Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

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.     

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.     

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.     

Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.     

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.     

Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum

The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 degrees C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 degrees C. The haemolytic activity of halibut sera was inactivated at 45 degrees C while sea bass sera were inactivated at 56 degrees C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at -80 degrees C, whereas the sea bass sera maintained fairly good activity after 1-year storage at -80 degrees C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.     

The ontogenic development of innate immune parameters of cod (Gadus morhua L.)

The aim of this study was to monitor the ontogenic development of innate immune parameters of cod (Gadus morhua L.) and to determine the presence of maternal IgM. The general protein composition and enzyme activity was also studied. At intervals, samples were collected of fertilized cod eggs and larvae from 3 days after fertilization until 57 days after hatching. Cell lysates were prepared and analysed by Western blotting using antibodies prepared against cod IgM, the complement component C3 and C-reactive protein (CRP) as well as against cod serum proteins and haemoglobin. Antibodies against salmon cathepsins and against several mammalian proteins of immunological significance were also used. Maternal IgM was not detected but C3 and the closely associated apolipoprotein A-I were present from the time of embryo organogenesis. C-reactive protein was not detected and none of the antibodies against mammalian immune parameters cross-reacted with the cod material. Protein and proteomic analysis showed that the major proteins of the egg samples were vitellogenin derived maternal proteins. Other non-vitellogenin maternal proteins, not yet identified, were also detected in the fertilized eggs. Cathepsin was present in all samples, but other enzyme activity was restricted to larval samples from 4 days after hatching when feeding had commenced. Haemoglobin was not detected until 10 days after hatching.     

The ontogeny of complement component C3 in Atlantic cod (Gadus morhua L.)--an immunohistochemical study

The complement system in fish is well developed and plays an important role in the immune response. Very little is known about the ontogeny of C3 in fish and no study has previously been done on the development of C3 in teleosts. In this study we have detected the presence of C3 in cod larvae from the age of 1 day post hatching (p.h.) till 57 days p.h., using immunohistochemistry. The specific primary antibodies used, were produced against the beta-chain of cod C3. Immunostaining on cod larvae sections revealed that C3 is detectable in the yolksac membrane from day 1 p.h., and in liver, brain, kidney and muscle from day 2 p.h. C3 was also detected in other organs such as eye, notochord, stomach, intestines, pancreas, heart and gills at different stages of cod larval development. These findings suggest that complement is not only important in immune defence against invading pathogens but may also play a role in the formation and generation of different organs.     

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.     

Experimental trophic ecology of juvenile largemouth bass,Micropterus salmoides,and blue tilapia,Oreochromis aureus

Synopsis The potential for feeding competition between largemouth bass, Micropterus salmoides, and blue tilapia, Oreochromis areus, in Lake Fairfield, Texas was evaluated experimentally. Largemouth bass and blue tilapia were grown in cages alone and in combination with each other. The fish were allowed to feed on the natural food within the lake. Largemouth bass grown in combination with blue tilapia were significantly shorter and weighed less than largemouth bass grown alone. Blue tilapia grown in combination with largemouth bass were statistically significantly longer and heavier than blue tilapia grown alone. Largemouth bass grown alone had diets (volume and number of food items) significantly different than the largemouth bass grown with the blue tilapia. Largemouth bass fed primarily on chironomid larvae and pupae, and odonates, whereas blue tilapia consumed vegetable matter, detritus, and chironomid larvae. Length and weight differences between large-mouth bass grown alone and in combination with blue tilapia, in conjunction with the largemouth bass diet shift, support the theory that these two species compete for food resources.     

It is the economy,stupid! Projecting the fate of fish populations using ecological–economic modeling

Four marine fish species are among the most important on the world market: cod, salmon, tuna, and sea bass. While the supply of North American and European markets for two of these species – Atlantic salmon and European sea bass – mainly comes from fish farming, Atlantic cod and tunas are mainly caught from wild stocks. We address the question what will be the status of these wild stocks in the midterm future, in the year 2048, to be specific. Whereas the effects of climate change and ecological driving forces on fish stocks have already gained much attention, our prime interest is in studying the effects of changing economic drivers, as well as the impact of variable management effectiveness. Using a process‐based ecological–economic multispecies optimization model, we assess the future stock status under different scenarios of change. We simulate (i) technological progress in fishing, (ii) increasing demand for fish, and (iii) increasing supply of farmed fish, as well as the interplay of these driving forces under different scenarios of (limited) fishery management effectiveness. We find that economic change has a substantial effect on fish populations. Increasing aquaculture production can dampen the fishing pressure on wild stocks, but this effect is likely to be overwhelmed by increasing demand and technological progress, both increasing fishing pressure. The only solution to avoid collapse of the majority of stocks is institutional change to improve management effectiveness significantly above the current state. We conclude that full recognition of economic drivers of change will be needed to successfully develop an integrated ecosystem management and to sustain the wild fish stocks until 2048 and beyond.     

Identification and distribution of heparan sulfate proteoglycans in the white muscle of Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor)

Heparan sulfate proteoglycans (HSPGs) were identified in pre-rigor muscle of two species of cold water fish, Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor) by biochemical and immunological methods. The distribution was described by immunohistology. Special emphasis was directed to the extracellular matrix (ECM) HSPGs perlecan and agrin. In vivo ³⁵S-sulfate labeling combined with ultracentrifugation in CsCl₂, DEAE chromatography and scintillation counting of the eluates, revealed that the content of ³⁵S-labeled PGs was much higher in wolffish than in cod. A considerable proportion of the ³⁵S-sulfated PGs in both species was HSPG, as judged by nitrous acid degradation. HSPG represented, however, a higher proportion of the ³⁵S-sulfated PGs in cod compared to wolffish. Dot blot and electrophoresis/western blot using two different HS-mAbs, 10E4 and HepSS-1 indicated structural differences in the HS-chains of the PGs present. This observation was strengthened by immunohistochemistry, showing that both mAbs detected epitopes in the pericellular area, but the staining patterns were not superimposable. Two different agrin isoforms were identified in both species. Furthermore, in the white muscle of both cod and wolffish, perlecan mAb (A7L6) showed positive staining restricted to the transition between myocommata and myofibers.     

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.     

Sulfated glycosaminoglycans in the extracellular matrix of muscle tissue in Atlantic cod (Gadus morhua) and Spotted wolffish (Anarhichas minor)

Two species of commercially important cold water fish were investigated for content of sulfated glycosaminoglycans (GAGs) in muscle tissue by use of in vivo 35S-sulfate labeling combined with different digestions (papain, chondroitinase ABC, keratanase and nitrous acid treatment), DEAE chromatography, SDS-PAGE and histology techniques. The species investigated in this study have different gaping properties. The non-gaping species, spotted wolffish (Anarhichas minor), contained 3-4 times more 35S-sulfated anionic components than the gaping species, Atlantic cod (Gadus morhua). The higher level of sulfation in wolffish was supported by light microscopy studies using Alcian blue staining with different concentrations of MgCl2 as critical electrolyte. Furthermore, the muscular connective tissue in the non-gaping species was dominated by chondroitin sulfate (CS)/dermatan sulfate (DS), whereas the gaping species was more dominated by heparan sulfate (HS). Moreover, structural differences were observed in the junctions between the myofibers, which were more pronounced in the wolffish. The histological studies revealed that the basement membrane area was rich in acidic mucopolysaccharides in both species.