Partial characterization of pyloric-duodenal lipase of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In the present study, we report the isolation and characterization of seabream Sparus aurata pyloric caeca-duodenal lipase. Optimum activity was found at pH 8.5 and salinity of 50 mM NaCl. Lipase activity was sensitive to divalent ions, and extreme pH values (4, 5, and 12), being more stable at alkaline than acid pH. Optimum temperature was found at 50°C, but lipase was stable at temperatures below 40°C. Lipase has a bile salt sodium taurocholate requirement for increased activity. Gradient PAGE electrophoresis revealed the presence of four isoforms with apparent molecular masses of 34, 50, 68, and 84 KDa, respectively. Pyloric-duodenal lipase was able to hydrolyze emulsified alimentary oils. Results confirm the presence of true lipases in Sparus aurata digestive tract.     

Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) to organophosphate pesticides

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K _m and V _max were calculated from the Michaelis–Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k _i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.     

Seasonality of nuclear abnormalities in gilthead sea bream <Emphasis Type="Italic">Sparus aurata</Emphasis> (L.) erythrocytes

The frequencies of erythrocyte nuclear abnormalities, such as irregularly shaped (ISN), vacuolated nuclei (VN), binucleated cells (BN), including micronuclei (MN), were monitored in the peripheral blood of cultured gilthead sea bream (Sparus aurata L.) from the southern Adriatic Sea (Croatia) and assessed for a relation to seasonality. Fish were sampled in February, April, June, and September. The lowest frequencies of ISN and MN were measured in April, and the lowest frequencies of VN and BN were found in February. The highest values of ISN and BN were detected in September. The highest values of MN and the most frequent occurrence of VN occurred in June and April, respectively. The Spearman rank order correlation test revealed a significant positive correlation between the frequencies of ISN, BN and MN; VN did not correlate with any of the measured parameters. The ISN had significantly higher nuclear surface area values than normal nuclei (P < 0.001). Our results demonstrate that the measured parameters displayed a distinct seasonality. Consequently, we conclude that seasonal variations should be taken into account when nuclear abnormalities are used as biomarkers. This variation should be well understood in order to successfully separate true contamination effects from seasonal factors that have the potential to affect the frequency and occurrence of nuclear abnormalities.     

Olive oil by-product as a natural antioxidant in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) nutrition

The aim of the study reported here was to evaluate the efficiency of a natural antioxidant substance in gilthead sea bream (Sparus aurata) feeds. An olive oil by-product, olive mill vegetation water (VW), contains polyphenols, which have a strong antioxidant activity. A 147-day growth trial was conducted (monofactorial balanced, 4 × 3) with diet as the experimental factor. Two diets [isonitrogenous (crude protein 40%) and isoenergetic (gross energy = 18MJ kg⁻¹ on a dry weight basis] were formulated with 1 and 5% of VW (VW1, VW5); the control diet did not contain VW. A total of 600 juvenile gilthead sea breams (mean body weight 114.1 ± 5.7 g) were utilised. Production parameters and somatic indexes were calculated at the end of the growth trial. Antioxidant activity in fish fillets was investigated using TBARS and DPPH assays. A number of haematological parameters and digestive enzyme patterns were measured in fish in the middle and at the end of the experiment. The TBARS values showed slight delays in the development of oxidation in the fillet of fish fed with VW. There was no statistical difference between fish fed with the experimental diet and the control group, except for maltase activity, which increased with increasing VW in the feed. We found that the use of VW in a gilthead sea bream diet did not have any detrimental effects on gilthead sea bream production and physiological parameters and slightly improved the conservation of the fish fillets.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Oxygen consumption and feeding rates of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) reveal lack of acclimation to cold

Low temperature has been implicated in inducing outbreaks of winter syndrome or winter disease in farmed gilthead sea bream (Sparus aurata). The responses of gilthead sea bream to reduced temperature followed by maintenance at low temperaturewere studied. In a first experiment, oxygen consumptionwas measured when water temperature was reduced from 18°C to 8 °C at either a rate of 1 °C· day-1 or as two sharp drops (from 18 °C to 12 °C, and from 12 °C to 8 °C). In a second experiment, the water temperature was reduced from 16 °C to 8 °C or 12 °C and then maintained for 20 days to study the fish acclimation to these temperatures. In both experiments, fish stopped feeding below 13 °C and did not resume feeding when maintained at low temperatures. The decrease in metabolic activity, expressed by the oxygen consumption rate, was directly related to the fall in water temperature: the Q10(18 °C-8 °C) values were between 2.2–2.5, independently of the descend rate in water temperature. However, we observed a more reduced metabolic rate when the water temperature was below 12 °C. Fish maintained at low temperatures showed only a partial recovery in oxygen consumption (15% at 8 °C and 20% at 12 °C) after 20 days. A higher metabolic rate together with a fasting-temperature condition meant that maintenance at 12 °C was more aggressive than at 8 °C, as revealed by the condition factor and energy needs. Data suggest that 12 .C could be a threshold temperature for the metabolic activity of gilthead sea bream. The relationship between low temperatures and their possible implication in the appearance of winter disease in gilthead sea bream is also discussed.     

