Retinal development in the gilthead seabream Sparus aurata

The retinal development of the gilthead seabream Sparus aurata has been analysed from late embryonic development to juvenile stages using classical histological and immunohistological methods. Five significant phases were established. Phases 1 and 2 comprise the late embryonic and hatching stages, respectively. The results indicate that during these early stages the retina is composed of a single neuroblastic layer that consists of undifferentiated retinal progenitor cells. Phase 3 (late prolarval stage) is characterized by the emergence of the retinal layers and the appearance of neurochemical profiles in differentiating photoreceptors, amacrine and ganglion cells. Phases 4 and 5 comprise the late larval and juvenile stages. In these stages, all the retinal cell types can be detected immunohistochemically. All the maturational events described are first detected in the central retina and, as development progresses, spread to the rest of the retina following a central‐to‐peripheral gradient. The results of this study suggest that S. aurata is an altricial teleost species that hatches with a morphologically undifferentiated retina. The most relevant processes involved in retinogenesis occur during the late prolarval stage (phase 3).     

Radiological characterization of gilthead seabream (Sparus aurata) by X-ray computed tomography

In recent years, the increasing use of fish as new animal models in scientific research and the growth of fish farming (mainly for human consumption) have highlighted the need for advanced technology to deepen our knowledge of fish biology. Hence, the present study was carried out to radiologically analyse the whole body of gilthead seabream (Sparus aurata) specimens using X-ray computed tomography (CT). Images were acquired in an Albira SPECT/PET/CT tri-modal preclinical-scanner. Segmentation, measurements and three-dimensional reconstruction were made using the Carestream Molecular imaging Albira CT system in conjunction with Pmod, AMIDE and Amira software packages. The results showed that the density values of gilthead seabream are in the range −700 to +2500 HU for the whole body. We also determined the density ranges that topographically coincide with the swim bladder, soft tissues, fat, skin and skeleton. This work describes, validates and demonstrates the application of a fully automated image analysis technique to study and quantify fish body composition, whether segmented or as a whole. In addition, the basis for applying this image technique in other in vivo studies is established.     

Characterization of digestive carbohydrase activity in the gilthead seabream (Sparus aurata)

Six species of Phallodrilinae are reported from Hainan Island in southern China. The mesopsammic Aktedrilus yiboi sp. nov. is new to science. It is characterized by a combination of (1) small, straight, conical, strongly cuticularized penis sheaths, (2) posterior prostate glands being associated with the most ectal parts of the atria, and (3) small spermathecal ampulla. Bathydrilus ampliductus Erséus, 1997 is recorded for the first time since it was originally described from Australia's Northern Territory.     

Induced swimming modified the antioxidant status of gilthead seabream (Sparus aurata)

Swimming has relevant physiological changes in farmed fish, although the potential link between swimming and oxidative stress remains poorly studied. We investigated the effects of different medium-term moderate swimming conditions for 6 h on the antioxidant status of gilthead seabream (Sparus aurata), analyzing the activity of enzymes related to oxidative stress in the liver and skeletal red and white muscle. Forty fish were induced to swim individually with the following conditions: steady low (SL, 0.8 body length (BL)·s⁻¹), steady high (SH, 2.3 BL·s⁻¹), oscillating low (OL, 0.2–0.8 BL·s⁻¹) and oscillating high (OH, 0.8–2.3 BL·s⁻¹) velocities, and a non-exercised group with minimal water flow (MF, < 0.1 BL·s⁻¹). All swimming conditions resulted in lower activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione-S-transferase (GST) in the liver compared to the MF group, while steady swimming (SL and SH) led to higher reduced glutathione/oxidized glutathione ratio (GSH/GSSG) compared to the MF condition. Swimming also differently modulated the antioxidant enzyme activities in red and white muscles. The OH condition increased lipid peroxidation (LPO), catalase (CAT) and glutathione peroxidase (GPx) activities in the red muscle, decreasing the GSH/GSSG ratio, whereas the SL condition led to increased GSH. Oscillating swimming conditions (OL and OH) led to lower CAT activity in the white muscle, although GPx activity was increased. The GSH/GSSG ratio in white muscle was increased in all swimming conditions. Liver and skeletal muscle antioxidant status was modulated by exercise, highlighting the importance of adequate swimming conditions to minimize oxidative stress in gilthead seabream.     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

Influence of cortisol on osmoregulation and energy metabolism in gilthead seabream Sparus aurata

Gilthead seabream Sparus aurata were injected intraperitoneally with slow-release implants of coconut oil alone or containing cortisol (50 and 100 microg x g(-1) body weight), and sampled after two, five, and seven days to assess the simultaneous effects of cortisol on both osmoregulation and energy metabolism. Plasma cortisol levels increased in treated fish to 50-70 ng x ml(-1). An enhanced hypoosmoregulatory capacity of cortisol-implanted fish is suggested by the increase observed in gill Na+, K+-ATPase activity, and the decrease observed in plasma ion concentration (Na+ and Cl-) and osmolality. Cortisol also elicited metabolic changes in liver (increased gluconeogenic potential suggested by elevated FBPase activity, and decreased potential of glycolysis and pentose-phosphate shunt, suggested by the decreased activities of both PK and G6PDH) supporting changes in levels of plasma metabolites suitable for use in other tissues. Thus in this study, we demonstrate for the first time in fish that cortisol treatments elicit changes in the use of exogenous glucose in gills (decreased HK activity) and an increased glycolytic and glycogenic potential in brain (increased GPase, PK and PFK activities).     

