Vaccination of Silver Sea Bream (Sparus sarba) against Vibrio alginolyticus: Protective Evaluation of Different Vaccinating Modalities.

In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.

PBDE-47 exposure causes gender specific effects on apoptosis and heat shock protein expression in marine medaka, Oryzias melastigma.

Marine medaka (Oryzias melastigma) was fed with a low and high dose of dietary 2,2',4,4'-tetra-bromodiphenyl ether (PBDE-47), over 21 days. Gender specific changes in caspases 3 and 8 in medaka were found as activities in male medaka were significantly increased in both liver and muscle at both low and high exposure levels whereas caspase activity in female medaka tissue remained unchanged. Results of HSP90 and HSP70 immunoassays also showed gender specific related changes as both HSP families were unchanged in liver and muscle of male medaka but significantly increased in liver and muscle of female medaka, following PBDE-47 exposure.

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba).

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes.

Aquaporin 1a expression in gill, intestine, and kidney of the euryhaline silver sea bream.

This study aimed to investigate the effects of chronic salinity acclimation, abrupt salinity transfer, and cortisol administration on aquaporin 1 (AQP1) expression in gill, intestine, and kidney of silver sea bream (Sparus sarba). An AQP1a cDNA was cloned and found to share 83-96% amino acid sequence identity with AQP1 genes from several fish species. Tissue distribution studies of AQP1a mRNA demonstrated that it was expressed in gill, liver, intestine, rectum, kidney, heart, urinary bladder, and whole blood.

Influence of dietary carbohydrate level on endocrine status and hepatic carbohydrate metabolism in the marine fish Sparus sarba.

Silver sea bream, Sparus sarba, were fed two diets of different carbohydrate levels (2 and 20% dextrin) for 4 weeks, and the effects on organ indices, liver composition, serum metabolite and hormone levels and gene expression profile of key enzymes of carbohydrate metabolism in the liver were investigated. By using real-time PCR, mRNA expression levels of carbohydrate metabolic enzymes including glucokinase (GK, glycolysis), glucose-6-phosphatase (G6Pase, gluconeogenesis), glycogen synthase (GS, glycogenesis), glycogen phosphorylase (GP, glycogenolysis) and glucose-6-phosphate dehydrogenase (G6PDH, pentose phosphate pathway) in liver of sea bream have been examined, and it was found that high dietary carbohydrate level increased mRNA level of GK but decreased mRNA levels of G6Pase and GP. However, mRNA levels of GS and G6PDH were not significantly influenced by dietary carbohydrate.

Effects of growth hormone, insulin-like growth factor I, triiodothyronine, thyroxine, and cortisol on gene expression of carbohydrate metabolic enzymes in sea bream hepatocytes.

The present study investigated the regulatory effects of growth hormone (GH), human insulin-like growth factor I (hIGF-I), thyroxine (T(4)), triiodothyronine (T(3)) and cortisol, on mRNA expression of key enzymes involved in carbohydrate metabolism, including glucokinase (GK), glucose-6-phosphatase (G6Pase), glycogen synthase (GS), glycogen phosphorylase (GP) and glucose-6-phosphate dehydrogenase (G6PDH) in hepatocytes isolated from silver sea bream. Genes encoding GK, G6Pase, GS and GP were partially cloned and characterized from silver sea bream liver and real-time PCR assays were developed for the quantification of the mRNA expression profiles of these genes in order to evaluate the potential of these carbohydrate metabolic pathways. GK mRNA level was elevated by GH and hIGF-I, implying that GH-induced stimulation of GK expression may be mediated via IGF-I.

Growth hormone attenuates branchial HSP70 expression in silver sea bream.

In this study the effects of growth hormone (GH) on silver sea bream branchial heat-shock protein 70 (HSP70) expression was investigated using in-vivo and in-vitro experiments. For in-vivo experiments, sea bream were administered recombinant bream GH or the GH secretagogue hexarelin. Pituitary levels of GH were unchanged in fish administered exogenous GH but decreased on hexarelin administration, in comparison with saline controls.

Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells.

Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH active-site mutant has lost all hemolytic and phospholipase activities as well as pathogenicity. In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated.

