Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Cellular responses in the skin of the gilthead sea bream Sparus aurata L. and the sea bass Dicentrarchus labrax (L.) exposed to high ammonia

Adult gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax were exposed for 24 and 48 h, respectively, to two concentrations of ammonia each (mean values of 3·34 and 13·10 mg l(-1) TA-N in S. aurata; 2·99 and 11·90 mg l(-1) TA-N in D. labrax). Light microscopy and computerized morphometry were used to evaluate ammonia-induced alterations in skin structure during exposure and following recovery in normal water. In S. aurata, ammonia exposure induced a concentration-dependent increase in the number (hyperplasia) of neutral mucous cells (mc), with peak values at 24 h recovery after exposure. An increase in the dispersion of melanosomes in skin melanocytes was also observed in the dermis and occasionally in the epidermis of S. aurata, with peak values at 24 h of ammonia exposure. Exposure of D. labrax to ammonia had, likewise, concentration-dependent effects on mucous secretion. Of the two types of mc in this species, there was an increase in the number of the neutral mc and a reduction in the much more numerous acid mc, with peak values at 24 and 48 h, respectively, of ammonia exposure. The more intense mucous secretion in D. labrax compared to S. aurata could be related to the lower tolerance to ammonia in D. labrax, as reported elsewhere. Finally, the increase in melanosome dispersion was less evident in D. labrax, due to highly variable control values. These morphological alterations to the skin could be useful indicators of non-specific stress in cultured fishes.     

Acute stress response in gilthead sea bream (Sparus aurata L.) is time-of-day dependent: Physiological and oxidative stress indicators

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream (Sparus aurata L.) submitted to air exposure for 30?s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4?h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm of corticotropin-releasing hormone (crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression of crh-binding protein (crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i) cytochrome c oxidase 4 showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii) peroxiredoxin (prdx) 3 and 5 (prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii) uncoupling protein 1 showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses.     

Effect of decreased environmental salinity on growth hormone cells in the gilthead sea bream (Sparus aurata)

The effect of decreased environmental salinity on growth hormone producing cells (GH cells) of the adenohypophysial proximal pars distalis has been studied in the gilthead sea bream ( Sparus aurata L.) adapted to sea water (SW, 980 mosmol kg ⁻¹) and brackish water (BW, 200 mosmol kg ⁻¹). A combined immunocytochemical, morphometric and electron microscopic study was carried out. GH cells offish adapted to BW occupied a greater hypophysial volume (about 21% of the total hypophysial volume in BW, 17% in SW) and had a larger nuclear area (mean 16 μm² in BW, 13 μm² in SW) than GH cells of SW-adapted fish. The immunoreactivity against a salmon GH-antiserum was lower in BW (mean optical density 142 in BW, 159 in SW). Ultrastructural characteristics of GH cells of BW-adapted fish were distended rough endo-plasmatic reticulum and large secretory granules (about 216 nm in diameter for BW, 209 nm in SW). Volumetric, densitometric and ultrastructural evidence suggested that the synthesis and release of GH were activated in S. aurata adapted to hypo-osmotic environment.     

The larval development of lateral musculature in gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax

Fibre-type differentiation of the lateral musculature has been studied in Sparus aurata (L.) and Dicentrarchus labrax (L.) during larval development. Histochemical and ultrastructural techniques show two presumptive muscle layers and two germinative zones of presumptive myoblasts. At hatching, myotomal muscle consists of a monolayer of thin undifferentiated cells near the skin (first germinative zone) overlying another mono-layer of small diameter fibres extending hypaxially and epaxially away from the transverse septum. Below this, there is a much thicker, deep layer of fibres, generally large in diameter and polygonal in shape. The presumptive myoblasts are located between these two layers of fibres in the second germinative zone. Initially, the superficial and deep muscle fibres show high and low myosin ATPase activity, respectively. Both layers grow by generating new fibres from the two mentioned germinative zones. At the end of larval life, the superficial layer changes its histochemical profile from high to low myosin ATPase activity and, at the same time, intermediate or pink muscle fibres can be observed by oxidative activity (the NADH-TR reaction). Morphometric analysis shows a significant increase in mean fibre diameter during successive ages, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibre population from the germinative zones (hyperplastic growth).     