A comparative study on the composition and importance of free amino acids in semen of gilthead sea bream, <Emphasis Type="Italic">Sparus aurata</Emphasis>, and perch, <Emphasis Type="Italic">Perca fluviatilis</Emphasis>

A comparative study was conducted on the free amino acid composition of gilthead sea bream, Sparus aurata, and perch, Perca fluviatilis. Also the effect of 21 free amino acids on sperm motility was investigated. Spermatozoa were incubated in species-specific motility-inhibiting saline solution containing the different amino acids for 48 h. Thereafter, the motility was activated and investigated using computer-assisted cell motility analysis. Twelve free amino acids, respectively, were detected in S. aurata and P. fluviatilis semen. Arginine, cysteine, glutamic acid, leucine, and methionine occurred in semen of both species. In S. aurata, arginine, glycine, hydroxyproline, lysine, and phenylalanine in concentrations of 1.25 and 2.50 mmol/l, methionine in a concentration of 2.5 mmol/l, and serine in a concentration of 1.25 mmol/l had a positive effect on the motility of spermatozoa. In P. fluviatilis, alanine, asparagine, cysteine, glycine, isoleucine, lysine, methionine, serine, threonine, and valine in concentrations of 2.50 mmol/l positively affected motility. From these data, it can be concluded that the amino acid composition and the effect on motility are species specific. Possible consequences for spermatozoa functionality are discussed.     

Influence of time,storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In this study, we tested the effects of long-term storage (2 years) at ?20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine–alanine peptidase) enzymes between 140 and 270 days of storage at ?20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at ?20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.     

Genetic assessment of three gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) populations along the Spanish coast and of three broodstocks managements

The gilthead sea bream (Sparus aurata L.) is one of the most important Sparid farmed in Europe, especially in the Mediterranean area. However, efficient breeding programs for this species are scarce and very little, and it is known concerning their population structure. The present study was mainly designed to genetically characterize, by microsatellite markers, three gilthead sea bream populations sampled along the Spanish coast (Cantabrian Sea, the Atlantic Ocean and Mediterranean Sea) and their progeny with the aim of studying its genetic variability and its genetic structure. Moreover, we evaluated different strategies of broodstocks management (breeders’ number, origin and sex combination) on parental contributions and on effective breeding numbers. That number of breeders in the stock was of great importance to the maximization of contribution since the larger broodstock resulted in larger proportion of parents contributing. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire was translated into reduced effective breeding numbers and consequently into expected increased inbreeding rates. Our results highlight the high genetic variability of the studied sea bream populations, as well as the existence of three genetically differentiated populations along the Spanish coast. These findings should be relevant for the establishment of successful breeding programs in aquaculture of the gilthead sea bream.     

Changes in muscle collagen content during <Emphasis Type="Italic">post mortem</Emphasis> storage of farmed sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>): influence on textural properties

The changes in collagen content and its solubility in sea bream muscle were studied for variable storage times following the death of the fish, and these variables were related to the evolution of physical parameters important for consumer acceptance: firmness and water-holding capacity (WHC). The results show that the collagen content in muscle diminished slightly over storage time and that this variable was directly related to firmness but inversely related to the water-holding capacity. With regard to collagen solubility, a decline was detected in acid-soluble collagen (ASC) in the first few post mortem hours, perhaps related to the end of rigor mortis that occurs at these stages. Pepsin-soluble collagen (PSC) increased, while insoluble collagen (ISC) decreased from 96 h, coinciding with a loss of firmness. This softening can be explained as a result of specific collagenases acting on the insoluble fraction of the collagen.     

Elevated whole brain arginine vasotocin with Aroclor 1254 exposure in two <Emphasis Type="Italic">Syngnathus</Emphasis> pipefishes

The neurohypophysial hormone arginine vasotocin (AVT) and the neuroendocrine system strongly interact with the rest of the teleostean endocrine system. The aim of this study is to investigate the effects of exposure to an endocrine disruptor on whole brain AVT concentrations for the pipefishes Syngnathus floridae and S. fuscus. Following treatment over the entire brood period, AVT concentrations were significantly higher for Aroclor 1254-exposed, post-brooding males compared to controls for both species. Considering both previously documented seventeen-fold increases in AVT for brooding males with embryos in some developmental stages and changes in parental nutrient concentrations after Aroclor 1254 exposure, these data begin to address potential physiological mechanisms that may underlie paternal activities in syngnathid males.     