Effect of extracellular osmolality and ionic levels on pituitary prolactin release in euryhaline silver sea bream (Sparus sarba).

In many euryhaline fish, prolactin (PRL) plays a key role in freshwater adaptation. Consistent with this function, the present study showed a remarkable reduction in pituitary PRL content of silver sea bream abruptly transferred to low salinity (6ppt). This reduction in pituitary PRL content followed closely the temporal changes in serum osmolality and ion levels.

Direct actions of cortisol, thyroxine and growth hormone on IGF-I mRNA expression in sea bream hepatocytes.

The present study aims to investigate potential regulatory effect of different growth-related hormones including growth hormone (GH), human insulin-like growth factor-I (hIGF-I), thyroxine (T(4)), triiodothyronine (T(3)) and cortisol, on insulin-like growth factor-I (IGF-I) mRNA expression of hepatocytes isolated from silver sea bream. By using real-time PCR, IGF-I mRNA expression profiles of hepatocytes in response to individual hormones were determined in vitro. Hepatocytes incubated with GH at concentrations of 10-1000 ng/mL showed significantly higher IGF-I expression, but the elevation was attenuated at high concentration of GH (1000 ng/mL).

Upregulation of metallothionein and glucose-6-phosphate dehydrogenase expression in silver sea bream, Sparus sarba exposed to sublethal levels of cadmium.

In this study, the induction of metallothionein (MT) and glucose-6-phosphate dehydrogenase (G6PDH) gene expression in response to exposure to cadmium (Cd(2+)) was investigated in silver sea bream (Sparus sarba) in vivo. In addition, a primary hepatocyte culture has been developed from silver sea bream liver in order to assess the changes in gene expression of MT and G6PDH in hepatocytes directly exposed to Cd(2+) in vitro. The sea bream metallothionein gene was cloned and characterized for the development of real-time PCR assays for quantification of MT transcript abundance.

Prolactin-releasing peptide, a possible modulator of prolactin in the euryhaline silver sea bream (Sparus sarba): A molecular study.

PRL and PrRP cDNAs have been isolated from euryhaline silver sea bream (Sparus sarba). The PRL cDNA consists of 1360bp encoding 212 amino acids whereas the PrRP cDNA contains 631bp encoding preproPrRP with 122 amino acids. The mature PrRP sequence within the preprohormone is identical to the PrRPs isolated from other fish species.

Impact of nitrite exposure on endocrine, osmoregulatory and cytoprotective functions in the marine teleost Sparus sarba.

The effects of nitrite, at varying concentrations (0, 25 and 50mg/l), on silver sea bream (Sparus sarba), was assessed after 7 days exposure. Nitrite exposure resulted in an elevated renosomatic index in parallel with increased kidney water content. Measurements of serum thyroid hormones demonstrated that levels of thyroxine (T(4)) were decreased upon nitrite exposure whereas triiodothyronine (T(3)) concentrations remained unchanged.

Effects of mitogenic hormones on HSP70 expression in a silver sea bream fibroblast cell line and a primary macrophage preparation.

This study was performed in order to elucidate the role and importance of three mitogenic hormones [growth hormone (GH), insulin-like growth factor-I (IGF-1) and prolactin (PRL)] on heat shock protein 70 (HSP70) expression in a silver sea bream fibroblast cell line and a primary macrophage preparation. Fibroblasts and macrophages that were exposed to GH at concentrations of 1-1000ng/ml did not exhibit modulated HSP70 expression, whereas GH at an exposure concentration of 10ng/ml lowered HSP70 levels in macrophages. Upon exposure to IGF-1 it was found that HSP70 expression remained unchanged in fibroblasts but was significantly decreased in macrophages at exposure concentrations of 1-10ng/ml.

Positive feedback of hepatic angiotensinogen expression in silver sea bream (Sparus sarba).

The renin-angiotensin system (RAS) is involved in the maintenance of fluid homeostasis in vertebrates. Production of the precursor protein, angiotensinogen, is regulated by other components within the RAS. Angiotensin II (Ang II) stimulates the production and secretion of angiotensinogen in many mammalian models.

Cortisol can be pro- or anti-apoptotic in sea bream cells: potential role of HSP70 induction for cytoprotection.

