Structure–function relationships in histidine-rich antimicrobial peptides from Atlantic cod

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure–function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH 7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH 5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.     

Probiotics–pathogen interactions elicit differential regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus morhua

Little is known on the cutaneous immune responses during probiotics–pathogen interactions in fish. Thus, this study employed Atlantic cod primary epidermal (EP) cell cultures as a model to understand this interaction. The probiotics–pathogen interactions in the EP cell cultures were elucidated using Vibrio anguillarum 2133 (VA) as the pathogen and two host-derived bacteria (GP21 and GP12) as the probiotics. There was a regional size difference on the EP cells; i.e., EP cells from the dorsal region were significantly larger than the EP cells at the ventral side. VA significantly decreased viability of EP cells. In the presence of probiotics, this inhibition was mitigated. The probiotics reduced VA-induced cellular apoptosis and the probiotics–pathogen interactions influenced cellular myeloperoxidase activity during the latter stage of co-incubation. The probiotics–pathogen interactions triggered differential regulation of immune-related genes and the effects of the interaction were dependent on the region where the cells were isolated and the length of the co-incubation period. In most cases, the presence of probiotics alone showed no significant change on the mRNA level of immune genes in the EP cells but triggered immunostimulatory activity when incubated with VA. This study showed that the virulence of VA in EP cells could be modulated by host-derived probiotics and the immunomodulatory characteristics of the two candidate probionts advanced their immune-related probiotic potential.     

Multiple linked β and α globin genes in Atlantic cod: A PCR based strategy of genomic exploration

Allozyme variation in Atlantic cod hemoglobins shows various signs of natural selection. We report a genomic exploration of globin genes in this non-model organism. Applying a PCR based strategy with a strict criterion of phylogenetically informative sites we estimate the number of linked β and α globin genes. We estimate PCR error rate by PCR of cloned DNA and recloning and by analysis of singleton variable sites among clones. Based on the error rate we exclude variable sites so that the remaining variation meets successively stricter criteria of doubleton and triplet variable site. Applying these criteria we find ten clusters of linked β/α globin genes in the genome of Atlantic cod. Six variable amino acid changes in both genes were found in linkage disequilibrium with silent nucleotide substitutions. A phylogenetic tree, based on our strictly phylogenetically informative sites among 57 clones from 19 individuals, is split into two major branches by an amino acid change in a β gene. This change is supported by extensive linkage disequilibrium between the amino acid change and numerous other phylogenetically informative silent nucleotide sites. The different gene sets in the genome may represent different loci encoding different globins and/or allelic variation at some loci.     

The significance of heart rate in free swimming cod,Gadus morhua: Some observations with ultra‐sonic tags

Ultra sonic transmitters (cylindrical, 1.5 cm diameter × 7 cm long) with three‐week battery life were used to examine the heart rate and electrocardiogram (ECG) of cod in swimming experiments. The maximum heart rate was observed when the period between the recovery (T) wave and the initial wave (P) of the ECG was zero and was limited by the P to T period which was not observed to vary in an individual. Low heart rates were observed in resting fish and maximum rates were associated with exhaustion and cruising swimming. Maximum rates continued during periods of oxygen debt repayment and during anaesthesia as long as oxygen was supplied to the gills. Brief inhibition of heart rate was associated with sudden stimuli and during maximum swimming exertion. The significance of rate change and its relation to the aerobic and anaerobic states is discussed.     

