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.     

Immune defence mechanisms presented in liver homogenates and bile of gilthead seabream (Sparus aurata)

Because the role of the liver of fishes in providing possible immunity remains largely unknown, the aim of this work was to identify and characterize different humoral defence mechanisms in the liver homogenates and bile of gilthead seabream (Sparus aurata) for the first time. Total protein levels and several immune parameters (complement activity, lysozyme and immunoglobulin M level) were studied. Furthermore, the activity of some lytic (proteases, antiproteases, esterase, alkaline phosphatase) and antioxidant (superoxide dismutase, catalase and peroxidase) enzymes was determined. Finally, bacteriostatic activity on three opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum and Photobacterium damselae) was measured. Lysozyme and antiprotease activity were undetected in liver and bile, while natural haemolytic complement activity was only detected in bile, and immunoglobulin M was detected in both samples. The levels of proteases, esterase and antioxidant enzymes were greater in bile than in liver homogenates, while the level of alkaline phosphatase was very low in both samples. In addition, while no bacteriostatic activity was detected on liver homogenates, the bile revealed a very potent bacteriostatic activity against all the tested pathogenic bacteria. These results corroborate that fish liver – especially fish bile – contains many factors involved in innate immunity that could be useful for better understanding the role of the liver as an organ involved in fish immune functions as well as the possible contribution of bile to gut mucosal immunity.     

Improving sperm cryopreservation with antifreeze proteins: effect on gilthead seabream (Sparus aurata) plasma membrane lipids

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.     

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.     

Chromosome set manipulations and hybridization experiments in gilthead seabream (Sparus aurata). II. Assessment of diploid and triploid hybrids between gilthead seabream and red seabream (Pagrus major)

The experiments were designed to evaluate the suitability for mariculture of the diploid and triploid hybrids of gilthead seabream Sparus aurata and red seabream Pagrus major , both of which are members of the Sparidae family. Performance testing of the hybrids was carried out in comparison with the parental species under the same controlled environment. The reciprocal diploid hybrids as well as the triploid hybrid did not exhibit any significant growth advantage over diploid parental species either before or after sexual maturity. The adults, both reciprocal diploid hybrids and triploid hybrid (female S. aurata × male P. major ), were clearly immature and had only vestigial gonads; neither ovaries nor released milts were observed. Histological examination of the gonads, carried out during associated periods of the first and second maturation cycles of the parental species, showed that all the hybrids were completely sterile. Despite their sterility, the growth of the hybrids did not display any positive effect, and for this reason their commercial culture appears to be questionable. On the other hand, the use of sterilization through hybridization and chromosome set manipulation may be important when there is a need to restrict the ecological impact on a wild population.     

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata)

Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoDgenes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoDexpression in adaxial cells.     

Thermal tolerance and standard metabolic rate of juvenile gilthead seabream (Sparus aurata) acclimated to four temperatures

Temperature variation affects the growth, maturation and distribution of fish species due to increasing constraints on physiological functions therefore, the aim of the present study is to evaluate effect of temperature on thermal tolerance and standard metabolic rate (SMR) of gilthead seabream (Sparus aurata). For this purpose, tolerable temperature ranges of juvenile gilthead seabream acclimated at 15, 20, 25, and 30 °C for 30 days were estimated using dynamic and static thermal methodologies. The SMRs of the fish were also determined based on oxygen consumption rate (OCR). The dynamic and static thermal tolerance zones of gilthead seabream were calculated as 737 °C² and 500 °C², respectively, with a resistance zone area of 155.5 °C². The SMR of the fish at the above acclimation temperatures (AT) was determined as 138, 257, 510, and 797 mg O₂ h⁻¹ kg⁻¹, respectively and were significantly different (P < 0.01, n = 10). The temperature quotient (Q₁₀) in relation to the SMR of the fish was calculated as 3.45, 3.91, and 2.44 for acclimation temperature ranges of 15–20, 20–25, and 25–30 °C, respectively. The fact that the SMR increased with rising temperatures and then decreased gradually after 25 °C indicates that the temperature preference of juvenile gilthead seabream lies between 25 and 30 °C. This study shows that gilthead seabream tolerates a relatively narrow temperature range, and consequently, a low capacity for acclimatisation to survive in aquatic systems characterised by temperature variations.     

