Expression of Developmental-Stage-Specific Genes in the Gilthead Sea Bream Sparus aurata L.

The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.     

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).     

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.     

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 .     

Effects of 2-deoxy-D-glucose on the immune system of seabream (Sparus aurata L.)

Stressful situations are a major problem in aquaculture because they affect the immune system. 2-Deoxy-d-glucose (2-DG) is a derivative of a glucose analogue that reduces the availability of energy, thereby inhibiting cell metabolism so that it is unable to enter the glycolysis pathway. In this paper, 2-DG has been administered in order to study if the immune function is compromised during metabolic stress. Blood glucose level was measured as an indicator of the inhibition of glycolysis, and the effects of intraperitoneal administration of 2-DG on the main parameters of the humoral (complement, IgM levels and peroxidase activity in blood plasma) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis activity) immune parameters of gilthead seabream (Sparus aurata, L) were evaluated. Furthermore, the expression levels of immune-associated genes (CSF-1R, NCCRP-1, Hep, TCR-β, IgM_H, MHC-IIα, C3 and IL-1β) were analyzed by real-time PCR in head-kidney. A total of 5 intraperitoneal injections were performed at 48 h intervals. Three experimental groups were established: a control group injected with phosphate buffer saline, group 2-DG 500 and group 2-DG 750 injected with 500 mg kg^?1 and 750 mg kg^?1 2-DG, respectively (N = 15). After the third and fourth injection, some specimens of both DG-treated groups died. Following the first and third injection, the blood glucose levels of both 2-DG treated groups increased to a statistically significant extent with respect to the control group. While the humoral immune parameters were not significantly affected as a consequence of 2-DG administration, the cellular activities of leucocytes were. The injection of 500 mg kg^?1 2-DG provoked up- or down-regulation of the immune-relevant genes analyzed, while the injection of 750 mg kg^?1 always caused down-regulation of these genes. The results suggest that 2-DG provokes metabolic stress, which reduces the activities carried out by immune cells (leucocytes) and induces down-regulation of the immune-relevant genes analyzed when the energy available to the cell decreases.     

Effects of inulin on gilthead seabream (Sparus aurata L.) innate immune parameters

Inulin, a fructooligossacharide, is a prebiotic that plays an important role in the immune function in mammals, but it has never been assayed in other vertebrate groups. Thus, we have studied the inulin effects on the gilthead seabream (Sparus aurata L.) innate immune response both in vitro and in vivo. For the in vitro study, head-kidney leucocytes were incubated with inulin (ranging from 0 to 1000 microg ml(-1)) for 30, 90, 180 and 300 min and 24h and any effect was observed on leucocyte viability or the main innate cellular immune responses (leucocyte peroxidase, phagocytic, respiratory burst and natural cytotoxic activities). For the in vivo study, seabream specimens were fed for 1 or 2 weeks with a commercial diet supplemented with inulin: 0 (control), 5 or 10 g inulin kg(-1) diet (0.5 and 1%, respectively). Inulin produced a significant inhibition in phagocytosis and respiratory burst in leucocytes from specimens fed diets containing 0.5% or 1% of inulin for 1 week. Based on the present results, inulin does not seem to be a good immunostimulant for seabream, though its effects in other species and combined with other immunostimulans (i.e. probiotics) might be of great interest.     

Juvenile Sparus aurata L. on the south coast of Ireland

Records of gilthead sea bream Sparus aurata L. in Irish and British waters since 1967 are provided together with the first observations on the ecology of 0 year group gilthead sea bream in these islands.     

Effects of different levels of dietary zinc on the gilthead,Sparus aurata during the growing season

Gilthead were fed three diets. Diet A was the control diet, whereas diets B and C were supplemented with 300 and 900 mg Zn/kg, respectively. Fish fed with diet C, at the end of the experiment, showed the lowest weight. Zinc concentrations presented the higher values in gills, liver, and kidney. Muscle and brain had the lower mean values and showed a tight control of zinc levels. These results reinforce the hypothesis that zinc in the CNS should be strictly controlled in order to maintain the functional role of the metal. Significant differences in tissue zinc concentrations were obtained between fish fed different amounts of zinc, the metal concentrations being higher in tissues of fish fed diet C. The tissue decrease of zinc, found at the end of the experiment, may depend on a lower feed consumption or on different zinc requirements during the cold season. These changes, even if not univocal among the three diets, may be associated with the life cycle of fish. Furthermore, copper concentrations were little affected by the different concentrations of zinc in the three diets; liver and kidney presented the highest concentrations; liver showed a significant decrease in copper content at the end of the experiment. We conclude that: zinc concentrations of the diet may affect the gilthead weights and the tissual metal content; and zinc concentrations in the diets, depending on the growth rate, may be varied depending on the season.     

