Water temperature affects osmoregulatory responses in gilthead sea bream (Sparus aurata L.)

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psμ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na⁺,K⁺-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na⁺,K⁺-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (V_max) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na⁺,K⁺-ATPase capacity appears fixed and activity appears to be determined by temperature.     

Osmoregulatory responses to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata L.)

• 1.1. Gilthead sea breams (Sparus aurata L.) adapted to sea water (SW, 39‰ salinity) and brackish water (BW, 7‰) were submitted to abrupt osmotic stress by transferring the specimens to 7‰ and 39‰, respectively.
• 2.2. Plasma osmolality, Na,⁺ Cl, ⁻ K, ⁺ Ca, ²⁺ cortisol and glucose were measured before and after the transfers.
• 3.3. The transfer from SW to BW led to transitory hypomineralization and hyperglycemia. In long-term adapted fish cortisol level increased, and osmolality slightly decreased.
• 4.4. Conversely, the transfer from BW to SW provoked transitory hypermineralization. In adapted fish, cortisol levels strongly decreased, and osmolality slightly increased.

     

Gill area and dimensions of gilthead sea bream Sparus aurata L.

Detailed measurements of gill area and constituent variables (total filament length, lamellar frequency and bilateral area) were performed on both hemibranchs of all eight arches in six specimens of gilthead sea bream Sparus aurata (mean ±s.e . 49·9 ± 0·2 g). Shrinkage was also quantified and results were corrected accordingly. Filament number decreased from the first to the fourth gill arch, and average bilateral area of secondary lamellae was higher in the second and third arches. Total and mean filament length, total number of secondary lamellae and total gill area (A_TG) were lower in posterior than in anterior hemibranchs of the second, third and fourth gill arches; while the opposite was observed for the first arch. Lamellar frequency was increased in posterior hemibranchs of all arches compared to that in anterior hemibranchs, especially at the fourth arch. Comparison of the actually measured A_TG and constituent variables with estimates revealed that the third gill arch is the most representative for appropriate measurements and that any of its components (even one hemibranch) approximates the best A_TG (within the range of 0·2–4·3%, P > 0·05) and related dimensions. Consequently, necessary measurements were restricted to the posterior hemibranch of the third gill arch, and A_TG and dimensions (y) were estimated in 21 specimens (23·5–217·6 g) and correlated to body mass (M) according to the allometric equation y = aM^b. As fish increased in size, A_TG (b= 0·664), total (b= 0·425) and mean (b= 0·323) filament length, total number of filaments (b= 0·103) and secondary lamellae (b= 0·377), as well as average lamellar bilateral area (b= 0·288), increased, while the opposite was observed for lamellar frequency (b=?0·049) and mass‐specific area (b=?0·336). Data obtained are discussed in relation to S. aurata activity and living ethology.     

Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA

In the current work, we have cloned and sequenced the full cDNA for a Mx protein in the gilthead sea bream (Sparus aurata) by RACE PCR. The Mx cDNA of 2182 bp contained an open reading frame of 1857 bp that codes for a protein of 618 aa. Within the coding sequence, characteristic features of Mx proteins were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD), the signature of the dynamin family, LPRG(S/K)GIVTR, and a sequence that codes for a leucine zipper at the C-terminal region of the protein. An RT-PCR was optimised to estimate the level of expression of Mx protein in sea bream. Through this method we determined that Mx is constitutively expressed in head kidney, liver, spleen, heart, gills, muscle and brain of healthy sea bream. Intramuscular challenge of sea bream with polyinosinic:polycytidylic acid (Poly I:C) up-regulated Mx expression in liver, head kidney, spleen and muscle. Constitutive expression was also found in isolated head kidney macrophages and blood leukocytes. This expression was significantly up-regulated by addition of Poly I:C. Mx was not constitutively expressed in the sea bream established cell line, SAF-1, but Poly I:C and nodavirus were also capable of inducing Mx expression in this cell line.     

Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata)

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism by virtue of its capacity to hydrolyze triglycerides circulating in the form of lipoprotein particles. Here we analyzed the fasting effects of LPL in gilthead sea bream (Sparus aurata) and also present the first study in fish of the role of insulin as a potential modulator of both LPL activity and expression. Fasting for 2 weeks provoked a clear decrease in adipose tissue LPL activity, concomitant with lower levels of plasma insulin, while no effects were observed in red muscle. To elucidate the specific role of insulin, increases of plasma insulin were experimentally induced by arginine and insulin injections. However, arginine predominantly stimulated glucagon over insulin secretion in this fish species while LPL activity did not change significantly in adipose tissue. Instead, insulin administration induced an increase in adipose tissue LPL activity 3 h after the injection, whereas LPL activity in red muscle was not affected. Changes in LPL activity were accompanied by an increase in LPL mRNA levels in the adipose tissue of insulin-injected gilthead sea bream, although changes in LPL expression were delayed in time with respect to variations in LPL activity. Finally, LPL mRNA levels in red muscle were similar between control and insulin-injected gilthead sea bream, suggesting that insulin does not play a direct role in the regulation of LPL in this tissue. The current study shows that LPL activity is regulated by nutritional condition and underscores the importance of insulin as a modulator of LPL activity and expression in the adipose tissue of gilthead sea bream.     

Inheritance of a darkened caudal peduncle and yellow body coloration in the gilthead sea bream (Sparus aurata L.)

This study presents data on inheritance of a darkened caudal peduncle (ebony) and yellow body coloration (yellow) in the gilthead sea bream (Sparus aurata). Fifteen progeny groups, obtained by crossing fish with three color phenotypes and of known origin, were analyzed. Analyzes of segregation in F₁ progeny involving groups from parental crosses of wild‐type colored × wild‐type colored; ebony × ebony; yellow × yellow, showed that the parents produced the offspring only with the same phenotypes (true breeding). Crosses involving F₁ wild‐type colored parents (that resulted from crosses of wild‐type parents with either ebony or yellow fish) showed in their F₂ progeny groups of which their phenotypic segregations did not differ significantly from a 3 : 1 Mendelian ratio. The progeny of back‐cross of ebony × (F₁ wild‐type colored × ebony) showed phenotypic segregations that did not differ significantly from the 1 : 1 Mendelian ratio. Overall, the results of the crossing experiments demonstrated that, similar to albinism described in a number of aquacultured species, ebony and yellow body coloration in S. aurata are both due to a single recessive allele. However, the yellow mutation of a gene controlling yellow pigment synthesis affects the yellow color of the whole fish body, whereas the ebony mutation causes production of melanin only in a specific area of the fish body, resulting in the development of a black coloration of the caudal peduncle. Experiments to assess culture performance showed that the color genes controlling ebony and yellow coloration had significant detrimental pleiotropic effects on growth, survival and body shape. Color mutations in the gilthead sea bream may be used as models for the study of: (i) genetic and physiological mechanisms of sterility, (ii) stress and disease resistance, (iii) effects of heterosis, (iv) genetic polymorphism in populations, and (v) methods of genetic protection in selected sea bream strains as well as in experiments on chromosome set manipulation.     

Visualizing mineralization in deformed opercular bones of larval gilthead sea bream (Sparus aurata)

During the rearing process of gilthead sea bream (Sparus aurata), abnormal development of the opercular bone is particularly common (Aquaculture 156, 1997, 165). In order to alleviate its occurrence in rearing facilities, it’s crucial to identify the very first physical signs of deviation in normal skeletal development. Nano‐CT‐scanning was tested for its applicability to quantify deviations in bone mineralization levels. Seven opercles were dissected from larvi of 65 days post hatching, randomly sampled at the commercial sea bream hatchery Maricoltura di Rosignano Solvay (Livorno, Italy). The samples were nano‐CT‐scanned and computationally reconstructed. Mineralization intensity was colorcoded using Amira software, resulting in a detailed visualization of opercular morphology and mineralization patterns. In conclusion, nano‐CT‐scanning promises to be a good tool to both describe morphology and detect mineralization levels in the early onset of deformities.     

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.     

