Eleven new microsatellites of the sea bass (Dicentrarchus labrax L.)

Eleven polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with an (AC)₁₂ probe. The loci showed different variation patterns in 21 unrelated sea bass individuals, with a mean number of alleles of 8.6 and a mean observed heterozygosity of 0.68. These microsatellite markers should be useful for population genetic analysis and biodiversity studies of sea bass.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) Tapasin

Mammalian tapasin (TPN) is a key member of the major histocompatibility complex (MHC) class I antigen presentation pathway, being part of the multi-protein complex called the peptide loading complex (PLC). Several studies describe its important roles in stabilizing empty MHC class I complexes, facilitating peptide loading and editing the repertoire of bound peptides, with impact on CD8⁺ T cell immune responses. In this work, the gene and cDNA of the sea bass (Dicentrarchus labrax) glycoprotein TPN have been isolated and characterized. The coding sequence has a 1329 bp ORF encoding a 442-residue precursor protein with a predicted 24-amino acid leader peptide, generating a 418-amino acid mature form that retains a conserved N-glycosylation site, three conserved mammalian tapasin motifs, two Ig superfamily domains, a transmembrane domain and an ER-retention di-lysine motif at the C-terminus, suggestive of a function similar to mammalian tapasins. Similar to the human counterpart, the sea bass TPN gene comprises 8 exons, some of which correspond to separate functional domains of the protein. A three-dimensional homology model of sea bass tapasin was calculated and is consistent with the structural features described for the human molecule. Together, these results support the concept that the basic structure of TPN has been maintained through evolution. Moreover, the present data provides information that will allow further studies on cell-mediated immunity and class I antigen presentation pathway in particular, in this important fish species.     

Not so much a sea bass: Divergent European sea bass (Dicentrarchus labrax L.) freshwater incursions

European sea bass (Dicentrarchus labrax [Linnaeus, 1758]) is a euryhaline marine migrant fish highly valuable for fisheries and aquaculture. Although juveniles are known to use estuaries and occasionally move to freshwater environments, these freshwater incursions had not been reported for adults. Recently, this behavior was observed in the Tagus River (Portugal) for adults occurring up to 150 km from the river mouth, about 80 km upstream from the tidal influence, suggesting the existence of a freshwater contingent. Fisheries management of sea bass should consider the putative existence of a freshwater contingent.     

Molecular cloning and characterization of sea bass (Dicentrarchus labrax,L.) calreticulin

Mammalian calreticulin (CRT) is a key molecular chaperone and regulator of Ca²⁺ homeostasis in endoplasmic reticulum (ER), also being implicated in a variety of physiological/pathological processes outside the ER. Importantly, it is involved in assembly of MHC class I molecules. In this work, sea bass (Dicentrarchus labrax) CRT (Dila-CRT) gene and cDNA have been isolated and characterized. The mature protein retains two conserved motifs, three structural/functional domains (N, P and C), three type 1 and 2 motifs repeated in tandem, a conserved pair of cysteines and ER-retention motif. It is a single-copy gene composed of 9 exons. Dila-CRT three-dimensional homology models are consistent with the structural features described for mammalian molecules. Together, these results are supportive of a highly conserved structure of CRT through evolution. Moreover, the present data provides information that will allow further studies on sea bass CRT involvement in immunity and in particular class I antigen presentation.     

More polymorphic microsatellite markers in the European sea bass (Dicentrarchus labrax L.)

We provide details of five microsatellite loci screened in 163 individual sea bass. Large numbers of alleles were observed at three loci (20–25) and heterozygosities ranged from 0.52 to 0.86. These loci should prove useful for population genetic studies and for the pedigree analysis and genetic management of this species in aquaculture.     

Influence of ATP and magnesium on phosphofructokinase from sea bass (Dicentrarchus labrax L.) liver

Glycolytic enzyme phosphofructokinase (PFK) from sea-bass liver shows inhibition for ATP4- and MG-ATP2-, and ATP4- is a competitive inhibitor with respect to MG-ATP2-. Free Mg2+ behaves as a mixed inhibitor on the kinetic with respect to the true enzyme substrate Mg-ATP2-, and eliminates the inhibition effect of this substrate. The kinetics with respect to Mg-ATP2- at non-inhibiting concentrations is not visibly affected by temperature of pH variation. The inhibiting effect of Mg-ATP2- is more marked at 22 and 10 degrees C (of three assayed temperatures 22, 15 and 10 degrees C and at physiological pH 6.8) as opposed to the maximum activity pH (8.0).     

Purification of 6-phosphogluconolactonase from bass (Dicentrarchus labrax L.)liver

The enzyme 6-phosphogluconolactonase (EC 3.1.1.31) is present at high levels in bass liver. The enzyme has been purified 1253-fold with an overall yield of 78% in a procedure involving dye-ligand and gel filtration chromatographies. The purified enzyme which is homogenous by all the usual criteria has a molecular weight of about 30,000. It exhibits a considerably high catalytic efficiency with Kcat/Km of 8.5 x 10(7) M-1.s-1 at 22 degrees C. Its activity illustrates the importance of glucose oxidation via the pentose phosphate pathway relative to glycolysis in this organism.     

A model for acute iron overload in sea bass (Dicentrarchus labrax L.)

Evaluation of several parameters involved in iron metabolism was carried out after intraperitoneal (i.p.) injection with iron dextran (IDx) in sea bass (Dicentrarchus labrax L.). After treatment, a rapid mobilization of IDx from the peritoneal cavity to other organs was observed. This was followed by a modification of normal peripheral blood iron parameters. Total iron (TI) and transferrin saturation (TS) rose rapidly, to 4.14 microg/ml and 83.7%, respectively, on day 3. In contrast, unsaturated iron binding capacity (UIBC) dropped from 3.19 microg/ml (at day 0) to 0.90 microg/ml on day 3. Tissue iron content was determined by atomic absorption spectometry (AAS). Three days post-IDx injection, values of iron concentration in liver, spleen and head kidney were significantly higher than control values (15, 6 and 9-fold increase, respectively). Samples of liver, spleen and head kidney were processed for routine histology, and the Perl's method was used for iron staining. Histological sections of the IDx-treated animals showed iron deposition in all tissues studied. In the liver, the iron was evenly distributed over the whole organ, being present in the hepatocytes. In the head kidney and spleen, the iron deposition was mainly observed in the melanomacrophage centres (MMCs). The present study characterizes several parameters involved in iron metabolism, and develops a fish model, of iron overload, which can be used in further studies of iron toxicity and iron-induced susceptibility to bacterial infections.     

Evidence of temperature-dependent sex determination in the European sea bass (Dicentrarchus labrax L.)

To test the hypothesis that sex determination in the European sea bass (Dicentrarchus labrax L.) Can be affected by the incubating temperature during the very early developmental stages, eggs from the same batch of spontaneously spawned broodstock were divided at the stage of half-epiboly into three groups according to rearing temperature: G13 = 13 degrees C, G15 = 15 degrees C, and G20 = 20 degrees C. Temperature treatment lasted until the middle of metamorphosis (17-18 mm total length, [TL]), and, with the exclusion of water temperature, all biotic and abiotic conditions were identical for the three experimental groups. The on-growing phase was performed under ambient photoperiod and temperature conditions for all groups. Sex proportions were determined by histological examination of the gonads of fish at 308, 467, and 568 days posthatch (DPH). At 308 DPH (TL: 135-201 mm), 100% of the specimens had differentiated into males and females. A significantly higher (P < 0.01) proportion of females was found in groups G13 (72-74%) and G15 (67-73%) than in group G20 (24-28%). At the final sampling there was no statistically significant difference in body weight between the experimental groups. However, in all groups, female fish were larger than males (P < 0.001). Results provide for the first time clear evidence that temperature during the very early developmental stages is the crucial factor affecting the process of sex differentiation of the sea bass, with low rearing temperatures (13 or 15 degrees C) resulting in sex proportions consistently skewed in favor of females.     

Ontogeny of B and T cells in sea bass (Dicentrarchus labrax, L.)