The effect of live feeds bathed with the red seaweed <Emphasis Type="Italic">Asparagopsis armata</Emphasis> on the survival,growth and physiology status of <Emphasis Type="Italic">Sparus aurata</Emphasis> larvae

Larval rearing is affected by a wide range of microorganisms that thrive in larviculture systems. Some seaweed species have metabolites capable of reducing the bacterial load. However, no studies have yet tested whether including seaweed metabolites on larval rearing systems has any effects on the larvae development. This work assessed the development of Sparus aurata larvae fed preys treated with an Asparagopsis armata product. Live prey, Brachionus spp. and Artemia sp., were immersed in a solution containing 0.5% of a commercial extract of A. armata (Ysaline 100, YSA) for 30 min, before being fed to seabream larvae (n = 4 each). In the control, the live feed was immersed in clear water. Larval parameters such as growth, survival, digestive capacity (structural-histology and functional-enzymatic activity), stress level (cortisol content), non-specific immune response (lysozyme activity), anti-bacterial activity (disc-diffusion assay) and microbiota quantification (fish larvae gut and rearing water) were monitored. Fish larvae digestive capacity, stress level and non-specific immune response were not affected by the use of YSA. The number of Vibrionaceae was significantly reduced both in water and larval gut when using YSA. Growth was enhanced for YSA treatment, but higher mortality was also observed, especially until 10 days after hatching (DAH). The mortality peak observed at 8 DAH for both treatments, but higher for YSA, indicates larval higher susceptibility at this development stage, suggesting that lower concentrations of YSA should be used until 10 DAH. The application of YSA after 10 DAH onwards promotes a safer rearing environment.     

Growth and feed utilization of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, L.) fed to satiation and restrictively at increasing dietary energy levels

Three isoproteic (47% protein) diets were formulated to contain graded levels of crude fat (diet D16:16%, diet D24: 24% and diet D32: 32%). Each diet was fed to satiation in three and to 80% satiation in two replicate groups of gilthead sea bream (Sparus aurata), having an initial body weight of 72–74 g. The trial lasted 81 days. Groups fed to satiation showed higher final body weight (FBW; 238.8–252.3 g vs. 218.0–229.3 g) and daily growth index (DGI; 2.49–2.65%/day vs. 2.27–2.34%/day) than those fed to 80% satiation. Feed intake was significantly different both for feeding level and for diet composition. Fish fed to satiation had higher feed conversion rate (FCR) compared to the 80% satiation groups (1.33–1.44 vs. 1.13–1.17; P ≤ 0.001). Within satiation groups, FCR was significantly lower in fish fed D16 compared to fish fed D32 (1.33 vs. 1.44, P ≤ 0.05), whereas no statistical differences were found within the 80% satiation groups. The increase in dietary lipid level did not improve growth performance, feed efficiency and protein utilization but decreased gross lipid efficiency. Conversely, a reduction in ration from satiation to 80% satiation decreased DGI, thus improving FCR. Feed costs were influenced by dietary energy level and feeding ratio, the lowest energy diet at 80% satiation being the most profitable combination among the variables.     

Origin of broodstock and effects on the deformities of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L. 1758) in a Mediterranean commercial hatchery

The use of broodstock of different origin as a method to improve fry production performance and consequently to minimize deformities was examined at industrial scale in a commercial gilthead sea bream hatchery. The outcome of fry production from three different broodstock groups (BA: broodfish (Mediterranean) with multiannual hatchery presence, BB: selected offspring originating from the BA group, and BC: broodfish of Atlantic origin) was investigated in the same rearing conditions and feeding protocol. Performance factors assessed were the survival and weaning of the larvae; the mortality rates from the “weaning until the end of the hatchery stage” of the larvae/fry; the percentage of fry without swim bladder; the percentage of fry with skeletal deformities and the feed conversion ratio. In all factors, no statistical differences among the experimental groups were detected. However, due to early rejection of the deformed individuals, benefits are expected from the decrease of the supplied amount of food and the reduced labor cost.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Social rank modulates brain arginine vasotocin immunoreactivity in false clown anemonefish (<Emphasis Type="Italic">Amphiprion ocellaris</Emphasis>)

The brain nanopeptide arginine vasotocin (AVT) and its mammalian homolog arginine vasopressin are involved in the regulation of social and reproductive behavior. We investigated the relationship between social rank formation and the brain AVT system in the false clown anemonefish (Amphiprion ocellaris), which forms a social rank that leads to sex differentiation in higher-ranked individuals. Tanks of three sexually immature fish were kept for 90 days and each fish’s behavior was observed once a month. The social rank of each individual was distinguishable by behavior, but gonadosomatic index (GSI) did not differ significantly. The number of AVT neurons in the magnocellular layer in the preoptic area (POA) increased in subordinate individuals and declined with increasing hierarchical dominance. These results suggest that social rank formation modulates AVT production in the brain of the clown anemonefish and may influence their later sex differentiation.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Gene expression of thyrotropin- and corticotrophin-releasing hormones is regulated by environmental salinity in the euryhaline teleost <Emphasis Type="Italic">Sparus aurata</Emphasis>

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65–81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments.