Effect of water temperature and dietary starch on growth and metabolic utilization of diets in gilthead sea bream (Sparus aurata) juveniles

We evaluated the effect of dietary starch level on growth performance, feed utilization, whole-body composition and activity of selected key enzymes of intermediary metabolism in gilthead sea bream juveniles reared at 18 and 25 degrees C. A diet was formulated to contain 48% crude protein, 12% lipids and 30% gelatinized maize starch (diet 30GS). Two other diets were formulated to include the same level of ingredients as diet 30GS except for the gelatinized starch, which was included at 20% (diet 20GS) or 10% (diet 10GS). No adjustment to diet composition was otherwise made. Each diet was fed to triplicate groups of gilthead sea bream (30 g initial mass) for 8 weeks, on a pair-feeding scheme. The higher temperature improved growth performance but the opposite was true for feed efficiency and protein efficiency ratio. Independently of temperature, growth performance, feed efficiency and protein efficiency ratio were lower in fish fed diet 30GS. No effect of temperature or dietary starch level on whole-body composition was noticed. Hepatosomatic index and liver glycogen were higher at 18 degrees C and, within each temperature, in fish fed diet 30GS. Glycemia was not affected by temperature, but was lower in fish fed diet 10GS. Data on enzyme activities showed that increasing water temperature enhances liver glucokinase (GK) and pyruvate kinase (PK) activities, suggesting that gilthead sea bream is more apt to use dietary starch at higher temperatures. No effect of temperature was noticed on hexokinase (HK), fructose-1,6-bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) activities. Dietary starch enhanced PK and FBPase activities while depressed GDH activity, suggesting a lack of significant regulation of hepatic glucose utilization and production in this species. HK, GK and G6PD activities were unaffected by dietary composition. Irrespectively of water temperature, gelatinized starch may be included up to 20% in diets for gilthead sea bream juveniles; at higher dietary levels, growth and efficiency of feed utilization are depressed.     

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.     

Rearing temperature enhances hepatic glucokinase but not glucose-6-phosphatase activities in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) juveniles fed with the same level of glucose

The aim of this work was to elucidate if the previous results observed in hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities in European sea bass and gilthead sea bream are due to temperature per se or to differences in feed intake at different water temperatures. For that purpose triplicate groups of fish (30 g initial body weight) were kept at 18 degrees C or 25 degrees C during two weeks and fed a fixed daily ration of a glucose-free or 20% glucose diet. At the end of the experimental period, plasma glucose levels in both species were not influenced by water temperature but were higher in fish fed the glucose diet. Higher hepatic GK activity was observed in the two fish species fed the glucose diet than the glucose-free diet. In the glucose fed groups, GK activity was higher at 25 degrees C than at 18 degrees C. Glucose-6-phosphatase activities in both species were not influenced by water temperature. In European sea bass and in contrast to gilthead sea bream it was observed an effect of dietary composition on G6Pase activities with surprising higher activities recorded in fish fed the glucose diet than in fish fed the glucose-free diet. Overall, our data strongly suggest that European sea bass and gilthead sea bream are apparently capable to strongly regulate glucose uptake by the liver but not glucose synthesis, which is even enhanced by dietary glucose in European sea bass. Within limits, increasing water temperature enhances liver GK but not G6Pase activities, suggesting that both species are more able to use dietary carbohydrates at higher rearing temperatures.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.     

Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo,and cross‐species amplification in Sparus aurata and Dicentrarchus labrax

Several new fish species are currently being included in breeding programmes. However, as specific molecular markers have not yet been developed, this represents a commercial handicap with respect to traditional aquaculture species such as gilthead sea bream or Atlantic salmon. In the present study, 12 new microsatellite loci were developed for blackspot sea bream (Pagellus bogaraveo) that show high levels of polymorphism, especially useful in parentage assignment and individual identification. In addition, cross‐amplification was obtained for two important species for Spanish aquaculture, gilthead sea bream and sea bass.     

Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures

The effects of carbohydrate sources/complexity and rearing temperature on hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities and gene expression were studied in gilthead sea bream juveniles. Two isonitrogenous (50% crude protein) and isolipidic (19% crude lipids) diets were formulated to contain 20% waxy maize starch or 20% glucose. Triplicate groups of fish (63.5 g initial body weight) were fed each diet to near satiation during four weeks at 18 degrees C or 25 degrees C. Growth, feed intake, feed efficiency and protein efficiency ratio, were higher at the higher water temperature. At each water temperatures fish growth and feed efficiency were higher with the glucose diet. Plasma glucose levels were not influenced by water temperature but were higher in fish fed the glucose diet. Hepatosomatic index and liver glycogen were higher at the lower water temperature and within each water temperature in fish fed the glucose diet. No effect of water temperature on enzymes activities was observed, except for hexokinase and GK which were higher at 25 degrees C. Hepatic hexokinase and pyruvate kinase activities were not influenced by diet composition, whereas glucose-6-phosphate dehydrogenase activity was higher in fish fed the glucose diet. Higher GK activity was observed in fish fed the glucose diet. GK gene expression was higher at 25 degrees C in fish fed the waxy maize starch diet while in fish fed the glucose diet, no temperature effect on GK gene expression was observed. Hepatic G6Pase activities and gene expression were neither influenced by dietary carbohydrates nor water temperature. Overall, our data suggest that in gilthead sea bream juveniles hepatocytes dietary carbohydrate source and temperature affect more intensively GK, the enzyme responsible for the first step of glucose uptake, than G6Pase the enzyme involved in the last step of glucose hepatic release.     

Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures.

The effects of carbohydrate sources/complexity and rearing temperature on hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities and gene expression were studied in gilthead sea bream juveniles. Two isonitrogenous (50% crude protein) and isolipidic (19% crude lipids) diets were formulated to contain 20% waxy maize starch or 20% glucose. Triplicate groups of fish (63.5 g initial body weight) were fed each diet to near satiation during four weeks at 18 degrees C or 25 degrees C. Growth, feed intake, feed efficiency and protein efficiency ratio, were higher at the higher water temperature. At each water temperatures fish growth and feed efficiency were higher with the glucose diet. Plasma glucose levels were not influenced by water temperature but were higher in fish fed the glucose diet. Hepatosomatic index and liver glycogen were higher at the lower water temperature and within each water temperature in fish fed the glucose diet. No effect of water temperature on enzymes activities was observed, except for hexokinase and GK which were higher at 25 degrees C. Hepatic hexokinase and pyruvate kinase activities were not influenced by diet composition, whereas glucose-6-phosphate dehydrogenase activity was higher in fish fed the glucose diet. Higher GK activity was observed in fish fed the glucose diet. GK gene expression was higher at 25 degrees C in fish fed the waxy maize starch diet while in fish fed the glucose diet, no temperature effect on GK gene expression was observed. Hepatic G6Pase activities and gene expression were neither influenced by dietary carbohydrates nor water temperature. Overall, our data suggest that in gilthead sea bream juveniles hepatocytes dietary carbohydrate source and temperature affect more intensively GK, the enzyme responsible for the first step of glucose uptake, than G6Pase the enzyme involved in the last step of glucose hepatic release.     

Effect of dietary ascorbic acid supplementation and chronic hypoxia on sea bream growth and vitamin C status

Hypoxia (55% DO) and ascorbic acid (AA) supplementation level (0, 10, 25 and 50 mg AA equivalent kg^—1 diet, supplied as ascorbyl polyphosphate) did not significantly alter juvenile sea bream Sparus aurata growth, body composition, survival and skin hydroxyproline concentration. Liver, head kidney and spleen AA concentrations significantly decreased with decreasing dietary AA level, but were not influenced by hypoxia. This study showed that sea bream AA requirements were not influenced by chronic hypoxic stress.     

