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.     

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.     

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.     

Embryonic development of the sea bass Dicentrarchus labrax

The embryonic development of the sea bass Dicentrarchus labrax during the endotrophic period is discussed. An 8 cells stage, not reported for other studied species, results from two rapid successive cleavages. Blastula occurs at the eighth division when the embryo is made of 128 cells. During gastrulation, the infolded blastoderm creates the endomesoblastic layer. The Kupffer??s vesicle is reported to drive the left/right patterning of brain, heart and digestive tract. Heart formation starts at 8 pairs of somites, differentiation of myotomes and sclerotomes starts at the stage 18 pairs of somites; main parts of the digestive tract are entirely formed at 25 pairs of somites. At 28 pairs of somites, a rectal region is detected, however, the digestive tube is closed at both ends, the jaw appears the fourth day after hatching, but the mouth is not opened before the fifth day. Although cardiac beating and blood circulation are observed, gills are not reported in newly hatched individuals; eye melanization appears concomitant with exotrophic behavior.     

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.     

Melatonin binding sites in the brain of European sea bass (Dicentrarchus labrax)

Characteristics, day-night changes, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[(125)I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radioligand binds to a single class of receptor site with the affinity (Kd) of 9.3 +/-0.6 pM and total binding capacity (Bmax) of 39.08 +/-0.86 fmol/mg protein (mean+/-SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPgammaS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.     

Double staining protocol for developing European sea bass (Dicentrarchus labrax) larvae

The alcian blue‐alizarin red technique was successfully adjusted to stain developing European sea bass (Dicentrarchus labrax) larvae. For an optimal staining protocol design both larval size and their morphological characteristics at each developmental stage were considered, since such parameters notably influence the staining of tissues. The incubation times of the different solutions were adjusted to allow the stain penetration for revealing the integrity of cartilaginous and bony tissues without significant tissue degradation. Three developmental windows were determined for an optimal staining procedure: (i) 4.5–6.4 mm, (ii) 6.7–8.7 mm, and (iii) 12.8–15.5 mm total length (TL). In order to validate the continuity of staining along the larval development, quantification of bone mineralization and osteocalcin gene expression were also monitored. Quantitative analysis revealed that ossification followed an exponential kinetic that was positively correlated with the osteocalcin gene expression pattern (Rs = 0.9762, P < 0.05). The mineralized tissue increased from 6.4 mm TL onwards, corresponding with the detection of the first ossified structures. The quantity of bony tissue increased gradually until 7.6 mm TL, since mineralization remained limited to the skull. From 8.3 to 15.5 mm TL, the mineralized bone was notable and nearly concerned the whole larval skeleton (skull, vertebral column and caudal complex). Since it was possible to detect the first cartilaginous and mineralized structures in specimens as small as 4.5 and 6.4 mm TL, respectively, this procedure is a useful tool to study the European sea bass skeletal ontogenesis, to precociously diagnose skeletal malformations in small larvae and eventually to better characterize the effect of different environmental and/or nutritional factors on the ossification status of specific skeletal components.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na⁺/K⁺‐ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na⁺/K⁺/2Cl^? cotransporters (NKCCs) and the chloride channel “cystic fibrosis transmembrane regulator” (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na⁺/K⁺-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na⁺/K⁺-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.     

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.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae ( Dicentrarchus labrax ) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila , Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.