Exploring neutral and adaptive processes in expanding populations of gilthead sea bream, Sparus aurata L., in the North-East Atlantic

Recent studies in empirical population genetics have highlighted the importance of taking into account both neutral and adaptive genetic variation in characterizing microevolutionary dynamics. Here, we explore the genetic population structure and the footprints of selection in four populations of the warm-temperate coastal fish, the gilthead sea bream (Sparus aurata), whose recent northward expansion has been linked to climate change. Samples were collected at four Atlantic locations, including Spain, Portugal, France and the South of Ireland, and genetically assayed using a suite of species-specific markers, including 15 putatively neutral microsatellites and 23 expressed sequence tag-linked markers, as well as a portion of the mitochondrial DNA (mtDNA) control region. Two of the putatively neutral markers, Bld-10 and Ad-10, bore signatures of strong directional selection, particularly in the newly established Irish population, although the potential 'surfing effect' of rare alleles at the edge of the expansion front was also considered. Analyses after the removal of these loci suggest low but significant population structure likely affected by some degree of gene flow counteracting random genetic drift. No signal of historic divergence was detected at mtDNA. BLAST searches conducted with all 38 markers used failed to identify specific genomic regions associated to adaptive functions. However, the availability of genomic resources for this commercially valuable species is rapidly increasing, bringing us closer to the understanding of the interplay between selective and neutral evolutionary forces, shaping population divergence of an expanding species in a heterogeneous milieu.     

Heterozygosity-fitness correlations in the gilthead sea bream Sparus aurata using microsatellite loci from unknown and gene-rich genomic locations

Heterozygosity-fitness correlations (HFC) were assessed for a sample of a gilthead sea bream Sparus aurata population. Two hundred and seventy-one fish were genotyped at 22 known and novel microsatellite loci, from which correlations between the multilocus heterozygosity index (I(MLH) ) and various fitness traits (fork length, mass and specific growth rates) were calculated. Significant global HFCs were found in this sample (0·02 ≤r(2) ≤ 0·08). In addition, all the significant correlations found in this work were negative, indicating that heterozygotes had lower fitness than their homozygote counterparts. Marker location could not explain the observed HFCs. Evidence of inbreeding, outbreeding or population and family structuring was not found in this work. The presence of undetected general effects that may lead to the appearance of HFCs, however, cannot be ruled out. These results seem to be best explained by the occurrence of local effects (due to linkage) or even by possible direct locus advantages.     

Oxygen uptake,heart rate and activities of locomotor muscles during a critical swimming speed protocol in the gilthead sea bream Sparus aurata

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue.     

Establishment of dominance relationships in gilthead sea bream Sparus aurata juveniles during feeding: effects on feeding behaviour, feed utilization and fish health

In gilthead sea bream Sparus aurata held in groups of two, five or 10 fish, social hierarchies were observed. Subordinate S. aurata were characterized by elevation of basal levels of plasma cortisol, together with a reduced immunological potential. Subordinate fish also showed lower feed intake, feed utilization and lower growth. Fatty acid composition was also affected by social status, with a lower content of saturated acids, oleic and eicosapentaenoic, in muscle and liver of fish considered as subordinate. Results show that social hierarchy acts as a stressor in S . aurata .     