Monoclonal antibodies specific to sea bass Ig heavy (WDI 1) and light (WDI 3) chains and T cells (DLT15) were used in an ontogenetic study of sea bass by flow cytometry and immunocytochemistry. The influence of weight and age, as well as season, on B cell development was studied in the fastest and slowest growing offspring from the same spawn (5-305 days post hatch: dph). Additionally, B and T cell development was followed in samples of different offspring (5-137 dph). The results suggest that DLT15 recognises very early (pre-?) T cells as well as mature T cells and that these very early T cells might have their origin in a different compartment and subsequently mature in the thymus. They also appeared much earlier in ontogeny (between 5-12 dph onwards) than pre-B cells having cytoplasmic Ig (from 52 dph onwards). With the monoclonal antibodies used, adult levels of T and B cells were both reached between 137-145 dph, suggesting that sea bass is immunologically mature from at least that age onwards. As in other teleosts, the thymus appears to be the primary organ for T lymphocytes and head kidney the primary organ for B lymphocytes. For sea bass, age seems to be more important in determining B cell maturation than body weight.     

Temperature effects on cranial deformities in European sea bass, Dicentrarchus labrax (L.)

The present study investigates the effect of water temperature on the development of deformities during embryonic and larval stages of European sea bass, Dicentrarchus labrax (L.). Two temperature conditions were examined in duplicate by studying the presence of skeletal deformities in a sample of 45 to 51 fish taken from each population at the end of the rearing trials [20–23 mm total length (TL)]. The results indicated that water temperature during the embryonic and larval phase has significant effects on deformation of the branchiostegal rays (P < 0.001), but not of the mouth and the fins (P > 0.05). At 15°C, 27.2–33.4% of the examined fish had branchiostegal rays of abnormal shape and/or orientation, whereas at 20°C this deformity had a frequency of only 4.0–4.1%. The frequency distribution graph of branchiostegal counts demonstrated a significant deviation in the deformed fish from the normal (seven rays on each side of the body) phenotype at both temperatures tested. This deviation was mainly expressed as a lack of one to four rays (56.7% of deformed fish), or the formation of one extra ray (26.7% of deformed fish) (P < 0.001, G‐test). The results are discussed in respect to the possible mechanisms of temperature effects on the development of skeletal deformities.     

Pathological and epidemiological observations on rickettsiosis in cultured sea bass (Dicentrarchus labrax L.) from Greece

A systemic infection of a Rickettsia‐like organism (RLO) in cultured sea bass is described for the first time. In hatcheries, clinical signs were lethargy, inappetence and discoloration. Twenty days after transfer to sea cages from hatcheries where the disease existed, fish showed erratic and abnormal swimming behaviour, loss of orientation, and lethargy. Cumulative mortality in colder months of the year reached 30% in hatcheries and 80% in cages. Surviving fish in cages did not show any clinical signs of RLO infection in the subsequent year. Evidence for a systemic distribution of RLO was supported by histolopathological lesions in both infected hatchery and caged fish, where the lesion profile included cranial sensory, central nervous, integumental and alimentary organ systems. Intracranial lesions were primarily characterized by an ascending histiocytic perineuritis and necrotizing congestive meningoencephalitis, with evidence for transfer of infective agents across the blood–brain barrier confirmed by the presence of RLOs within capillary endothelium and histiocytes in inflamed regions of the optic tectum and the cerebellum. In the most severe cases, infection spread to the statoacoustical (semicircular) canal system and the ependymal lining of ventricles, with marked rickettsial‐laden histiocytic infiltration of the canal lumen. Integumental lesions were restricted to the oral submucosa, nares and integumental dermis of the cranium. Alimentary lesions were noted in both the liver parenchyma and mucosa/submucosa of the stomach. In all affected organs the RLOs were found by immunohistochemistry to be related to Piscirickettsia salmonis.     