Genetic and morphological variation in Echinorhynchus gadi Zoega in Müller, 1776 (Acanthocephala: Echinorhynchidae) from Atlantic cod Gadus morhua L

Previous studies have shown considerable variability in morphological features and the existence of genetically distinct sibling species in the acanthocephalan Echinorhynchus gadi Zoega in Müller, 1776. The aim of the present study was to follow up and extend those earlier studies by using a combination of DNA analysis and morphometrics to investigate differences between samples of E. gadi from Atlantic cod Gadus morhua L. caught at five fishing grounds in the Baltic Sea and three in different parts of the North Atlantic. Twelve morphological features were measured in 431 specimens of E. gadi, 99 individuals were studied by polymerase chain reaction-restriction fragment length polymorphosm (PCR-RFLP), and selected PCR products were sequenced. The molecular analyses showed the nucleotide sequences of E. gadi rDNA from cod caught at all the sampling sites to be identical. The comparative morphological study, in contrast, revealed significant differences between samples of E. gadi from different sampling sites and showed the separation of E. gadi into two groups corresponding approximately to the systematic classification of cod into the two subspecies, Atlantic G. morhua morhua and Baltic G. morhua callarias. The E. gadi infrapopulation size had a significant effect on some of the morphological features. The results are discussed in relation to cod population biology, the hydrography of the study area and the history of the Baltic Sea formation.     

Recent trends in the abundance of plaice Pleuronectes platessa and cod Gadus morhua in shallow coastal waters of the Northeastern Atlantic continental shelf – a review

Shallow, near-shore water habitats on the continental shelf of the Northeast Atlantic have been productive fishing areas in the past. Here, we review the present knowledge about (i) recent trends in the abundance of plaice and cod in these habitats and (ii) hypotheses regarding the factors responsible for any trends. At present, only a few studies exist on the trends of abundance of plaice or cod, namely from the Bay of Biscay, the North Sea and the Skagerrak/Kattegat. They suggest a declining abundance in coastal, shallow areas and – at least for plaice – a latitudinal gradient with an erosion of the southern distribution boundary in the Bay of Biscay and deepening of stocks in the North Sea. In contrast, no trend in shallow water abundance of plaice similar to a decline in deep-water stocks during the 1970s and their slow recovery during the 2000s is apparent in the Skagerrak/Kattegat. Although shallow habitats fundamentally differ from deeper areas by the prevalence of juvenile stages, the declining trends coincide with decreasing abundance/landings and spatial stock relocations in the deeper areas. Whether this indicates a common trend pointing at connectivity between shallow and deep water remains open. Fundamental differences exist in the suggested causes of the trends in different geographical areas. High fishing pressure together with low local recruitment apparently prevents the recovery of overexploited plaice and cod stocks in the Skagerrak/Kattegat. In contrast, the responses of juveniles and adult fish to increasing seawater temperature are the main hypotheses for changes in distribution and abundance of both fish species in the North Sea/Bay of Biscay. However, temperature alone cannot explain the observed decline of fish in coastal areas, and the causes may be more complex, involving nutrient loading, primary productivity or food availability, although at present, knowledge of these factors is insufficient.     

The isolation of Atlantic cod, Gadus morhua (Gadiformes), populations in Northern Meromictic lakes—A recurrent arctic phenomenon

Although it has previously been considered to be a rare phenomenon, this paper provides evidence of eight occurrences of Atlantic cod in northern coastal saline lakes in Arctic Canada, Greenland, Norway, and Russia. Historic and current habitat features that are necessary for Atlantic cod to colonize and persist in Arctic meromictic lakes are discussed. Data analyses confirm that the persistence of Atlantic cod in Arctic lakes is a recurrent phenomenon, and these populations represent an important component of intraspecific biodiversity. Published in Russian in Voprosy Ikhtiologii, 2008, Vol. 48, No. 2, pp. 179–190. The text was submitted by the authors in English.     

Why does the immune system of Atlantic cod lack MHC II?

MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).     

Growth,starvation and enzyme activity in white muscle of atlantic cod: at what point do muscle metabolic capacities change?

