Feeding European sea bass (Dicentrarchus labrax) juveniles with a functional synbiotic additive (mannan oligosaccharides and Pediococcus acidilactici): An effective tool to reduce low fishmeal and fish oil gut health effects?

The aim of this study was to assess the effects of dietary mannan oligosaccharides (MOS), Pediococcus acidilactici or their conjunction as a synbiotic in low fish meal (FM) and fish oil (FO) based diets on European sea bass (Dicentrarchus labrax) disease resistance and gut health. For that purpose, sea bass juveniles were fed one of 6 diets containing different combinations of MOS (Biomos® and Actigen©; Alltech, Inc., Kentucky, USA) and Pediococcus acidilactici (BAC, Bactocell®; Lallemand Inc., Cardiff, UK) replacing standard carbohydrates as follows (MOS (%)/BAC (commercial recommendation): high prebiotic level (HP) = 0.6/0, low prebiotic level (LP) = 0.3/0, only probiotic (B) = 0/+, high prebiotic level plus probiotic (HPB) = 0.6/+, low prebiotic level plus probiotic (LPB) = 0.3/+, control (C) = 0/0 for 90 days. After 60 and 90 days of feeding trial, fish were subjected to an experimental infection against Vibrio anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity and major histocompatibility complex class II (MHCII), transforming growth factor ß (TGF-ß), regulatory T-cell subset (CD4+T lymphocytes) and effector T cell (CD8α+T lymphocytes) gene expression patterns in gut by in situ hybridization were evaluated after 90 days of feeding. The effects of both additives on posterior gut through Gut Associated Lymphoid Tissue (GALT) gene expression was also studied. Fish fed the prebiotic and its combination with P. acidilactici presented increased weight regardless of the dose supplemented after 90 days of feeding, however no effect was detected on somatic indexes. For posterior gut, morphometric patterns and goblet cells density was not affected by MOS, P. acidilactici or its combination. Anti-iNOS and anti-TNFα gut immunopositivity patterns were mainly influenced by MOS supplementation and not by its combination with P. acidilactici. MHCII-ß, TCR-ß, CD4 and CD8-α positive cells distribution and incidence was not affected by diet. Fish fed HP dose presented a clear up-regulation of TNF-α, cyclooxygenase-2 (COX-2), CD4 and IL10, whereas P. acidilactici dietary supplementation increased the number of interleukin-1ß (IL1ß) and COX-2 gene transcripts. Synbiotic supplementation resulted in a reduction of MOS-induced gut humoral proinflammatory response by increasing the expression of some cellular-immune system related genes. Fish mortality after V. anguillarum infection was reduced in fish fed LPB and LP diets compared to fish fed the non-suppelmented diet after 90 days of feeding. Thus, overall pointing to the combination of a low dose of MOS and P. acidilactici as synbiont (LPB) as a viable tool to potentiate European sea bass juvenile's growth and disease resistance when supplemented in low FM and FO diets.

Molecular cloning and characterization of the matricellular protein Sparc/osteonectin in flatfish, Scophthalmus maximus, and its developmental stage-dependent transcriptional regulation during metamorphosis.

SPARC/osteonectin is a multifunctional matricellular glycoprotein, which is expressed in embryonic and adult tissues that undergo active proliferation and dynamic morphogenesis. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during development are largely unknown. In this report, we describe the isolation, characterization, post-embryonic developmental expression and environmental thermal regulation of sparc in turbot. The full-length turbot sparc cDNA contains 930 bp and encodes a protein of 310 amino acids, which shares 77, 75 and 80% identity with human, frog and zebrafish, respectively. Results of whole-mount in situ hybridization reveal a dynamic expression profile during post-embryonic turbot development. Sparc is expressed differentially in the cranioencephalic region; mainly in jaws, branchial arches, fin folds and rays of caudal, dorsal and anal fins. Furthermore, ontogenetic studies demonstrated that Sparc gene expression is dynamically regulated during post-embryonic turbot development, with high expression during stage-specific post-embryonic remodeling. Additionally, the effect of thermal environmental conditions on turbot development and on ontogenetic sparc expression was evaluated.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes.

Whole animal studies have indicated that Ca(2+) uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1-34)PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The amino-terminal peptides (2-34)PTHrP, (3-34)PTHrP and (7-34)PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1-34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca(2+) uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor.

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein.

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1-125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1-125) synthesis was induced by addition of 1mM isopropyl-beta-d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1-125) as judged by SDS-PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1-125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1-34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled (125)I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro.

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide.

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.