The spotted wolffish (Anarhichas minor Olafsen) complement component C3: isolation,characterisation and tissue distribution

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M(r)65 and 115kDa, respectively. The high molecular weight alpha-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the beta-alpha junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.     

Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish (Anarhichas minor Olafsen) juveniles

To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A. salmonicida bacterin. Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent. The A-protein was the most immunogenic bacterial component. In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish. Plasma cells were also present in gut and gills in equal numbers irrespective of treatment. No plasma cells were found in the skin. Finally, the frequencies of expressed V(H)families and C(L)isotypes of wolffish immunoglobulins were shown by PCR. The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A. salmonicida bacterin.     

Susceptibility of spotted wolffish Anarhichas minor to experimental infection with nodavirus and infectious pancreatic necrosis virus

The spotted wolffish Anarhichas minor is a promising new species for cold-water aquaculture. The broad host-range of piscine nodavirus (NV) and infectious pancreatic necrosis virus (IPNV) makes them potentially pathogenic to new fish species in aquaculture. IPNV and NV strains highly pathogenic in farmed Atlantic salmon Salmo salar and halibut Hippoglossus hippoglassus, respectively, in Norway were used for the challenge of spotted wolffish. In general, water-borne infection with IPNV and NV resulted in significant mortality among juveniles <1 g. Cumulative mortality after bath-challenge and cohabitation was 60 to 75% in the smallest juveniles (0.3 g). Intramuscular and intraperitoneal injection of NV was 100% lethal to wolffish of 10 g, and the groups at 12 degrees C died before those at 7 degrees C. No cohabitants of this size died, but NV was still detectable in these individuals after 10 wk. A persistent IPNV infection with low mortality developed in bath-challenged juveniles of 0.7 g, in which IPNV was still detectable 4 mo later. This study comprises a demonstration of experimental viral infections in cultured spotted wolffish, although to date no natural outbreaks of viral diseases have been reported in this species.     

Seasonal modulation of plasma antifreeze protein levels in Atlantic (Anarhichas lupus) and spotted wolffish (A. minor)

The Atlantic and spotted wolffish (Anarhichas lupus and A. minor, respectively) inhabit the cold waters of the northeast Atlantic Ocean. Although both species experience subzero water temperatures during winter, the Atlantic wolffish, which occupies shallower waters than the spotted wolffish, faces the greater threat of coming into contact with ice and freezing. This laboratory study was designed to determine whether these species differed in their abilities to resist freezing by examining the seasonal changes in blood plasma freezing points, antifreeze protein (AFP) activity and Na⁺ and Cl⁻ concentrations when exposed to seasonally cycling water temperatures and photoperiod. The plasma of both species showed distinct seasonal cycles in all parameters with the highest values occurring during the winter. However, of the two species, only the Atlantic wolffish produced sufficient AFP to protect the fish down to the freezing point of seawater (− 1.80 °C). The levels of AFP in the spotted wolffish were too low to impart any significant improvement in their resistance to freezing (approximately − 0.8 °C).When wolffish were maintained in warm water under a seasonally changing photoperiod, the amplitude of the seasonal cycle in AFP activity was greatly reduced, indicating that low water temperatures are necessary to maximize plasma AFP levels. However, despite being maintained in warm water, plasma levels of AFP activity began to increase over summer values at the same time of year as did the fish exposed to seasonally changing water temperatures. This suggests that photoperiod plays a major role in the timing of the annual AFP cycle.     

Effects of reduced salinities on growth, food conversion efficiency and osmoregulatory status in the spotted wolffish

No significant differences in mean mass between groups were found at any time in spotted wolffish Anarhichas minor , mean (±S.D.) initial mass 76 (±21) g, reared at salinities of 12, 17, 25 and 34‰ for 12 weeks at 8° C. Salinity did not have a significant effect on daily feeding rates, total food consumption, food conversion efficiency and protein efficiency ratio. Growth trajectories varied between groups, but no overall difference in growth was found. Plasma osmolality and plasma chloride levels decreased with salinity in a 48 h abrupt exposure trial, and in the growth experiment the low salinity groups (12 and 17‰) exhibited significantly lower values compared with 25 and 34‰. The decrease was moderate and concentrations were well within the range described for other marine species. The results indicate that the spotted wolffish is a strong osmoregulator which could be reared at various salinity levels.     

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.     