Genetic structure of gilthead seabream, Sparus aurata, in the Central Mediterranean Sea

The gilthead seabream, Sparus aurata, represents an important economic resource for Mediterranean aquaculture. In spite of its wide geographic distribution and economic importance, only recently studies have been carried out on the genetic composition of natural populations, which have revealed a picture of a heterogeneous degree of genetic differentiation among S. aurata populations. In this study an allozyme analysis of samples from six different collecting sites along the Italian and Croatian coasts was carried out, covering an area in the Central Mediterranean sea that has yet to be investigated through gene-enzyme systems. Data on 26 gene loci, 10 of which are polymorphic, indicate a slight but significant genetic structure (F_ST = 0.0167) of the species. A hierarchical analysis of population subdivision made it possible to identify three different assemblages found in the Adriatic Sea, Tyrrhenian Sea and Sardinian Channel, though an isolation by distance model can be rejected. The results are discussed in the light of previous literature and taking conservation into consideration.     

Characterization of gonadotropin-releasing hormone binding to pituitary receptors in the gilthead seabream (Sparus aurata).

Receptors for GnRH in the pituitary of Sparus aurata were characterized using iodinated [D-Ala6-Pro9-NEt]-LHRH (GnRHa). Equilibrium binding of the ligand to the receptor was achieved after 1 h at 4 degrees C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of one-sixteenth to three-fourths pituitary per tube. Displacement experiments with salmon GnRH (sGnRH), GnRHa, as well as unrelated peptides demonstrated the specificity of the receptors. Binding was found to be saturable at ligand concentrations of 4 x 10(-9) M. Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity sites (Ka = 0.567 +/- 0.136 x 10(9) M-1, Bmax = 1091 +/- 207 fmol/mg protein).     

The first radiation hybrid map of a perch-like fish: the gilthead seabream (Sparus aurata L)

Among Teleosts, Perciformes are the largest order of fishes and include numerous species of commercial importance. Perciformes also comprise species of primary interest for evolutionary studies and analysis of the sex determination systems and sex chromosome plasticity. Unfortunately, genomics tools and resources for Perciformes remain to be developed. Here, we report the production of a seabream whole-genome radiation hybrid (RH) panel in which quality was ascertained by the construction of a 2-Mb-resolution RH map. The map encompasses 440 markers (288 microsatellites, 82 gene-based markers, and 70 STS) suitable for linkage analysis and comparative mapping studies. Achievement of a RH panel and a whole-genome RH map should contribute to establishing seabream as a fish model among the Perciformes and should be of importance in aquaculture for marker-assisted selection, improvement of growth performance, and disease management. Development of RH maps in a cost-effective manner for other fishes with the described methodology will offer a powerful approach in aquaculture and will provide extended capabilities for comparing vertebrate genome evolution.     

Identification of a major locus determining a pigmentation defect in cultivated gilthead seabream (Sparus aurata)

The gilthead seabream (Sparus aurata) is an important cultivated species in the Mediterranean area. A major problem for the gilthead seabream aquaculture sector derives from the high frequency of phenotypic abnormalities, including discolorations. In this study, we applied a whole-genome resequencing approach to identify a genomic region affecting a pigmentation defect that occurred in a cultivated S. aurata population. Two equimolar DNA pools were constructed using DNA extracted from 30 normally coloured and 21 non-pigmented fish collected among the offspring of the same broodstock nucleus. Whole-genome resequencing reads from the two DNA pools were aligned to the S. aurata draft genome and variant calling was performed. A whole-genome heterozygosity scan from single pool sequencing data highlighted a peak of reduced heterozygosity of approximately 5 Mbp on chromosome 6 in the non-pigmented pool that was not present in the normally coloured pool. The comparison of the non-pigmented with the normally coloured fish using a whole-genome F_ST analysis detected three main regions within the coordinates previously detected with the heterozygosity analysis. The results support the presence of a major locus affecting this discoloration defect in this fish population. The results of this study have practical applications, including the possibility of eliminating this defect from the breeding stock, with direct economic advantages derived from the reduction of discarded fry. Other studies are needed to identify the candidate gene and the causative mutation, which could add information to understand the complex biology of fish pigmentation.     