Muscle protein decreases only during prolonged starvation of Atlantic cod (Gadus morhua, Gadidae), but in the absence of protein renewal, muscle metabolic capacities may decrease before marked loss of muscle protein. This study aimed at elucidating the threshold at which decreases in growth and condition reduce muscle metabolic capacities, as well as identifying the indicators that best explain changes in metabolic capacities. To generate a wide spectrum of individual growth rates, condition factors and proximate compositions, cod showing different initial condition were fed or starved for different periods of time. The relationships between muscle proteins and metabolic enzyme activities (LDH and CCO) on one hand, and growth rate, condition factor, hepato- and gonadosomatic index and muscle and liver water and energy contents, on the other hand, were examined through linear regression models. Multiple linear regressions explained a large proportion of the observed variability in proteins and enzyme activities. Changes in LDH and CCO activities were not driven by changes in growth rate. Muscle water was the only significant correlate for both enzymes. Enzyme activities decreased as soon as muscle water began to rise. Increases in water content from 79 to 92% resulted in a 10-fold decrease in LDH and CCO activities.     

Thermal acclimation to 4 or 10°C imparts minimal benefit on swimming performance in Atlantic cod (Gadus morhua L.)

Thermal acclimation is frequently cited as a means by which ectothermic animals improve their Darwinian fitness, i.e. the beneficial acclimation hypothesis. As the critical swimming speed (U _crit) test is often used as a proxy measure of fitness, we acclimated Atlantic cod (Gadus morhua) to 4 and 10°C and then assessed their U _crit swimming performance at their respective acclimation temperatures and during acute temperature reversal. Because phenotypic differences exist between different populations of cod, we undertook these experiments in two different populations, North Sea cod and North East Arctic cod. Acclimation to 4 or 10°C had a minimal effect on swimming performance or U _crit, however test temperature did, with all groups having a 10–17% higher U _crit at 10°C. The swimming efficiency was significantly lower in all groups at 4°C arguably due to the compression of the muscle fibre recruitment order. This also led to a reduction in the duration of “kick and glide” swimming at 4°C. No significant differences were seen between the two populations in any of the measured parameters, due possibly to the extended acclimation period. Our data indicate that acclimation imparts little benefit on U _crit swimming test in Atlantic cod. Further efforts need to identify the functional consequences of the long-term thermal acclimation process.     

Characterization of MHC class I and β2-microglobulin sequences in Atlantic cod reveals an unusually high number of expressed class I genes

 Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and β₂-microglobulin (b ₂ m) genes in Atlantic cod (Gadus morhua L.). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic α1-, α2-, and α3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the α2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an α3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an α1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b ₂ m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean β₂m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b ₂ m locus in Atlantic cod. Received: 10 March 1999 / Revised: 1 June 1999     

Is the poleward expansion by Atlantic cod and haddock threatening native polar cod, <Emphasis Type="Italic">Boreogadus saida</Emphasis>?

During a recent period of increased influx of warm Atlantic water to the western coast of Svalbard, we have observed a northward expansion of boreal Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) into areas dominated by the native polar cod (Boreogadus saida). To determine the potential impact of new ecological interactions, we studied the diet of co-occurring juvenile gadoids in fjords, open water, and sea ice around Svalbard. We also reviewed the available literature on polar cod feeding in different habitats across the Arctic to determine whether region, habitat, or fish size may influence diet. Feeding by polar cod in the pelagic zone was size dependent, with small fish primarily consuming Calanus spp. and smaller copepods, with an increasing ration of Themisto spp. at larger sizes. In benthic habitats, diets were more varied and included considerably more unidentified material and sediment. Less than 40% dietary overlap was detected among the three species when they were found together. Stable isotope analyses indicated these patterns were representative of longer-term assimilation. The low interspecific dietary overlap suggests little direct competition. Future increases in abundance and the high predation potential of the boreal taxa, however, may impact the persistence of polar cod on some Arctic shelves.     

