Josep Rotllant

Background colour influence on the stress response in cultured red porgy Pagrus pagrus

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

Expression of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis genes during zebrafish Danio rerio early embryogenesis

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes, many of which are particularly vital during embryonic development. This study investigated the expression of genes encoding enzymes involved in LC-PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, during early embryonic development of zebrafish. First, zebrafish elovl2 cDNA was isolated and functionally characterised in yeast, showing high specificity towards C20- and C22-PUFAs, compared to C18 substrates. Second, spatial-temporal expression for elovl2 and the previously cloned fad and elovl5 were studied during zebrafish early embryonic development. Temporal expression shows that all three genes are expressed from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. However, a complete activation of the biosynthetic pathway seems to be delayed until 12 hpf, when noticeable increases of fad and elovl2 transcripts were observed, in parallel with high docosahexaenoic acid levels in the embryo. Spatial expression was studied by whole-mount in situ hybridisation in 24 hpf embryos, showing that fad and elovl2 are highly expressed in the head area where neuronal tissues are developing. Interestingly, elovl5 shows specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues analysed, with liver, intestine and brain showing the highest expression.

Characterization of a C3 and a factor B-like in the carpet-shell clam, Ruditapes decussatus.

The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.

Fish melanocortin system

Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC(1)-MC(5)) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC(5) receptor, while pufferfish have 4 receptors with no melanocortin MC(3) receptor and one copy of melanocortin MC(5) receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC(2) receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC(1) receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC(1) receptor are responsible for reduced melanization. Both melanocortin MC(4) and MC(5) receptor are expressed in the hypothalamus, and central melanocortin MC(4) receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC(2) and MC(5) receptors. Both sea bass melanocortin MC(1) and MC(4) receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively.

Expression and role of Elovl4 elongases in biosynthesis of very long-chain fatty acids during zebrafish Danio rerio early embryonic development

Elovl4 is a fatty acyl elongase that participates in the biosynthesis of very long-chain fatty acids (>/=C24), which are relatively abundant in skin (saturated chains), or retina, brain and testes (polyunsaturated chains) of mammals. In the present study we characterised two Elovl4 proteins, Elovl4a and Elovl4b, from zebrafish Danio rerio, and investigated their expression patterns during embryonic development. Heterologous expression in bakers yeast showed that both zebrafish Elovl4 proteins efficiently elongated saturated fatty acids up to C36, with 26:0 appearing the preferred substrate as reported for human ELOVL4. Interestingly, activity for the elongation of PUFA substrates was only shown by Elovl4b, which effectively converted eicosapentaenoic (20:5n-3) and arachidonic (20:4n-6) acids to elongated polyenoic products up to C36. Furthermore, zebrafish Elovl4b may be involved in the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) as it had the capacity to elongate 22:5n-3 to 24:5n-3 which can be subsequently desaturated and chain shortened to DHA in peroxisomes. The distinct functional roles of zebrafish Elovl4 proteins were also reflected in their spatial-temporal expression patterns during ontogeny. Analyses by whole-mount in situ hybridisation in zebrafish embryos showed that elovl4a was expressed in neuronal tissues (wide-spread distribution in the head area), with elovl4b specifically expressed in epiphysis (pineal gland) and photoreceptor cells in the retina. Similarly, tissue distribution in adults revealed that elovl4a transcripts were found in most tissues analysed, whereas elovl4b expression was essentially restricted to eye and gonads. Overall, the results suggest that zebrafish elovl4b resembles other mammalian orthologues in terms of function and expression patterns, whereas elovl4a may represent an alternative elongase not previously described in vertebrates.

Sparc (Osteonectin) functions in morphogenesis of the pharyngeal skeleton and inner ear

Sparc (Osteonectin), a matricellular glycoprotein expressed by many differentiated cells, is a major non-collagenous constituent of vertebrate bones. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during embryogenesis are largely unknown. We cloned zebrafish sparc and investigated its role during development, using a mo rpholino antisense oligonucleotide-based knockdown approach. Consistent with its strong expression in the otic vesicle and developing pharyngeal cartilages, knockdown of Sparc function resulted in specific inner ear and cartilage defects that are highlighted by changes in gene expression, morphology and behavior. We rescued the knockdown phenotypes by co-injecting sparc mRNA, providing evidence that the knockdown phenotype is due specifically to impairment of Sparc function. A comparison of the phenotypes of Sparc knockdown and known zebrafish mutants with similar defects places Sparc downstream of sox9 in the genetic network that regulates development of the pharyngeal skeleton and inner ear of vertebrates.

Isolation and characterization of piscine osteonectin and downregulation of Its expression by PTH-related protein

The skeleton is the main source of osteonectin mRNA in adults of the seawater teleost sea bream Sparus auratus. It is expressed by cells forming the basement membrane of calcifying tissue indicating that, as in mammals, it may play a role in osteoblast differentiation. PTHrP induced downregulation of osteonectin mRNA in vitro in scales, a mineralizing tissue with bone‐like metabolism. This indicates a means to redirect calcium to activities such as vitellogenesis when this ion is in high demand.

Novel bioactive parathyroid hormone and related peptides in teleost fish

We report the identification, gene expression and biological activity of two parathyroid hormones (PTH; PTHA and PTHB), two PTH‐related peptides (PTHrP; PTHrPA and PTHrPB) and a PTH‐like ligand (PTH‐L) with hybrid characteristics in puffer fishes (Takifugu rubripes and Tetraodon fluviatilis). Experimental data are consistent with PTH‐L and PTHrPA having calciotropic activities equivalent, respectively, to tetrapod PTH and PTHrP. We hypothesise on the basis of phylogenetic and functional analysis that PTH‐L could be a fish relic of an ancestral PTH/PTHrP gene.

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.

Calcium handling in Sparus auratus: effects of water and dietary calcium levels on mineral composition, cortisol and PTHrP levels.

SUMMARY Juvenile gilthead sea bream (Sparus auratus L.; 10–40 g body mass) were acclimatized in the laboratory to full strength (34‰) or dilute (2.5‰) seawater and fed normal, calcium-sufficient or calcium-deficient diet for nine weeks. Mean growth rate, whole-body calcium and phosphorus content and accumulation rates were determined, as well as plasma levels of ionic and total calcium, cortisol and parathyroid hormone related protein (PTHrP; a hypercalcemic hormone in fish). When confronted with limited calcium access (low salinity and calcium-deficient diet), sea bream show growth arrest. Both plasma cortisol and PTHrP increase when calcium is limited in water or diet, and a positive relationship was found between plasma PTHrP and plasma ionic calcium (R2=0.29, N=18, P<0.05). Furthermore, a strong correlation was found between net calcium and phosphorus accumulation (R2=0.92, N=16, P<0.01) and between body mass and whole-body calcium (R2=0.84, N=25, P<0.01) and phosphorus (R2=0.88, N=24, P<0.01) content. Phosphorus accumulation is strongly calcium dependent, as phosphorus accumulation decreases in parallel to calcium accumulation when the diet is calcium deficient but phosphorus sufficient. We conclude that PTHrP and cortisol are involved in the regulation of the hydromineral balance of these fish, with growth-related calcium accumulation as an important target.