The effect of hypoxia on intermediary metabolism and oxidative status in gilthead sea bream (Sparus aurata) fed on diets supplemented with methionine and white tea

The present study evaluates the influence of previous nutritional status, fish fed on diets supplemented with tea and methionine, on acute hypoxia tolerance and subsequent recovery of Sparus aurata juveniles. Four isonitrogenous (45% of protein) and isolipidic (18% lipid) diets were formulated to contain 0.3% methionine, 2.9% white tea dry leaves or 2.9% of white tea dry leaves+0.3% methionine. An unsupplemented diet was used as control. Hepatic key enzymes of intermediary metabolism and antioxidant status, superoxide dismutase isoenzyme profile, glutathione (total, reduced and oxidized) and oxidative damage markers were determined under normoxia, hypoxia challenge and during normoxia recovery. Dietary white tea inclusion decreased plasma glucose levels under normoxia and seemed to induce an increase in anaerobic pathways as showed by enhanced liver lactate dehydrogenase activity. Hypoxia challenge reversed some of the responses induced by diet tea supplementation. Hypoxia decreased plasma glucose levels, increased glucose 6-P-dehydrogeanse activity, decreased superoxide dismutase activity (especially Mn-SOD and CuZn-SOD isoforms) and increased glutathione peroxidase activity in all dietary treatments. Catalase activity during hypoxia varied with dietary treatments and glutathione reductase was not modified. Antioxidant defenses were insufficient to avoid an oxidative stress condition under hypoxia, independently of dietary treatment. In general, pre-challenge values were recovered for almost all parameters within 6 h recovery time.     

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

Leptotheca sparidarum n. sp. (Myxosporea: Bivalvulida), a parasite from cultured common dentex (Dentex dentex L.) and gilthead sea bream (Sparus aurata L.) (Teleostei: Sparidae)

A new myxosporean, Leptotheca sparidarum n. sp., is described from the trunk kidney of two sparid fish, Sparus aurata and Dentex dentex, in several culture facilities from the Western Mediterranean coasts. It is distinguished from all the previously described species by spore morphometrics. Spores with two equal polar capsules and one binucleated sporoplasm. Spore measurements from D. dentex were 5-7.1 micron long x 8.8-12.3 micron thick x 5.88-6.18 micron wide; polar capsules 2.6-3.5 micron in diam. Spore measurements from S. aurata were 5.1-8.24 micron long x 9.41-11.76 micron thick; polar capsules 2.4-3.2 micron in diam. No significant differences were detected between either host. Prevalence could reach 21.4% in D. dentex and 19% in some stock of S. aurata. Renal tubules were the typical site of infection, which was also found in ureters and glomeruli, but seldom in the epiepithelial position of the gut. Spores were formed in disporous sporoblasts, and spore maturation seemed to proceed from the host epithelium towards the lumen. Trophozoites were attached to the host epithelial cells of renal tubules by pseudopodial-like projections, which were inserted into gaps between epithelial cells. Also, cell junctions were observed between primary cells and between neighboring spores.     

Dietary oils mediate cortisol kinetics and the hepatic mRNA expression profile of stress-responsive genes in gilthead sea bream (Sparus aurata) exposed to crowding stress. Implications on energy homeostasis and stress susceptibility

Juveniles of gilthead sea bream were fed with plant protein-based diets with fish oil (FO diet) or vegetable oils (66VO diet) as dietary lipid sources. No differences in growth performance were found between both groups, and fish with an average body mass of 65–70 g were crowded (90–100 kg/m³) to assess the stress response within the 72 h after the onset of stressor. The rise in plasma cortisol and glucose levels was higher in stressed fish of group 66VO (66VO-S) than in FO group (FO-S), but the former stressed group regained more quickly the cortisol resting values of the corresponding non-stressed diet group. The cell–tissue repair response represented by derlin-1, 75 kDa glucose-regulated protein and 170 kDa glucose-regulated protein was triggered at a lower level in 66VO-S than in FO-S fish. This occurred in concert with a long-lasting up-regulation of glucocorticoid receptors, antioxidant enzymes, enzyme subunits of the mitochondrial respiratory chain, and enzymes involved in tissue fatty acid uptake and β-oxidation. This gene expression pattern allows a metabolic phenotype that is prone to “high power” mitochondria, which would support the replacement of fish oil with vegetable oils when theoretical requirements in essential fatty acids for normal growth are met by diet.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Homologous growth hormone (GH) binding in gilthead sea bream (Sparus aurata). Effect of fasting and refeeding on hepatic GH-binding and plasma somatomedin-like immunoreactivity