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata)

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.     

Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.)

The effects of the oral administration of heat-inactivated Lactobacillus delbrüeckii ssp. lactis and Bacillus subtilis, individually or combined, on gilthead seabream immune responses were investigated both systemically and locally in the gut. In a first experiment, seabream (65 g) were fed for 3 weeks different diets supplemented with 1 x 10(7)CFU g(-1)Lactobacillus, 1 x 10(7)CFU g(-1)Bacillus, or 0.5 x 10(7)CFU g(-1)Lactobacillus plus 0.5 x 10(7)CFU g(-1)Bacillus. Controls were fed non-supplemented diet. Six fish per group were sampled at the end of the trial and some humoral and cellular systemic innate immune parameters were evaluated. Feeding the mixture of the two killed bacteria species significantly increased natural complement, serum peroxidase and phagocytic activities compared with controls. In a second experiment, juvenile seabream (13 g) were fed for 3 weeks the same experimental diets and total serum IgM and numbers of gut IgM(+) cells and acidophilic granulocytes were evaluated. All these parameters were significantly higher in the multispecies probiotic group compared to monospecies and control fed groups. The advantages provided by administration of killed probiotic bacteria as well as multispecies versus monospecies formulations are discussed in light of the results obtained and for their possible application in aquacultural practices.     

Feeding habits of the gilthead seabream (Sparus aurata) from the Ria Formosa (southern Portugal) as compared to the black seabream (Spondyliosoma cantharus) and the annular seabream (Diplodus annularis)

The feeding habits of Sparus aurata L., Diplodus annularis L. and Spondyliosoma cantharus L. in the Ria Formosa (southern Portugal) lagoon system were studied using three simple methods (frequency of occurrence, numeric percentage and percentage weight) and a composite index [index of relative importance (IRI)]. The Ivlev index was used to evaluate diet selectivity, while the Schoener overlap index was used to compare diets, and diet diversity was characterized by the Simpson index. The diets of the three species consist of a wide variety of food organisms, nevertheless S. aurata seems to be the most specialized. No significant dietary overlap was found, with S. aurata preferentially selecting gastropods and bivalves, while S. cantharus preferentially selected a wide variety of crustaceans and D. sargus a wider array, including crustaceans, gastropods and bivalves.     

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.     

Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the ‘MedFish’ SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4–99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Effect of dietary supplementation with yeast Saccharomyces cerevisiae on skin,serum and liver of gilthead seabream (Sparus aurata L)

The effect of dietary supplementation with Saccharomyces cerevisiae on gilthead seabream (Sparus aurata L.) was studied. Four replicates of fish (n = 6) were fed with a commercial diet containing 0 (control, no yeast added) or 10 mg per kilogram of heat-killed (30 min, 60°C) S. cerevisiae. After 4 weeks, half of the fish (two replicates) were injured and continued with the same diet. At 3 and 7 days post-wounding, samples of blood, skin mucus, skin and liver were obtained from each fish. The results showed that calcium concentrations were significantly higher (with respect to control fish) in the serum from fish sampled at 3 days post-wounding, whereas antioxidant enzymes in the skin mucus were altered after wounding (at both 3 and 7 days). Histological analyses revealed oedema, signs of inflammation and white cell recruitment together with a reduction in the epidermis layer in the wounded regions of fish fed control diet. Yeast supplementation did not change growth performance and helped maintain the normal serum calcium concentrations in wounded fish. Furthermore, a reduction in inflammation around wounds in the animals fed yeast with respect to that fed control diet was evident in the histological study. Furthermore, increased levels of stress-related gene expression in liver and skin from wounded fish were obtained. Overall, yeast supplementation seemed to be a functional and appropriate dietary additive to improve skin recovery reducing the stress resulting from wounds.