food deprivation. This may reflect a negligible difference in the cost/benefit equation for fed vs. food-deprived larvae in performing an escape response when under attack.     

Effect of somatic and otolith growth rate on stable isotopic composition of early juvenile cod (Gadus morhua L) otoliths

The relative amounts of the stable isotopes of carbon and oxygen in fish otoliths can be used to reveal the environmental history experienced by the fish. This requires that the relative amounts of the isotopes are deposited in equilibrium with the surrounding environment, or that the offset from this equilibrium is known and can be quantified. It is known that carbon isotopes in biogenic carbonates are a mixture of carbon from the seawater and metabolically derived carbon, but the effect of the somatic growth rate of the fish is still unclear. The possible effect of otolith growth rate and fractionation of both carbon and oxygen isotopes are also not established. We carried out a controlled laboratory experiment where we reared cod (Gadus morhua L.) larvae and early juveniles at two temperatures (6 and 10 °C) and generated different growth rates within each temperature by manipulation of prey levels. The otoliths of the resulting fish were analysed for carbon and oxygen isotopes. We found no effect of otolith precipitation rate on fractionation of either carbon or oxygen isotopes. However, there was a depletion of ¹³C in the otoliths of fish with elevated metabolism. The proportion of metabolically derived carbon in the otoliths was estimated to be 28-32%. Our results suggest that measurements of oxygen isotopes in otoliths can be a reliable tool to estimate ambient temperature since the oxygen isotopes seem to be deposited in the otoliths independently of kinetic and metabolic effects. Fractionation of carbon isotopes in otoliths on the other hand can give valuable insight into metabolism and feeding pattern of fish.     

Characterization of a collagenolytic serine proteinase from the Atlantic cod (gadus morhua)

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH₄)₂SO₄ fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (±0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50°C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30°C.     

Structural studies of the O-antigenic oligosaccharide from Vibrio salmonicida strain C2 isolated from Atlantic cod, Gadus morhua L

We report the chemical structure of the oligosaccharide part of Vibrio salmonicida lipopolysaccharide serotype C2, isolated from Atlantic cod (Gadus morhua L.). The structure was established by NMR spectroscopy and mass spectrometry. It is concluded that the oligosaccharide has the following structure in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, and PEA is phosphoethanolamine. [chemical structure: see text]     

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.     

Microscopic rate-constants for substrate binding and acylation in cold-adaptation of trypsin I from Atlantic cod

Temperature imposes limits on where life can thrive and this is evident in the evolution of the basic structural properties of proteins. Cold-adaptation of enzymes is one example, where the catalytic rate constant (k(cat)) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (k(cat)/K(m)) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin. Here, the individual rate-constants for association of substrate (k(1)), dissociation of substrate (k(-1)), and acylation of the enzyme (k(2)) have been determined using benzoyl-Arg-p-nitroanilide or benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide as substrates. Rather unexpectedly, by far the largest difference (37-fold increase) was observed in k(1), the rate constant for binding of substrate. The cold-adaptation of the dissociation and catalytic steps were not as prominent (increased by 3.7-fold). The length of substrate did have an effect by increasing the reaction rate by 70-fold, and again, the step most affected was the initial binding-step.     

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Specific growth rate and proximate body composition of Atlantic cod (Gadus morhua L.)

The effect of growth rate and maturation on the proximate composition and energy content of Atlantic cod, Gadus morhua L., was investigated over 10 months for each of two consecutive years, 1978–1980 at 5 and 8 °C. Relative energy and lipid content of whole cod increased with specific growth rate for all three sampling periods (November, January, March), each at 5 and 8 °C. Relative water content decreased with specific growth rate and temperature, and was lower in March than in January and November. Relative protein content was positively correlated with specific growth rate, but to a lesser degree than with temperature and age. Relative ash content was negatively correlated with specific growth rate. The effect of season and temperature on the proximate content of gonad, liver, muscle, and carcass was also determined. The major energy and lipid source in cod was the liver. Energy, lipid, and water were highly correlated to each other, and regressions are provided to allow for their prediction, given one of the components. Energy budgets for cod at 5 and 8 °C are calculated and the effect of increased ration size on the budget is estimated. The prediction of short-term specific growth rates of cod from the proximate composition is proposed. The proximate composition of cod is affected by growth rate and thus feeding level, and in turn directly affects behaviour. The relative proximate content of maturing and immature 3-yr-old cod was not found to be significantly different. Keywords: specific growth rate; proximate; bioenergetics; Atlantic cod; energy budget; temperature; season     

A molecular analysis of some Eastern Atlantic grouper from the Epinephelus and Mycteroperca genus

Mitochondrial cytochrome b (397 bp) and 16S rDNA (516 bp) sequences analysis was used to investigate the phylogenetic relationships among some Eastern Atlantic Epinephelinae species. Six species of Epinephelus (E. aeneus, E. caninus, E. costae, E. haifensis, E. marginatus and E. tauvina) and two species of Mycteroperca (M. rubra and M. fusca) were analysed. Neighbour-joining and maximum-parsimony analysis support the paraphyletic grouping of the Epinephelus and Mycteroperca analysed. The maximum pairwise nucleotide divergence value in cyt b among all taxa was 0.196 between E. aeneus and E. marginatus and the minimum value was 0.006 between E. costae and M. rubra. Meanwhile, in 16S sequence analysis, the maximum value is 0.093 between E. aeneus and E. tauvina and the minimum value is 0.011 between E. marginatus and M. rubra. Molecular clock estimates for the species suggest a divergence time of 20-24 mya, which coincides with the Miocene period. A molecular analysis was also conducted, using other Epinephelinae sequences from GenBank in order to improve our understanding of the phyletic status of the Epinephelus and Mycteroperca species analysed.     

Cross-shoal variability in the feeding habits of migrating Atlantic cod (Gadus morhua)

Prey intake and selection were related to within-shoal position for Atlantic cod (Gadus morhua) engaged in annual migration across the Newfoundland shelf in the northwest Atlantic. Comparisons made among fish occupying five regions from the front to rear of a large (>10 km across) migrating shoal indicated that leading fish, or scouts, were larger, ate more food by weight, and had a more varied diet than did fish at other positions. Also, scouts consumed more preferred prey types (fish and pelagic invertebrates) than did fish at other positions. In contrast, trailing fish consumed few fish prey but a larger proportion of benthic invertebrates. Our results are the first to document systematic heterogeneous feeding success among members of a free-ranging and migrating fish shoal in the open ocean.     

Linking population genetics and growth properties of Atlantic cod

It is strongly implicated that cod in the NorthAtlantic Ocean is sub-structured at a smallgeographic scale exemplified by studies fromCanadian, Icelandic, and Norwegian waters. Inthe first part of this review, we reviewedpopulation genetics studies in these threeareas and our conclusion is that, despite someinconsistencies in the numerous genetic studiesof cod in Norwegian and Icelandic waters, andthe northwest Atlantic, these studiesillustrate that cod in the investigated areasconsists of several distinct populations, bothwithin and between areas. However, tounderstand the contradictory results obtainedin some of the studies discussed in thisreview, more knowledge about the influence ofnatural selection, mutation, and genetic drifton the genetic material of cod is necessary.Such knowledge could guide us to the markersgiving the best illustration of the geneticstructure in these areas. Identifying andgenetically characterizing wild stocks areessential steps for their conservation, sinceoverexploitation of genetically differentpopulations can lead to the loss of geneticvariability and productivity in subsequentgenerations.Motivated by the hypothesis that growthpatterns may reflect specific genotypeadaptations, we reviewed stock specificresponses on growth in the second part of thisreview and try to link these with the differentlife histories within the different stock unitsindicated in the first part of the review. Anexample of genetically-based differencesbetween population units at two spawninglocalities off south Iceland is discussed.Studies have shown conflicting results,depending on which side of the Atlantic theproblem has been investigated. We propose thata common-garden meta-analysis with several codstocks from both sides of the Atlantic isneeded to give any reasonable answer to thequestion of genetically-based growthdifferences.In this review, we have not tried to quantifyhow large the environmental part of growthregulation is versus the genetic part, as thisinformation is not available in the publishedliterature on cod. Based on recent research ontwo flatfish species (turbot and Atlantichalibut), approximately 30% of growthvariation is caused by genetic factors, but itremains to be seen if this is similar in cod.     

Activity patterns of juvenile Atlantic cod (Gadus morhua) in Buckley Cove,Newfoundland

We monitored swimming speed of 2–3 year-old juvenile Atlantic cod (Gadus morhua) from August to December 1999, using a 2-D location finding acoustic telemetry system in a coastal area of Newfoundland, Canada. We concurrently monitored the locations of 22–41 individuals by triangulation using a fixed hydrophone array. We estimated average swimming speeds at intervals of 60–120 s and compared them over a 1 to 17 °C thermal range, three diel periods, and five substrates (sand, gravel, sand-sparse boulder, boulder, and kelp). However, cod did not exhibit a change in swimming speed over the temperature range studied. Increased activity and foraging rates (expressed as swimming speeds) were expected to increase at elevated temperatures due to increased metabolic demands. Activity did vary significantly with diel cycle and substrate. Swimming speeds were significantly lower at night during September and October. Results for August and November were inconclusive, while swimming speed was significantly lower during the day in December. We observed significantly reduced average swimming speeds in structurally complex substrates (e.g. rock, cobble and kelp) in September and October. Our results suggest that activity of juvenile cod in the wild does not vary with temperature as predicted from studies in the laboratory. Instead, activity varied with diel cycles and structural complexity, variables that influence an individual's ability to forage and seek refuge, potentially altering individual fitness.     

Isolation and distribution of iridescent Cellulophaga and other iridescent marine bacteria from the Charente-Maritime coast,French Atlantic

An intense colored marine bacterium, identified as Cellulophaga lytica, was isolated previously from a sea anemone surface on the Charente-Maritime rocky shore (Atlantic Coast, France), and iridescence of its colonies under direct light was recently described. In addition, iridescence intensities were found to differ strongly between C. lytica strains from different culture collections. However, importantly, the occurrence and distribution of iridescent bacteria in the marine environment were still unknown. Therefore, in this study, a search was undertaken for marine iridescent bacterial strains in different biotopes of the Charente-Maritime coast. Various marine samples (water, sediment, macroalgae, other macroorganisms and detritus) were collected from seven biotopes using a direct plate inoculation method. As a result, 34 iridescent strains related to the genus Cellulophaga, as well as 63 iridescent strains affiliated to the genera Tenacibaculum and Aquimarina, were isolated. Iridescent colors were different according to the genera but iridescent marine bacteria were widely distributed. However, a majority of strains were isolated from rocky shores and, in particular, red seaweed surfaces and mollusks. The data from the study suggested that isolates with iridescent properties were well conserved in stressful environments such as the coastal shoreline. This origin may provide an insight into the ecological and biological functions of iridescence.