Gonadotropin-Activated Androgen-Dependent and Independent Pathways Regulate Aquaporin Expression during Teleost (Sparus aurata) Spermatogenesis

The mediation of fluid homeostasis by multiple classes of aquaporins has been suggested to be essential during spermatogenesis and spermiation. In the marine teleost gilthead seabream (Sparus aurata), seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the spermiating testis, but the endocrine regulation of these channels during germ cell development is unknown. In this study, we investigated the in vivo developmental expression of aquaporins in the seabream testis together with plasma androgen concentrations. We then examined the in vitro regulatory effects of recombinant piscine gonadotropins, follicle-stimulating (rFsh) and luteinizing (rLh) hormones, and sex steroids on aquaporin mRNA levels during the spermatogenic cycle. During the resting phase, when plasma levels of androgens were low, the testis exclusively contained proliferating spermatogonia expressing Aqp1ab, whereas Aqp10b and -9b were localized in Sertoli and Leydig cells, respectively. At the onset of spermatogenesis and during spermiation, the increase of androgen plasma levels correlated with the additional appearance of Aqp0a and -7 in Sertoli cells, Aqp0a in spermatogonia and spermatocytes, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and spermatozoa. Short-term in vitro incubation of testis explants indicated that most aquaporins in Sertoli cells and early germ cells were upregulated by rFsh and/or rLh through androgen-dependent pathways, although Aqp1ab in proliferating spermatogonia was also activated by estrogens. However, expression of Aqp9b in Leydig cells, and of Aqp1aa and -7 in spermatocytes and spermatids, was also directly stimulated by rLh. These results reveal a complex gonadotropic control of aquaporin expression during seabream germ cell development, apparently involving both androgen-dependent and independent pathways, which may assure the fine tuning of aquaporin-mediated fluid secretion and absorption mechanisms in the seabream testis.     

Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.)

Anaesthesia may depress the immune system in mammals, but there is no available information on this topic in fish. In the present work, four anaesthetics that are used in aquaculture, MS222 (0 19 mM), benzocaine (0.21 mM), 2-phenoxyethanol (16 mM) and quinaldine sulphate (0.083 mM), were tested in order to observe their effects on the gilthead seabream (Sparus aurata L.) innate immune system. The results showed that the four anaesthetics produced increased blood glucose levels after an hour. In addition, benzocaine and 2-phenoxyethanol depressed complement activity and phagocytosis, while MS222 and quinaldine sulphate did not. Some anaesthesia is a common practice in aquaculture, the data obtained should be taken into account to avoid possible immunodepression in farmed fish.     

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).     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.     

Effects of Acute Cortisol Administration on Perceptual Priming of Trauma-Related Material

Intrusive memories are a hallmark symptom of posttraumatic stress disorder (PTSD). They reflect excessive and uncontrolled retrieval of the traumatic memory. Acute elevations of cortisol are known to impair the retrieval of already stored memory information. Thus, continuous cortisol administration might help in reducing intrusive memories in PTSD. Strong perceptual priming for neutral stimuli associated with a “traumatic” context has been shown to be one important learning mechanism that leads to intrusive memories. However, the memory modulating effects of cortisol have only been shown for explicit declarative memory processes. Thus, in our double blind, placebo controlled study we aimed to investigate whether cortisol influences perceptual priming of neutral stimuli that appeared in a “traumatic” context. Two groups of healthy volunteers (N = 160) watched either neutral or “traumatic” picture stories on a computer screen. Neutral objects were presented in between the pictures. Memory for these neutral objects was tested after 24 hours with a perceptual priming task and an explicit memory task. Prior to memory testing half of the participants in each group received 25 mg of cortisol, the other half received placebo. In the placebo group participants in the “traumatic” stories condition showed more perceptual priming for the neutral objects than participants in the neutral stories condition, indicating a strong perceptual priming effect for neutral stimuli presented in a “traumatic” context. In the cortisol group this effect was not present: Participants in the neutral stories and participants in the “traumatic” stories condition in the cortisol group showed comparable priming effects for the neutral objects. Our findings show that cortisol inhibits perceptual priming for neutral stimuli that appeared in a “traumatic” context. These findings indicate that cortisol influences PTSD-relevant memory processes and thus further support the idea that administration of cortisol might be an effective treatment strategy in reducing intrusive reexperiencing.     

Microsatellite variability of wild and farmed populations of Sparus aurata

The genetic diversity of Greek wild and farmed populations of Sparus aurata was investigated using seven microsatellite markers. Selective breeding programmes and founder effects have altered the composition of farmed populations leading to significant population differentiation between wild and cultured populations and lower allelic richness in farmed populations.     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.