A review of the culture potential of spotted wolffish <Emphasis Type="Italic">Anarhichas minor</Emphasis> Olafsen

The first articially fertilized spotted wolffish eggs hatched only 10 years ago, and today the species is considered a very promising candidate species for cold water aquaculture in the North Atlantic. Recent research has focused on identifying key biological parameters in spotted wolffish aquaculture in order to establish a full production line for the species, and basic aspects of reproduction and larval development are now understood, controlled, and no longer limiting production. Spotted wolffish eggs (5–6 mm) have a protracted incubation period (800–1000 D°) and newly hatched individuals (20–25 mm) are well developed, with the only larval characteristic remaining being a relatively small yolk sac which is completely resorbed after 3–4 weeks. The species can be weaned directly on formulated feed, and high specific growth rates have been obtained in land-based culture facilities using shallow raceways. Adaptive immune responses are present early after hatching and few potential disease problems have been identified. Only one bacterial disease, atypical furunculosis, has been reported in farmed fish, but oil-emulsified vaccines have displayed efficient protection both in juvenile and adult fish. Ectoparasites may, however, constitute a problem during parts of the year when sea-water temperature increases.Optimal temperature for growth decreases with increasing fish size and is 10–12 °C for early juveniles and 4–6 °C for adult fish and broodstock. Spotted wolffish is a very robust species, and juveniles thrive at high densities and may be reared at a wide range of salinity levels. The species has further displayed a high tolerance to environmental changes in water quality parameters such as oxygen, carbon dioxide and un-ionized ammonia. Currently, the possibility of rearing spotted wolffish in flat-bottom net cages with shelves in the sea is being investigated. Preliminary results suggest that sea-based production may be a viable alternative to land-based rearing of the species in certain areas.     

Unusual motility characteristics of sperm of the spotted wolffish

Unlike the sperm of most teleosts, that of the spotted wolffish Anarhichas minor is motile on stripping, remains motile for at least 2 days and loses motility when exposed to sea water. Computer assisted sperm analysis (CASA) was used to quantitatively examine the motility characteristics of spotted wolffish sperm. Straight line velocity (VSL), beat cross frequency (BCF) and percentage motility were the most sensitive indicators of movement. Sperm trajectories were very different to those of other teleosts examined, showing large side-to-side movements of the sperm head and a more 'wiggly' behaviour which may be an adaptation to swimming in the viscous gelatinous egg mass. VSL was not altered by pH from 5·0 to 9·0, but was lower at pH 4·5. It was highest at 200 to 500 mOsm and decreased rapidly at <200 mOsm and more slowly at >500 mOsm. It is suggested that the unusual characteristics of spotted wolffish sperm in its trajectory and duration of motility, its release in a fully activated state and its greatly decreased motility in both fresh and sea water are related to a spawning strategy involving mixing of sperm with eggs contained in a gelatinous mass rather than release directly into water in proximity to the ova.     

EROD activity in gill filaments of anadromous and marine fish as a biomarker of dioxin-like pollutants

The applicability of a gill filament-based ethoxyresorufin O-deethylase (EROD) assay, originally developed in rainbow trout, was examined in Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens) and spotted wolffish (Anarhichas minor). All species but spotted wolffish showed strong EROD induction in tip pieces of gill filaments following 48 h of exposure to waterborne beta-naphthoflavone. Atlantic salmon parr, smolts held in freshwater and smolts transferred to seawater showed EROD induction of similar magnitude. Arctic charr, differing 11-fold in body weight, showed similar EROD activities as expressed per gill filament tip. Laboratory exposure of saithe to water and sediments collected at polluted sites, resulted in strong EROD induction. In conclusion, the gill filament assay seems useful for monitoring exposure to aryl hydrocarbon receptor agonists in various species. Furthermore, smoltification status, water salinity and body size proved to have minor influence on gill filament EROD activity. However, the results in spotted wolffish show that some species may be less suitable for monitoring using the gill assay. Assessment of gill filament EROD activity in fish exposed to polluted water and sediments in the laboratory proved to be an easy and cost-effective way to survey pollution with dioxin-like chemicals.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

Microsatellite markers discriminate three species of North Atlantic wolffishes (Anarhichas spp.)

