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.     

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.     

Reduced gut bacterial translocation in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides (MOS)

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg^?1 dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

Summary The development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.     

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.     

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.     

Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides

The objective of this study was to determine the effect of two levels of inclusion of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on growth, feed utilization, immune status and disease resistance of European sea bass (Dicentrarchus labrax). Specimens of 35 g at initial density of 3 kg/m3 were fed during 67 days at 0 per thousand, 2 per thousand and 4 per thousand dietary MOS level of inclusion in a commercial sea bass diet. Food conversion rate, specific growth rate, whole body biochemical composition, phagocyctic index of head kidney macrophages, NBT index, lysozyme and alternative complement pathway (ACP) activities as well as gut and liver histological structure were evaluated. Growth significantly increased at both MOS dietary inclusion levels. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients. No differences were found on gut histological evaluation. Statistical differences (P<0.05) on the phagocytic index were denoted with the inclusion of 4 per thousand Bio-Mos group. A positive correlation was found between the levels of lysozyme and alternative complement pathway activities in blood and the level of inclusion of MOS in diets. After the feeding trial, a cohabitation challenge test and direct gut inoculation were also performed with the pathogen Vibrio alginolyticus in a ratio 3:1. Twenty-one days post-challenge the number of cohabitant fish infected in the control group reached 33% comparing with none on the 0.4 per thousand MOS group. Finally, new fish were infected with V. alginolyticus by gut canalisation. After 24h post-infection no significant difference was denoted between groups and 48 h post-infection total infected fish in the control group was twice that of the 2 per thousand and 4 per thousand MOS groups.     

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.     

Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus

Naïve sea bass juveniles (38.4 ± 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-β, TCRβ, CD4, CD8α, IgM, by using a quantitative PCR array system developed for sea bass.The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable “in vitro” increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-β and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.     

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.     

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.     

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.     

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.     

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.     

Imbalanced dietary ascorbic acid alters molecular pathways involved in skeletogenesis of developing European sea bass (Dicentrarchus labrax)

The influence of dietary ascorbic acid (AA) on growth and morphogenesis during the larval development of European sea bass (Dicentrarchus labrax) was evaluated until 45days post hatching. Diets incorporated 0, 5, 15, 30, 50 or 400mg AA per kg diet to give AA-0, AA-5, AA-15, AA-30, AA-50 and AA-400 dietary treatments, respectively. Dietary AA levels lower than 15mg/kg reduced larval growth and survival was affected in specimens fed diets devoid of AA. Globally, disruption of the expression of genes involved in AA and calcium absorption in the intestine (SVCT-1, TRPV-6), skeletogenesis (BMP-4, IGF-1, RARγ) and bone mineralization (VDRβ, osteocalcin) were observed in groups fed doses lower and higher than 50mg AA/kg diet. Such disturbances detected at molecular level were associated with disruptions of the ossification process and the appearance of skeletal abnormalities.