Specific binding of gilthead sea bream growth hormone (sbGH) to liver membrane preparations was a time and temperature dependent process, and was saturable by increasing amounts of membrane proteins. Scatchard analysis evidenced a single class of high-affinity and lowcapacity binding sites. Ovine prolactin, recombinant tilapia prolactin, carp gonadotropin and chinook salmon gonadotropin did not compete for the¹²⁵I-sbGH binding sites, while recombinant trout GH, bovine GH and human GH displaced iodinated sbGH in a dose dependent-manner. IGF-I-like immunoreactivity was detected after acidification of plasma and removal of IGF-I binding activity. A parallel displacement to the rhIGF-1 standard was observed with extracted plasma samples. Free and total hepatic GH-binding decreased during long-term starvation (3–9 weeks), returning to control values during the refeeding period. Plasma IGF-I-like immunoreactivity showed a similar trend. To our knowledge, this is the first report that indicates a coordinated regulation of GH-binding and plasma somatomedin-like activity in a typical marine fish.     

In vitro effect of peas, Pisum pisum, and chickpeas, Cicer arietinum, on the immune system of gilthead seabream, Sparus aurata

The future for a sustainable aquaculture relies on the formulation of feed including alternatives to fish meal and fish oil that do not impair fish growth and that improve fish health status. Grain legumes such as field peas and chickpeas offer good sources of proteins, carbohydrates, fibers, vitamins, and minerals. The effect of peas and chickpeas on the immune system of seabream was assessed in vitro in order to detect any potential immunosuppressing problem. Peas was determined to be a better fishmeal alternative than chickpeas as they induced higher respiratory burst measured by the nitro blue tetrazolium assay and primed the Phorbol 12-myristate 13-acetate (PMA)-stimulated intracellular respiratory burst whereas chickpeas neither directly stimulated respiratory burst nor primed it. However, when the intra- and extracellular respiratory burst activities were taken into account, high concentrations of peas inhibited the zymosan- and PMA-triggered chemiluminescence. This apparent reduction of the production of reactive oxygen species may reflect in fact the antioxidant activity of legumes. This, together with the absence of effect on the phagocytosis activity, suggested that peas are not immunosuppressing gilthead seabream. Further in vivo studies preferably comporting a bacterial challenge will have to ascertain the absence of immunosuppressing effect of these legumes.     

Changes in adipocyte cell size, gene expression of lipid metabolism markers, and lipolytic responses induced by dietary fish oil replacement in gilthead sea bream (Sparus aurata L.)

The effects of fish oil (FO) substitution by 66% vegetable oils in a diet with already 75% vegetable protein (66VO) on adipose tissue lipid metabolism of gilthead sea bream were analysed after a 14-month feeding trial. In the last 3 months of the experiment, a FO diet was administrated to a 66VO group (group 66VO/FO) as a finishing diet. Hormone-sensitive lipase (HSL) activity was measured in adipose tissue and adipocyte size, and HSL, lipoprotein lipase and liver X receptor gene expression in isolated adipocytes, on which lipolysis and glucose uptake experiments were also performed. Lipolysis was measured after incubation with tumour necrosis factor-α (TNFα), linoleic acid, and two conjugated linoleic acid isomers. Glucose uptake was analysed after TNFα or insulin administration. Our results show that FO replacement increased lipolytic activity and adipocyte cell size. The higher proportion of large cells observed in the 66VO group could be involved in their observed lower response to fatty acid treatments and lower insulin sensitivity. The 66VO/FO group showed a moderate return to the FO conditions. Therefore, FO replacement can affect the morphology and metabolism of gilthead sea bream adipocytes which could potentially affect other organs such as the liver.