Lordotic vertebrae in sea bass (Dicentrarchus labrax L.) are adapted to increased loads

Lordosis in fish is an abnormal ventral curvature of the vertebral column, accompanied by abnormal calcification of the afflicted vertebrae. Incidences of lordosis are a major problem in aquaculture and often correlate with increased swimming activity. To understand the biomechanical causes and consequences of lordosis, we mapped the morphological changes that occur in the vertebrae of European sea bass during their development from larva to juvenile. Our micro-CT analysis of lordotic and non-lordotic vertebrae revealed significant differences in their micro-architecture. Lordotic vertebrae have a larger bone volume, flattened dorsal zygapophyses and extra lateral ridges. They also have a larger second moment of area (both lateral and dorso-ventral) than non-lordotic vertebrae. This morphology suggests lordotic vertebrae to be adapted to an increased bending moment, caused by the axial musculature during increased swimming activity. We hypothesize the increase in swimming activity to have a two-fold effect in animals that become lordotic. The first effect is buckling failure of the axial skeleton due to an increased compressive load. The second effect is extra bone deposition as an adaptive response of the vertebrae at the cellular level, caused by an increased strain and strain rate in these vertebrae. Lordosis thus comprises both a buckling failure of the vertebral column and a molecular response that adapts the lordotic vertebrae to a new loading regime.     

Parasites of wild sea bass Dicentrarchus labrax from Norway

Thirteen wild sea bass from the Oslo fjord in south-eastern Norway were examined for parasites. Nineteen species were found, comprising 5 protozoans, 1 monogenean, 8 digeneans, 1 cestode, 2 nematodes and 2 crustaceans. Based on the similarity to the parasitic fauna of Mediterranean sea bass, it is predicted that sea bass farmers in Northern Europe will experience the same parasite problems as sea bass farmers in warmer regions.     

Neutral calcium-activated proteases from European sea bass (Dicentrarchus labrax L.) muscle: polymorphism and biochemical studies

Calcium-dependent proteinases or calpains were studied in fish muscle. Hydrophobic chromatography, followed by anion-exchange chromatography of the soluble fraction of sea bass white muscle proteins, resulted in three peaks of calcium-dependent protease activity at neutral pH (A, B and C). They are all neutral cysteine calcium-activated proteinases and can, therefore, be classified as calpain-like enzymes. From the Ca2+ concentration required for activity, A is a mu-calpain, and B and C are m-calpains. They share many properties with calpains from other vertebrate cells but differ in native mass, subunit composition, and the unusual numbers in which they are present. Their specific pattern of expression throughout the year could be of great importance to the resulting rate and extent of degradation of fish flesh after death.     

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II β -antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.     

Occurrence of two NOS isoforms in the developing gut of sea bass Dicentrarchus labrax (L.)

In this work we have examined the appearance and distribution of nitric oxide synthase (NOS), with histochemical, immunohistochemical and biochemical methods, during development of the sea bass (Dicentrarchus labrax) gut. The data showed that both the calcium-calmodulin dependent neuronal isoform (nNOS) and calcium-independent inducible isoform (iNOS) are present in the larval gut of sea bass. The nNOS-immunoreactivity was present in the epithelial cells and enteric nerve cells of gut both in the 8-day-old specimens and in the 24-day-old-larvae. In the adult nNOS-immunoreactivity disappeared from epithelial cells, remaining in the wall intramural neurons and fibers. The iNOS-immunoreactivity was present in the epithelial cells of 24-day-old-larvae and was not detectable in the adult gut. Western blot analysis and determination of NOS activity also demonstrated the presence of the two NOS isoforms, nNOS and iNOS, in the gut of 24-day-old specimens. The presumably different roles played by the two isoforms of enzyme are discussed. The presence of nNOS isoform in the gut enteric neurons of the same larval stages of D. labrax in which we previously demonstrated the presence of substance P and Vasoactive Intestinal Polypeptide (VIP), may suggest that all these three components of the motility control system are already present in the larval phase. Nitric oxide (NO) may be also involved in the early immune response. The present results on the occurrence of iNOS isoform in epithelial gut cells of the same regions in which the gut-associated lymphoid tissue (GALT) will differentiate, may suggest for NO a role in early defence mechanisms, before the establishment of immune responses in GALT. Finally, the developmental and regional differences in nNOS and iNOS expression also suggest a regulatory role in development and differentiation of the sea bass gut.     

Alternative stress indicators in sea bass Dicentrarchus labrax, L.

Stress indicators (plasma cortisol, glucose and lactate) were evaluated in sea bass Dicentrarchus labrax submitted to different pre-slaughter conditions, and cortisol levels in plasma were found to correlate with those detected in mucus, gut contents and muscle. The results demonstrate new possibilities for monitoring stress status in fishes in which blood sampling is difficult or impossible.