Seasonal variations of cellular stress response of the gilthead sea bream (Sparus aurata)

The present study aimed to investigate the seasonal cellular stress response in vital organs, like the heart, the liver, the whole blood and the skeletal (red and white) muscles of the Mediterranean fish Sparus aurata during a 1-year acclimatization period in the field, in two examined depths (0–2 m and 10–12 m). Processes studied included heat shock protein expression and protein kinase activation. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). The induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs in the examined five tissues of the gilthead sea bream indicated a cellular stress response under the prism of a seasonal pattern which was characterized by distinct tissue specificity. Specifically, Hsp induction and MAPK activation occurred before peak summer water temperatures, with no further increases in their levels despite increases in water temperatures. Moreover, although water temperature did not vary significantly with depth of immersion, significant effects of depth on cellular stress response were observed, probably caused by different light regime. The expression and the activation of these certain proteins can be used as tools to define the extreme thermal limits of the gilthead sea bream.     

Quantitative trait loci for resistance to fish pasteurellosis in gilthead sea bream (Sparus aurata)

Fish pasteurellosis is a bacterial disease causing important losses in farmed fish, including gilthead sea bream, a teleost fish of great relevance in marine aquaculture. We report in this study a QTL analysis for resistance to fish pasteurellosis in this species. An experimental population of 500 offspring originating from eight sires and six dams in a single mass‐spawning event was subjected to a disease challenge with Photobacterium damselae subsp. piscicida (Phdp), the causative agent of fish pasteurellosis. A total of 151 microsatellite loci were genotyped in the experimental population, and half‐sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5–8% of the phenotypic variation.     

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.     

Changes in the electrophoretic pattern of yolk proteins during vitellogenesis in the gilthead sea bream,Sparus aurata L.

• 1.1. To some extent, oocyte growth within a follicle is due, as well as to the accumulation of normal cytoplasmic components, to that of the cortical alveoli, and of hepatic-derived protein (vitellogenins).
• 2.2. Yolk proteins of pre-maturational oocytes at different stages and ovulated eggs were compared by SDS-gel electrophoresis. The largest components stained by Coomassie Blue and those stained by Stains-all, which had formed during vitellogenesis, either disappeared or diminished, whilst smaller components appeared.
• 3.3. The distinct changes in yolk-protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins.

     

Molecular cloning and sequence of gilthead sea bream (Sparus aurata) alpha-skeletal actin: tissue and developmental expression

In the present study, the Sparus aurata alpha-skeletal actin was cloned from a mixed larvae complementary DNA library. The clone isolated was 1523 bp long with an open reading frame of 1134 bp coding for a 377-amino acid protein. The deduced amino acid sequence of sea bream alpha-actin is identical to Fugu alpha-actin-1. The expression of alpha-actin was initiated at the onset of segmentation. In adult fish, alpha-actin is expressed predominantly in white and red muscle.     

Settlement of gilthead sea bream Sparus aurata L. in a southern Irish Sea coastal habitat

For the first time, early juvenile stages of gilthead sea bream Sparus aurata were consistently collected in a brackish estuarine area in the southern Irish Sea over a period of 4 months. This finding, in connection with other recent evidence, raises the possibility that S. aurata may currently spawn, or at least successfully settle, in more northern locations than previously known.     

Intestinal l-methionine transport in the cultured gilthead bream (Sparus aurata)

• 1.1. The influx and transepithelial movements of l-methionine and its effects on the electrophysiology and Na-Cl-transport in upper and lower intestine of the cultured fish, Spanis aurata, were measured.
• 2.2. The K_m and V_max of l-methionine influx into the tissues were higher in lower intestine than in upper intestine. A prominent diffusion-like transport component was also measured in both segments during influx experiments.
• 3.3. Net transepithelial fluxes of l-methionine (1 mM) were observed in both upper and lower intestine, this transport being Na⁺-dependent.
• 4.4. The two intestinal segments exhibited an electrical potential difference (PD) and a short circuit current (I_sc) serosa negative or near zero. Tissue conductance (G_t) was higher in posterior than in lower intestine.
• 5.5. Addition of l-methionine to the mucosal side of lower or upper intestine did not induce changes in PD in either part.
• 6.6. Isotopic fluxes of Cl⁻ or Na⁺ measurements under short circuit conditions showed that there were no net Cl⁻ or Na⁺ transport in either part.
• 7.7. l-Methionine additions to the mucosa did not induce changes in unidirectional fluxes of Cl⁻ or Na⁺ or in the (I_sc) in either the anterior or posterior intestine.