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The Atlantic wolffish (AW) and the spotted wolffish (SW) are long-lived fish found in the North Atlantic and Arctic oceans and are respectively classified as special concern and threatened species, mainly due to fisheries bycatch. To better understand health issues associated with the care of these species in public aquaria, reports from all necropsies performed in 2 zoological institutions between 2009 and 2019 were reviewed (31 AW and 8 SW). These wolffish were fed with a similar fish-based diet and kept in multi-species exhibits with comparable environmental parameters. The most frequent necropsy findings were the presence of xanthomas (AW: 41.9%; SW: 75.0%), nephrocalcinosis (AW: 42.9%; SW: 75.0%) and urocystoliths (AW: 6.5%; SW: 62.5%). Xanthomas were mostly located at the base of pectoral fins and were characterized by extensive granulomatous inflammation centered on accumulations of partly mineralized degenerate fatty material, mainly composed of cholesterol crystals. Nephrocalcinosis was characterized by the deposition of calcium salts within the renal tubules and was commonly associated with tubular necrosis. The aquarium-housed wolffish were fed a coldwater fish-based diet. However, the natural diet of wolffish is composed mostly of invertebrates such as urchins and crustaceans. Differences in nutrient composition between these diets, such as lipid and mineral content, may have contributed to the development of xanthomatosis, nephrocalcinosis and urocystolithiasis in wolffish housed in these institutions.

Spotted wolffishAnarhichas minorreproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

Physiological indicators such as lipid peroxidation and enzymes involved in aerobic/anaerobic energy metabolism were measured in pure and hybrid lines of wolffish juveniles (Anarhichas minor, ♀ A. minor ⨯ ♀ A. lupus). Interspecific hybridization was introduced as a powerful tool to promote augmentation of phenotypic variability and consequently improve our capacity for the detection of clear physiological relationships amongst oxidative stress and metabolic characters. Mitochondrial sequence genes analysis between the Atlantic (A. lupus) and spotted wolffish (A. minor) were later performed to further assess the extent of divergence between A. lupus and A. minor mitochondrial haplotypes. The observed divergences (2.0% and the number of nonsynonymous substitution n=23) do not seem to be sufficient to generate detectable differences in ETS complexes catalytic capacities. Total LDH activity was however higher in hybrids than in the pure strands and tightly correlated to TBARS levels which suggest that LDH actively participates to the regulation of cellular redox status. Our results are discussed in comparison with a similar study on a charr hybrid (Salvelinus alpinus × S. fontinalis).

The purpose of this study was to determine the cortisol response of spotted wolffish (Anarhichas minor) after exposure to air immersion and netting (acute stress) under a range of increasing densities (chronic stressor). In addition, the presence of a cumulative negative impact from chronic stress was assessed by the application of an additional acute stressor following rearing at various densities, and proxies of behavior were evaluated (feeding and aggression data). The stress response to air immersion and netting led to increasing plasma cortisol values at 0.5, 13, and 37 h post-stress, with the highest value achieved after 13 h. At 168 h, cortisol concentrations returned to similar pre-stress value. Changes in cortisol concentrations were relatively low after acute stress (five to six fold increases) and recovery time long lasting (>37 h). Prolonged rearing at 20, 30, and 40 kg m−2 showed a non-significant trend of increasing cortisol values with increasing density, and the cortisol response after the...

The effect of temperature and mass on specific growth rate (G) was examined in spotted wolffish Anarhichas minor of different size classes (ranging from 60 to 1500 g) acclimated at different temperatures (4, 8 and 12 degrees C). The relationship between G and 20S proteasome activity in heart ventricle, liver and white muscle tissue was then assessed in fish acclimated at 4 and 12 degrees C to determine if protein degradation via the proteasome pathway could be imposing a limitation on somatic growth. Cardiac 20S proteasome activity was not affected by acclimation temperature nor fish mass and had no correlation with G. Hepatic 20S proteasome activity was higher at 12 degrees C but did not show any relationship with G. Partial correlation analysis showed that white muscle 20S proteasome activity was negatively correlated to G (partial Pearson's r = -0.609) but only at cold acclimation temperature (4 degrees C). It is suggested that acclimation to cold temperature involves compensation of the mitochondrial oxidative capacity which would in turn lead to increased production of oxidatively damaged proteins that are degraded by the proteasome pathway and ultimately negatively affects G at cold temperature.