Sixteen tetranucleotide and dinucleotide microsatellite markers were isolated from Atlantic wolffish, Anarhichas lupus , following a microsatellite enrichment procedure using probe-labelled magnetic beads. These microsatellites were intended for use in Atlantic wolffish as well as in two closely related species, spotted wolffish, Anarhichas minor , and northern wolffish, Anarhichas denticulatus . As all three species are of conservation concern in Canadian waters and as forensic wildlife cases may arise for this genus, microsatellite markers were assessed to determine how well they differentiate these species from one another and to estimate probability values that could be expected for identification of individuals to species.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Dietary protein hydrolysate and trypsin inhibitor effects on digestive capacities and performances during early-stages of spotted wolffish: suggested mechanisms

Growth rate is dependent upon adequate provision of amino acids especially in newly-hatched fish which experience very high growth rate. The replacement of a fraction of protein content by partially hydrolyzed (pre-digested) proteins was carried out and the digestive capacities and performances of larval/juvenile spotted wolffish (Anarhichas minor) were measured. The goal of this study was to verify whether the scope for growth is principally dictated by the proteolytic capacity of the digestive system by examining the effect of protein hydrolysates (PH) and trypsin inhibitor dietary inclusion on protein digestion/assimilation capacities, growth and survival. Four experimental diets were examined: C (control) I (supplemented with 750 mg/kg soybean trypsin inhibitor (SBTI)) H (supplemented with 20% PH) and HI (supplemented with 20% PH and 750 mg/kg SBTI). Protein hydrolysate supplementation gave significantly higher body mass than control at day 15 post-hatching. Unexpectedly, at day 30 and 60, fish administered diet HI (containing trypsin inhibitor) were heavier than the other groups. Suggested mechanisms are presented and discussed. The main conclusions of this study are that wolffish larval stage lasts roughly 15 days and that juvenile growth is linked to proteolytic capacity, but also very likely to absorption capacity of peptides and amino acids.     

Nesting behavior of greater eelpout (Lycodes esmarkii), identified through a predation event by spotted wolffish (Anarhichas minor)

The stomach of a spotted wolffish (Anarhichas minor) caught in Icelandic waters was found to contain ~727 greater eelpout larvae (Lycodes esmarkii). All the larvae were of similar size and at a similar state of digestion, indicating they were all consumed together. The likely explanation for this observation is that greater eelpout lay their eggs in a nest, with the larvae remaining in the nest for a short period after hatching. The larvae were then predated upon by the spotted wolffish while still in the nest. This study sheds new light on greater eelpout in Icelandic waters, with recently hatched larvae being present in March, breeding at a depth of ~200–250 m, and likely exhibiting nesting behavior, which has not previously been documented.     

Effects of ischemia on lung macrophages

Angiogenesis after pulmonary ischemia is initiated by reactive O(2) species and is dependent on CXC chemokine growth factors, and its magnitude is correlated with the number of lavaged macrophages. After complete obstruction of the left pulmonary artery in mice, the left lung is isolated from the peripheral circulation until 5-7 days later, when a new systemic vasculature invades the lung parenchyma. Consequently, this model offers a unique opportunity to study the differentiation and/or proliferation of monocyte-derived cells within the lung. In this study, we questioned whether macrophage subpopulations were differentially expressed and which subset contributed to growth factor release. We characterized the change in number of all macrophages (MHCII(int), CD11C+), alveolar macrophages (MHCII(int), CD11C+, CD11B-) and mature lung macrophages (MHCII(int), CD11C+, CD11B+) in left lungs from mice immediately (0 h) or 24 h after left pulmonary artery ligation (LPAL). In left lung homogenates, only lung macrophages increased 24 h after LPAL (vs. 0 h; p<0.05). No changes in proliferation were seen in any subset by PCNA expression (0 h vs. 24 h lungs). When the number of monocytic cells was reduced with clodronate liposomes, systemic blood flow to the left lung 14 days after LPAL decreased by 42% (p<0.01) compared to vehicle controls. Furthermore, when alveolar macrophages and lung macrophages were sorted and studied in vitro, only lung macrophages secreted the chemokine MIP-2α (ELISA). These data suggest that ischemic stress within the lung contributes to the differentiation of immature monocytes to lung macrophages within the first 24 h after LPAL. Lung macrophages but not alveolar macrophages increase and secrete the proangiogenic chemokine MIP-2α. Overall, an increase in the number of lung macrophages appears to be critical for neovascularization in the lung, since clodronate treatment decreased their number and attenuated functional angiogenesis.