Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.     

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.     

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.     

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.     

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.)

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.     

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.     

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.     

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.     

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.     

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).     

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.     

Lateral line deformities in wild and farmed sea bass (Dicentrarchus labrax,L.) and sea bream (Sparus aurata,L.)

The lateral line of aquaculture fishes has rarely been studied although it is a very important anatomical organ that could serve as an inexpensive and easy tool to distinguish farmed from wild individuals. In the present study, lateral line deformities were examined in both wild and farmed sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) specimens to try to detail all possible differences between them. In order to do so, the morphology of the trunk lateral line in wild and farmed adults was examined whereby two major deformities were observed in both species: the ‘scale pocket’ deformity (14–40% incidence in all groups) where the specialized scales are missing but the canal underneath is present and the scale print is obvious, and the ‘somatic scales’ deformity (14–56% incidence in farmed individuals only) where the missing lateral line is covered with normal somatic scales. Histological examination confirmed the macroscopic observations in that the lateral line mechanism was present – although damaged – beneath the scale pocket deformity and completely absent beneath the somatic scales deformity. It is argued that the scale pocket deformity is a result of an accident during the life of the fish whereas the somatic scales deformity is an actual deformity in development.     

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.     

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.     

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.     

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.     

Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-alpha (TNF-alpha)

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.