The spotted wolffish (Anarhichas minor) is considered a promising species for coldwater aquaculture. It is a sedentary, bottom-dwelling fish which exhibits a calm and “non-stressed” behaviour in captivity. There are, however, no reports on the physiological responses to stressors in this species. In the present study we investigated primary (cortisol secretion) and secondary (glucose mobilization) responses to common aquaculture stressors like disturbance and hypoxia. Pre-stress plasma cortisol levels were within those generally considered representative for unstressed fish (~ 10 ng ml− 1), whereas basal glucose levels were unusually low (0.3–0.4 mM). After exposure to a short, but severe disturbance challenge (emptying the tank of water for 10 min), the increase in plasma cortisol level was slow and relatively weak, reaching a peak level of 25 ng ml− 1 4 to 8 h after disturbance. When the fish were exposed to a gradually decreasing oxygen level in the tank, a significantly elevated plasma cortisol level (35 ng ml− 1) was seen in the fish that remained in the tank until oxygen saturation had decreased to 20% oxygen saturation after 2.5 h. However, a two-fold higher plasma cortisol level (~ 70 ng ml− 1) was seen in all fish exposed to reduced oxygen levels (60, 40 and 20% oxygen saturation) after 3.5 h recovery in normoxic water. Plasma glucose levels showed only moderate increases (~ 70%) following disturbance and hypoxia challenges. An in vivo injection of ACTH caused a strong elevation of plasma cortisol (peak level ~ 170 ng ml− 1), demonstrating a high capacity for interrenal steroidogenesis in the spotted wolffish. The slow and relatively weak cortisol response to stressors, and low plasma glucose levels, may relate to the sedentary lifestyle of the spotted wolffish. The stress-response is characterised by a passive (reactive) coping style, which is considered adaptive for farming of this species.

The purpose of this work was to compare the microstructure of muscle connective tissue in cod and wolffish and to study its degradation during ice-storage to determine which structural alterations are related to the gaping phenomenon. Gaping is very often found in fillets of codfish, but not in wolffish. The results showed that detachments between myofibres occurred prior to the myotomes to myocommata detachments, which occurred more rapidly and to a larger extent in cod than in wolffish. Changes at the ultrastructural level in myocommata proper were mainly related to gaps between collagen fibres and between collagen fibres and cells in the extracellular matrix (ECM). The mean collagen fibre diameter was smaller and the collagen network in myocommata proper was denser in wolffish than in cod. After 7 days of ice-storage the collagen fibres, particular in cod, appeared sparsely packed. The results implied that the increased gaping during ice-storage might be due to degradation of proteoglycans and glycoproteins, important for the spatial organization of the collagen fibres and in anchoring cells in the ECM to the collagen network.

The effect of feeding two different diets, containing 38 and 45% protein and 35 and 28% lipid, respectively, on growth as well as performance of the digestive proteases (total protease at pH 1·5, 7·0, 9·0 and 10·0) and carbohydrases (total carbohydrase, α-amylase and α-glucosidase) of a new candidate species for aquaculture, the carnivorous spotted wolffish Anarhichas minor (Teleostei, Anarhichadidae), was investigated. No significant growth differences were observed when feeding high protein (HP) or low protein (LP) levels (P > 0·05), although the LP group exhibited slightly higher protease activity and capacity (P > 0·05), mostly at the anterior end of the intestine and at alkaline pH. Carbohydrase levels were generally low, apart from α-glucosidase, which was significantly increased for the HP group (P < 0·05). Implications concerning the performance of digestive enzymes under varying dietary macronutrient levels and effects of digestive enzyme activity on the formulation of efficient artificial diets for carnivorous fishes are discussed in this study.

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.