Cortisol, and heat shock protein 70 (HSP70) are known to perform key roles as part of the fish stress response. In the present study, two in vitro systems were used to investigate a possible cortisol-HSP70-apoptosis regulatory relationship. Using a developed silver sea bream fibroblast cell line (SSF), cortisol was found to induce HSP70 synthesis with a concomitant protection against camptothecin induced apoptosis.

Tissue distribution, effects of salinity acclimation, and ontogeny of aquaporin 3 in the marine teleost, silver sea bream (Sparus sarba).

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood.

Differential status of the renin-angiotensin system of silver sea bream (Sparus sarba) in different salinities.

Silver sea bream (Sparus sarba) is extremely euryhaline and can survive in a wide range of salinities (0-70 per thousand). The status of the renin-angiotensin system (RAS) in sea bream adapted to different salinities was studied. As indicated by plasma Ang II levels, a suppressed status of the RAS was found to occur under brackish water conditions; while under hypersaline conditions, an activated RAS prevailed, especially in fish adapted to double strength seawater (70 per thousand).

Impact of heavy metals and organochlorines on hsp70 and hsc70 gene expression in black sea bream fibroblasts.

The aim of the present study was to investigate the effects of environmentally important heavy metals and organochlorines on the transcriptional profiles of genes coding for heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) in a black sea bream fibroblast cell line. Using the nucleotide sequence information, from the cloned genes, specific reverse transcriptase-polymerase chain reaction (RT-PCR) methods were devised to test the effects of heavy metals (Cd2+, Cu2+ and Ni2+) and organochlorines (aroclor 1254, hexachlorobenzene and 2-4-dichloroaniline) on the cell stress response. Hsp70 was induced in fibroblasts upon heavy metal exposure concentrations as low as 0.

Seabream ghrelin: cDNA cloning, genomic organization and promoter studies.

Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels.

Molecular cloning of growth hormone from silver sea bream: effects of abiotic and biotic stress on transcriptional and translational expression.

The pituitary growth hormone (GH) gene of silver sea bream (Sparus sarba) was cloned and characterized and found to be 615 base pairs encoding a protein of 204 amino acids. Using a bacterial expression system, recombinant protein was prepared and rabbit polyclonal antibody was raised. Transcript and protein amounts of GH were measured in fish that were adapted to a range of salinities, acclimated to different temperatures, or undergoing a natural time course of Vibrio alginolyticus infection.

Expression studies on glucose-6-phosphate dehydrogenase in sea bream: effects of growth hormone, somatostatin, salinity and temperature.

In the present study, the gene encoding hepatic glucose-6-phosphate dehydrogenase (G6PDH) was cloned and characterized from silver sea bream (Sparus sarba). The deduced amino acid sequence from sea bream g6pdh shared 78-84% homology with deduced amino acid sequences from previously cloned teleost g6pdh genes. A reverse transcriptase polymerase chain reaction (RT-PCR) coupled with radioisotope hybridization method was used for assessment of g6pdh expression and it was found that administration of growth hormone to sea bream increased g6pdh transcript and G6PDH activity whereas injections of somatostatin decreased both of these parameters.

Upregulation of the somatotropic axis is correlated with increased G6PDH expression in Black Sea bream adapted to iso-osmotic salinity.

Black sea bream (Mylio macrocephalus) were adapted to salinities of 0 ppt (freshwater), 6 ppt (hypo-osmotic), 12 ppt (iso-osmotic), 33 ppt (seawater), and 50 ppt (hypersaline) for 1 month. Using RT-PCR assays, the expression of pituitary growth hormone (GH) and hepatic insulin-like growth factor 1 (IGF-1) genes were studied. It was found that the transcripts for both of these genes were highest in fish maintained at iso-osmotic salinity.

Growth hormone increases hsc70/hsp70 expression and protects against apoptosis in whole blood preparations from silver sea bream.

Preparations of whole blood, from silver sea bream (Sparus sarba), were used for heat shock protein 70 (hsp70) and apoptosis studies. It was found that the expression of both gene members of the hsp70 family (hsc70 and hsp70) were upregulated during acute heat shock. The transcript abundance of both of these genes was increased when cells were exposed to growth hormone (GH) at concentrations of 10 and 100 ng/mL.