The abundance and patterns of the egg and larval spatial distribution of Eastern Baltic cod Gadus morhua callarias (Gadidae) in 1931–1996

According to ichthyoplankton surveys of AtlantNIRO and published sources, the data on abundance of eggs and larvae of eastern Baltic cod Gadus morhua callarias at different spawning grounds of the Baltic Sea in 1931–1938, 1949–1956, and 1991–1996 are considered. We show that a similar type of spatial distribution of eggs and larvae of cod with localization of the greatest amount of its early ontogenetic stages in the Bornholm Basin in the southwest of the sea was observed in the 1930s and 1990s. The extension of the area of distribution was accompanied by an increase in the numbers of eggs and larvae of cod in all areas of the sea, and its decline was followed by a decrease in numbers, in the first turn, at northeastern spawning grounds in the Gotland Basin. The period of high numbers of eggs in 1949–1956 coincided with the century maximum of salinity of the near-bottom water layer.     

The occurrence of some metazoan parasites of Atlantic cod, Gadus morhua L., in relation to age and sex of the host in Balsfjord (70°N), North Norway

Age and sex differences in the occurrence of some metazoan parasites were investigated in 243 cod (Gadus morhua L.) from Balsfjord, a subarctic fjord in northern Norway. Thirteen species of parasite were studied, comprising seven nematodes, three parasitic copepods, one acanthocephalan, one cestode and one digenean. The nematode Anisakis simplex showed significant increases in both prevalence and mean abundance with host age, while the digenean Hemiurus levinseni showed a significant increase in mean abundance with age. The increases probably result from a long parasite life-span for A. simplex and changes in the feeding pattern of cod with age for H. levinseni. Prevalence of the nematode Contracaecum sp. and mean abundance of the acanthocephalan Echinorhynchus gadi were significantly higher in male than in female cod, whereas mean abundance of H. levinseni was significantly higher in female cod. These results suggest that there may be differences in feeding behaviour between male and female cod in Balsfjord. Accepted: 14 November 1999     

Drivers of growth for Atlantic cod (Gadus morhua L.) in Icelandic waters – A Bayesian approach to determine spatiotemporal variation and its causes

This study assesses spatiotemporal and sex-specific growth of Atlantic cod Gadus morhua in Icelandic waters. We use a Bayesian approach which lends itself to fitting and comparing nested models such as these. We then compare fitted parameters of these models to potential explanatory variables using a redundancy analysis (RDA) to look for drivers of growth in G. morhua. Results indicate that models that incorporate differences in growth among time, space and sex are the best-fitting models according to deviance information criterion (DIC). Results from RDA indicate that capelin Mallotus villosus recruitment and biomass is highly correlated with deviations in the von Bertalannfy growth parameter k and that L_∞ is correlated with G. morhua landings in the model that uses year to account for time-varying growth and estimated G. morhua recruitment in the model that uses cohort to account for time-varying growth.     

Are low but statistically significant levels of genetic differentiation in marine fishes ‘biologically meaningful’? A case study of coastal Atlantic cod

A key question in many genetic studies on marine organisms is how to interpret a low but statistically significant level of genetic differentiation. Do such observations reflect a real phenomenon, or are they caused by confounding factors such as unrepresentative sampling or selective forces acting on the marker loci? Further, are low levels of differentiation biologically trivial, or can they represent a meaningful and perhaps important finding? We explored these issues in an empirical study on coastal Atlantic cod, combining temporally replicated genetic samples over a 10‐year period with an extensive capture–mark–recapture study of individual mobility and population size. The genetic analyses revealed a pattern of differentiation between the inner part of the fjord and the open skerries area at the fjord entrance. Overall, genetic differentiation was weak (average F_ST = 0.0037), but nevertheless highly statistical significant and did not depend on particular loci that could be subject to selection. This spatial component dominated over temporal change, and temporal replicates clustered together throughout the 10‐year period. Consistent with genetic results, the majority of the recaptured fish were found close to the point of release, with <1% of recaptured individuals dispersing between the inner fjord and outer skerries. We conclude that low levels of genetic differentiation in this marine fish can indeed be biologically meaningful, corresponding to separate, temporally persistent, local populations. We estimated the genetically effective sizes (N_e) of the two coastal cod populations to 198 and 542 and found a N_e/N (spawner) ratio of 0.14.