Two egg batches of spotted wolffish, Anarhichas minor Olafsen, were incubated at 4, 6 and 8 degrees C. Embryo samples were fixed and compared on each 100th daydegree until hatching (up to 1000 daydegrees). Embryos, yolk sacs and chorions were dissected and the sizes, wet and dry weights were recorded separately. Comparisons of gross morphologies and measured parameters showed increasing and generally significant differences with time between the incubation temperatures. Lower temperatures produced longer and more differentiated larvae at hatch with a smaller yolk sac. Even though some unexpected deviations were registered among batches and experimental groups, it was clear that temperature affected embryo survival and time of hatching. Overall survival was best at 6 degrees C, in agreement with results from earlier studies. Yolk conversion efficiencies measured around the hatching point were generally high, ranging from 60% to 78%, varied between the two batches and probably reflected the developmental variations between embryos and larvae at the respective ages (daydegrees). The hatching process was apparently an energy-demanding period; yolk conversion efficiencies of unhatched embryos of similar age at each temperature were always higher. Temperature is one environmental factor that can be manipulated in hatcheries to induce hatching of viable larvae at an optimal stage of differentiation with respect to first-feeding success and early survival.

Non-specific mechanisms are important in the defence of all multicellular animals against pathogenic microorganisms. Macrophages and granulocytes play a central role in this respect. It is thus pertinent to develop methods for obtaining and cultivation of macrophages and assessing their functions in the spotted wolffish, a cold water species of current interest for the aquaculture industry. Kidney macrophages from the spotted wolffish ( Anarhichas minor Olafsen) were isolated by density sedimentation using Percoll. The cells were highly phagocytic and possessed typical macrophage morphology evaluated by transmission and scanning electron microscopy. Using electron microscopic analysis, the size of the macrophages, collected from the Percoll density interface, was 5–9 μm. The viability in vitro was highest (87.1%) when the cells were kept at 13 °C with the addition of synthetic serum replacement (SSR-2) when measured 24 h after seeding. One day old cells were not significantly activated by addition of bacterial lipopolysaccharide (LPS) for 24 h when measured by reduction of nitroblue tetrazolium compared to control cells. The cells were negative in respect to synthesis and contents of complement component C3.

The effects of dissolved oxygen (DO) concentration combined with sublethal doses of ammonia on growth and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish. Fish with a mean (S.D.) initial weight of 95.4 (18.0) g were reared in shallow raceways (initial stocking density 10 kg m −2 ) at normoxic (9.6 mg l −1 ) and hyperoxic (14.5 mg l −1 ) conditions, and also under both normoxia and hyperoxia with an additional sublethal concentration of unionised ammonia (0.17 mg NH 3 l −1 ) added, for 8 weeks at 8 °C. There was an interacting effect of oxygen saturation and ammonia level on growth, as growth rate was significantly higher in the hyperoxic/NH 3 group compared to the normoxic/NH 3 group ( P 2 consumption was significantly higher ( P 2 kg −1 h −1 ) compared to normoxic (79.9 mg O 2 kg −1 h −1 ) conditions. A clear diurnal rhythm in the O 2 consumption pattern was seen at both O 2 saturations, with the most striking observation being a dramatic increase in the morning when the light was turned on. Overall, our results support earlier findings of a high capacity to adapt to variations in environmental water quality parameters, and to restore normal growth rate in the spotted wolffish.

Growth performance and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish (Anarhichas minor Olafsen), mean (S.D.) initial weight 15.7 (4.8) g, reared at four levels of carbon dioxide (CO2(aq)) for 10 weeks at 6 °C and 33‰. CO2 levels averaged 1.1 (control), 18.1 (low), 33.5 (medium) and 59.4 (high) mg l−1, with corresponding pH values of 8.10, 6.98, 6.71 and 6.45, respectively. In addition, kidneys from sampled fish were examined macroscopically for gross signs of calcareous deposits, i.e. nephrocalcinosis, at the start and end of the experiment. Growth was significantly reduced at the highest concentration (P

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried α2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.