Induction of triploidy in offspring of gilthead seabream (Sparus aurata) by means of heat shock

By using heat shock, we have induced triploidy in the offspring of gilthead seabream ( Sparus aurata ). We have verified that the effectiveness of the treatment in this marine species depends not only on the three factors habitually considered (incubation time, treatment intensity and treatment time), but also on the salinity of the treatment water. The most effective treatment to provoke triploidy in this species is to apply a heat shock of 34 °C for 10 min without an incubation period and with water salinity at 30:1000.     

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.     

Tumouricidal activity of gilthead seabream (Sparus aurata L.) natural cytotoxic cells: the role played in vitro and in vivo by retinol acetate

The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was evaluated after in vitro incubation with retinol acetate as vitamin A source, and in samples taken from specimens receiving an intraperitoneal injection or a diet supplemented with this vitamin. Isolated leucocytes were incubated with 0 to 10(-10)m all-trans-retinol acetate-supplemented culture medium for 0, 6 or 24h and assayed for their tumouricidal activity which was found to increase for all the assayed concentrations and incubation times. Seabream specimens were intraperitoneally injected with 0 (control), 1.75 or 5.25 micro g retinol acetate 100 g(-1) biomass and sampled 1, 3 or 5 days post-injection. Leucocyte natural cytotoxic activity increased in a dose-dependent manner 1 and 3 days post-injection. When fish were fed a commercial diet supplemented with 0 (control), 50, 150 or 300 mg retinol acetate kg(-1) diet for 1, 2, 4 or 6 weeks, only fish which had been fed the highest supplement for 2 weeks showed any increase in head-kidney leucocyte cytotoxic activity. Serum was isolated and analysed for all-trans-retinol concentration by reverse-phase high-pressure-liquid-chromatography. The normal level was about 0.4 micro g ml(-1) serum, while treatment for 1 to 4 weeks with vitamin A increased this level.In conclusion, retinol acetate increases gilthead seabream head-kidney leucocyte cytotoxic activity both in vitro and in vivo.     

Histopathological alterations and induction of cytochrome P-450 1A in the liver and gills of the gilthead seabream (Sparus aurata) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated in the seabream Sparus aurata specimens. Liver presented hepatocytic alterations, with an increase of lipid droplets and glycogen granules. Ultrastructural modifications of hepatocytes included RER fractionation, glycogen augmentation, as well as a rise in the number of lipid droplets, vacuoles and secondary lysosomes. In the gills, secondary lamellar epithelium showed hyperplasia, hypertrophy and lamellar fusion on the edge of the filaments. At the end of the exposure period (1 pg1(-1) TCDD for 20 days), some organelles in epithelial cells of the secondary lamellae and the tubular system of the chloride cells appeared altered. In the liver of TCDD-exposed specimens, immunoreactive cytochrome P-450 1A was concentrated close to the cytoplasmic and nuclear membranes, and positive granules were also evident throughout cytoplasm of the hepatocytes. Significant cytochrome P-450 staining was especially evident in endothelium of the hepatic vascular system. At the beginning of the exposure (1 pg 1(-1) TCDD, for 5 and 10 days), cytochrome P-450 immunostaining was observed in the cytoplasm of scarce hepatic cells and after 20 days of treatment, specific immunostained cytoplasmic granules were detected in most hepatocytes. In gills of TCDD-treated specimens, pillar-endothelial cells showed a cytochrome P-450 1A immunostaining concentrated close to the base of gill filaments and dispersed through the gill lamellae. There was also significant cytochrome staining of the endothelium of the branchial vascular system. However, no cytochrome immunoreactivity was observed in epithelial-respiratory cells.     

Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.     

Cytochemical characterization of leucocytes from the seawater teleost,gilthead seabream (Sparus aurata L.)

The cytochemical characterization of head-kidney and peripheral blood leucocytes of gilthead seabream (Sparus aurata L.) was studied by light and electron microscopy. Neutrophilic granulocytes show some cytoplasmic granules, which are positive for alkaline phosphatase and peroxidase but acid phosphatase negative. The scarce granules found in the cytoplasm of the circulating neutrophils and their cytochemical features seem to be indicative of an immature stage. Acidophils are also alkaline phosphatase and peroxidase positive at pH 11.0. They are strongly positive for acid phosphatase and acid phosphatase activity may thus be considered a cytochemical marker to characterize and differentiate neutrophilic from acidophilic granulocytes in this fish species. Three granule populations are characterized in the cytoplasm of the gilthead seabream acidophils: the first is positive only for peroxidase and the second contains a dense core with acid and alkaline phosphatase activities, surrounded by a thin peroxidase positive electron-dense halo. The third granule type contains an eccentric core, which is strongly positive for acid and alkaline phosphatase and peroxidase. As regards their cytochemical features, the first and second granule types seem to correspond respectively to the azurophilic and specific granules found in acidophils of mammals and could be involved in phagocytic processes, thus playing an important microbicidal role in this species. The monocytes, monocyte-macrophages and macrophages show different cytochemical features. The first have scarce acid phosphatase-positive lysosomes, while blood monocyte-macrophages and macrophages are positive for acid and alkaline phosphatases and for peroxidase; the monocyte-macrophages show scarce lysosomes.