Comparative Study on the Cellular and Systemic Nutrient Sensing and Intermediary Metabolism after Partial Replacement of Fishmeal by Meat and Bone Meal in the Diet of Turbot (Scophthalmus maximus L.)

This study was designed to examine the cellular and systemic nutrient sensing mechanisms as well as the intermediary metabolism responses in turbot (Scophthalmus maximus L.) fed with fishmeal diet (FM diet), 45% of FM replaced by meat and bone meal diet (MBM diet) or MBM diet supplemented with essential amino acids to match the amino acid profile of FM diet (MBM+AA diet). During the one month feeding trial, feed intake was not affected by the different diets. However, MBM diet caused significant reduction of specific growth rate and nutrient retentions. Compared with the FM diet, MBM diet down-regulated target of rapamycin (TOR) and insulin-like growth factor (IGFs) signaling pathways, whereas up-regulated the amino acid response (AAR) signaling pathway. Moreover, MBM diet significantly decreased glucose and lipid anabolism, while increased muscle protein degradation and lipid catabolism in liver. MBM+AA diet had no effects on improvement of MBM diet deficiencies. Compared with fasted, re-feeding markedly activated the TOR signaling pathway, IGF signaling pathway and glucose, lipid metabolism, while significantly depressed the protein degradation signaling pathway. These results thus provided a comprehensive display of molecular responses and a better explanation of deficiencies generated after fishmeal replacement by other protein sources.     

Macrophage aggregates in gilthead sea bream fed copper, iron and zinc enriched diets

Gilthead sea bream Sparus aurata from consecutive year classes (0+ and 1+) and from the same parent stock were fed four diets, three of which were fortified with copper, iron and zinc. Concentrations of these elements were little affected by the diet. Hepatic, renal and splenic tissues sections were examined to detect the influence of the diet on the number and morphology of macrophage aggregates (MAs); in particular their structure was examined in the spleen. Three different types of MAs were detected: (1) unstructured, (2) partially structured and scarcely defined and (3) fully structured and well defined. Melanin was the most abundant pigment in the pigmented macrophages which form MAs and the amount of this pigment was influenced by season. Ferritin, in contrast with previous data obtained in other fish species, was more abundant in renal than in splenic MAs. Significant differences in splenic MA numbers among fish fed different diets and among different periods of the year were detected. The results suggest that a polyfactorial regulation could act on the splenic MA number in gilthead sea bream.     

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.     

Nutritional assessment of somatolactin function in gilthead sea bream (Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream (Sparus aurata) has been analysed. First, a down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH) administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 microg/g fish) evoked a short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 micromol/g fish) on plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a daily and seasonal signal of abundant energy at a precise calendar time.     

Feeding Responses of Hatchery-Reared Gilthead Sea Bream (Sparus aurata L.) to a Commercial Diet and Natural Prey Items

Many fish species have evolved feeding mechanisms and behaviours enabling them to feed on specific prey. However, such mechanisms may not be optimal for feeding on commercial-pelleted diets in aquaculture. Gilthead sea bream chew and occasionally eject pellets or parts of pellets from the mouth when feeding on commercial diets. This may result in an increase in nutritional waste from the intensive culture of this species. In this study we examined the prevalence of this food processing behaviour in two sizes of sea bream, feeding on three types of natural prey items in comparison to a commercial pellet, to give an insight into the circumstances in which excess chewing and ejection of food items from the mouth occurred. These included two hard-textured food items (commercial pellet and hard-shelled prey) and two soft-textured food items (larvae and small crustacean). Both sizes of sea bream frequently consumed the soft-textured food types, however large sea bream also frequently consumed hard-textured pellets. Hard-textured food required longer handling times and elicited more chewing and the ejection of food items from the mouth. These results suggest that future investigations on the food processing behaviour and consequent waste when fed commercial diets differing in texture could give an insight into improving diets and feeding efficiency for intensively cultivated gilthead sea bream.