The effect of water oxygen content on growth and food conversion efficiency was evaluated for juvenile spotted wolffish, mean (± SD) initial weight 68.5 (± 17.5) g, reared at oxygen levels of 4.0, 6.0 (hypoxia), 9.6 (normoxia) and 14.5 (hyperoxia) mg L−1 for 11 weeks at 8 °C. Mean weights and total food consumption were significantly higher in the control and hyperoxic groups compared with the hypoxic groups at the end of the experiment. The 9.6 and 14.5 mg L−1 groups exhibited significantly higher overall specific growth rates (0.90 and 0.86% day−1 respectively) compared with the groups on 4.0 and 6.0 mg L−1 (0.46 and 0.71% day−1 respectively). In the hyperoxic group, growth was only limited in the first period and, in the hypoxic groups, growth rates increased throughout the experiment, with the 6.0 mg L−1 group performing equally well compared with the control in the last period. Overall, our findings suggest that the species will adapt to both high and low ambient water oxygen content given a period of adaptation. After the adaptation phase, growth and food conversion efficiency are comparable in the oxygen level range of 6.0–14.5 mg L−1.

Full scale experiments in tanks were conducted to elucidate the effect of photoperiod regime, dietary fat level and stocking density on growth in spotted wolffish. The study showed that continuous light (LD24:0) did not give a higher final mean weight or faster overall growth rate in spotted wolffish compared to constant 8 h light and 16 h darkness (LD8:16). However, there were indications that the fish needed time to adapt to a new photoperiod regime. The feeding experiment indicated a negative relation between dietary fat level and growth, where fish given 15% fat in the diet had a 13% higher final mean weight compared to fish on a diet with 20% fat. However, no conclusions could be made. The final mean weight was 10% higher at 25 kg/m2 compared to 40 kg/m2, indicating a negative impact of high stocking density on spotted wolffish.

Wolffish Anarhichas lupus (Anarhichadidae) hatch from large demersal eggs (c. 5-6 mm) after a long incubation period (c. 800-1000 degree days) at 20-25 mm L T with a juvenile appearance: a small yolk sac, pigmented but partly transparent skin densely supplied with mucous cells, well developed body musculature and a cartilaginous skeleton. The results of histomorphological studies illustrate the apparent functionality of tissues and organs at hatching. The mucosa of the oral cavity and various parts of the digestive channel are well differentiated and the cellular characteristics are described within each region. A stomach with gastric glands is established before hatching. The pancreas has well developed endo- and exocrine tissue and the hepatocytes are vacuolized. The kidney tissue has numerous tubuli and haematopoietic tissue in the bilobed head region. The spleen, thymus and thyroid glands also appear mature. Cones are dominant in the visual cell layer of the retina.

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.

Temperature influenced the developmental rate, survival and early growth of eggs and embryos of spotted wolffish, Anarhichas minor (Olafsen), an interesting candidate for cold water cultivation. The total incubation period decreased from 220 days at 4 °C (880 daydegrees), to 177 days at 6 °C (1062 daydegrees) and 150 days at 8 °C (1200 daydegrees) in these experiments. The proportion of normal embryos and survival of eggs until hatching were highest when the eggs were incubated at 6 °C. During the incubation period, the embryo and yolk sac size at 280 daydegrees was not significantly different but at 850 daydegrees the embryo size was inversely related to temperature and the remaining yolk sac size positively correlated with the incubation temperature. The transformation of yolk to body mass during incubation appeared to be most efficient at 4 °C, and the embryos hatched with a larger visible yolk sac at 6 and 8 °C. The largest larvae (wet-weight) hatched from the largest eggs and the egg groups incubated at the lowest temperature (4 °C). There was no effect of temperature on meristic characters. During 6 weeks post-hatching, all larvae from the three temperature groups were fed formulated dry feed in excess at 8 °C in low water-level raceway systems. During startfeeding, the larvae from eggs incubated at the lowest temperature (4 °C) showed the highest growth rates (SGR). Best survival of larvae was noted among batches incubated at 6 °C.

Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl2/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2–787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.

International audience; Three groups of newly hatched spotted wolffish (Anarhichas minor) were held at three different temperatures in order to determine relationships between metabolic, digestive and growth response in rapidly developing larvae. Growth rates were successfully modulated by temperature (5, 8 and 12 degrees C). Activity levels of trypsin and lactate dehydrogenase (LDH) were positively linked to specific growth rates at all temperatures. Trypsin showed a positive compensation (higher activity at lower temperature) whereas glycolytic enzymes (pyruvate kinase and Lactate dehydrogenase) and aspartate aminotransferase (AST) showed a negative compensation (lower activity at lower temperature). Citrate synthase was not affected by growth rate, indicating that the level of aerobic capacity was adequate in sustaining the high energetic needs associated with rapid growth early in the life of the spotted wolffish. In light of our results, it is suggested that protein digestion, as demonstrated by the activity profile of trypsin in relation to growth rate and temperature, is likely a key growth-limiting agent during the early-life stages of wolffishes. Our results are discussed in comparison with A. lupus, a closely related species displaying different temperature preferences and growth capacities.

Atlantic cod and spotted wolffish fry were fed high-M alginate containing feed for 59 and 55 days, respectively. During this period the fry showed a higher specific growth rate compared to controls. Uptake and distribution of alginate was studied by inclusion of the 125 I-labelled molecule in the feed. The stomach and intestine contained the highest amount while the kidney, liver and spleen contained some, indicating that the alginate was taken up by the gut and transported to internal organs. Cod fry fed 0.06% and 0.1% high-M alginate showed a death rate of 51.4% and 53.3%, respectively. The lowest mortality, 48.1%, was found in fry fed 0.01% high-M alginate. Controls showed a mortality rate of 49.0%. Differences were, however, not statistically significant. Challenge of the immunostimulated fry (fed 0.02% and 0.06% alginate for 62 days) with atypical Aeromonas salmonicida bacteria resulted in accumulated mortalities of 56% and 49%, respectively, 47 days after infection. The group that received 0.06% alginate for a shorter period (47 days) and then control feed until challenged, and the group that received alginate by bath reached a cumulative mortality of 59% and 60%, respectively. Lowest mortality (44%) was seen in the control group. Numerous microabscesses were found in both immunostimulated and control fish in secondary lamellae of the gills, haematopoietic tissues of the kidneys, the submucosa and mucosa of the intestine, the spleen, the liver and the myocardium of the heart.

Spotted wolffish Anarhichas minor (approx. 0.7 g) were found to be susceptible to infection with a nodavirus isolated from Atlantic halibut (AHNV) by bath-challenge. During the acute stage of infection, 4 to 8 wk post-challenge, viral encephalopathy and retinopathy (VER) were diagnosed by histopathology, immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR). Accumulated mortality was 52% in the challenged group. The surviving fish were sampled 16 wk post-challenge, by which time they had grown to approximately 17 g. No clinical signs of VER were observed in these fish. RT-PCR examination revealed the presence of nodavirus in several organs of the survivors, but no immunopositive cells were detected by IHC. Nodavirus was reisolated from fish at the last sampling in SSN-1 cells, showing that nodavirus retains virulence in persistently infected wolffish for at least 16 wk post-bath-challenge.

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 VHfamilies and CLisotypes 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.

Incubation of follicular cells from postvitellogenic spotted wolffish ovaries with tritiated steroid precursors revealed that granulosa cells were able to convert 17-hydroxyprogesterone (17-P) to 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and androstenedione. Theca cells had limited ability to synthesise additional steroids from 17-P but converted 17,20β-P to 17,20β-P sulphate. Neither of the two cell layers was able to synthesise 5β-pregnane-3α,17,20β-triol-20-sulphate (3α,17,20β-P-5β-S) which is found in high concentrations in plasma. 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), 17,20β-dihydroxy-5β-pregnan-3-one (17,20β-P-5β) and 17,20β-P were most potent in inducing germinal vesicle breakdown (GVBD). Sulphation of 17,20β-P resulted in loss of GVBD inducing activity.