Betanodavirus ability to infect juvenile European sea bass,Dicentrarchus labrax,at different water salinity

Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT‐PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock‐infected group. VERv presence was confirmed by rRT‐PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.     

Phosphorus requirement of European sea bass (Dicentrarchus labrax L.) juveniles

A growth trial was conducted to estimate the phosphorus requirement of European sea bass juveniles. Six experimental isonitrogenous and isoenergetic semi‐purified diets (casein based) were formulated to contain 0.48%, 0.65%, 0.77%, 0.86%, 1.05% and 1.25% phosphorus (diets D1, D2, D3, D4, D5 and D6 respectively). Dicalcium phosphate was used as dietary phosphorus source. Twelve groups of 25 fish of 10 g initial body weight were allocated to 55‐L tanks in a thermoregulated water recirculating system. Each experimental diet was assigned to duplicate groups of these fish. The trial lasted for 10 weeks and fish were fed two times a day, 6 days a week, to apparent visual satiation. At the end of the trial, final weight of fish fed diet D1 was significantly lower than that of the other groups, except of fish fed diet D4. Mortality of fish fed diet D1 was significantly higher than that of fish fed the other diets, except for diet D6. Feed efficiency and protein efficiency ratios were significantly lower with diet D1 than with the other diets. Nitrogen retention (% nitrogen intake) of fish fed diet D1 was significantly lower than in the other groups, except that of fish fed diet D4. Energy retention (% energy intake) was not significantly different among groups. At the end of the trial there were no differences in whole body composition among groups. Whole body phosphorus content averaged 0.72% (on a fresh weight basis) and was not significantly affected by dietary phosphorus content. Phosphorus retention averaged 6.1 g kg^?1 weight gain and was not significantly different among groups. Phosphorus retention (% phosphorus intake) was significantly higher in fish fed diets D2 and D3 than in fish fed higher dietary phosphorus levels. Expressed per unit body weight per day, phosphorus retention was not significantly different among groups fed diets D2 to D6, while phosphorus losses linearly increased with dietary phosphorus intake. Results of this trial indicate that the phosphorus requirement of sea bass juveniles was satisfied with a diet containing 0.65% of phosphorus.     

Evaluation of zinc supplementation in European sea bass (Dicentrarchus labrax) juvenile diets

This study aimed to investigate the effect of increased zinc dietary levels on the growth performance, feed utilization, immune status and induced wound healing in European sea bass (Dicentrarchus labrax). Fish weighing 10 g were fed five diets containing organic zinc at 30, 70, 110 and 150 mg kg^?1 diet and one inorganic zinc source (zinc oxide) at 150 mg kg^?1 diet for a period of 12 weeks. No significant (P>0.05) differences were found in the growth performance parameters (final weight, specific growth rate), immunological indices tested (respiratory burst activity in whole blood) and the wound‐healing process. Zinc accumulation in the skin, vertebrae and liver increased significantly (P<0.01) with increased dietary zinc levels but not in muscle. The highest zinc concentrations were obtained in skin tissue, followed by vertebrae, liver and muscle tissue. Using the concentration of zinc in skin as a response criterion, broken‐line analysis showed that the supplementation of 148 mg organic Zn kg^?1 diet seemed to be the optimum dietary zinc supplementation level for sea bass juveniles.     

Feed discrimination and selection in self-fed European sea bass Dicentrarchus labrax L.

Abstract This study was conducted to evaluate feed discrimination and preference of European sea bass Dicentrarchus labrax L., taking into account the effects of feed location, previous feeding experience and light conditions. Fish (63 g) were held in tanks and exposed to a 12L:12D photoperiod and ambient temperature. The following feeding practices were applied in triplicate: three‐choice feeding (self‐ feeding with feeds formulated for carnivorous, herbivorous and omnivorous fish rotated on a weekly basis) and monofeeding (self‐feeding with one of these feeds). After 9 weeks (phase I), fish previously held in monofeeding, were subjected to three‐choice feeding for 2 weeks (phase II). In phase I, fish discriminated between feeds after their rotational displacements and preferred the feed formulated for carnivorous species. This also supported the best growth. Fish used in phase II preferred the same feed. There was no preference for any location within tanks. Feeding was generally diurnal, although in some tests with the feeds for herbivores and omnivores, diurnal and nocturnal feed demands were similar and fewer in number than with the feed formulated for carnivores.     

Antipredator responses to overhead fright stimuli in hatchery-reared and wild European sea bass (Dicentrarchus labrax L.) juveniles

In this study, shoals of hatchery‐reared and wild sea bass juveniles (Dicentrarchus labrax L.) were tested for differences in their antipredator responses towards a visual (shadow) and a mechanical (dummy bill) overhead stimulus. Two behavioural variables – distance from the bottom and freezing duration – were measured during post‐stimulus phases of each test and compared between wild and hatchery‐reared shoals, composed of 10 juveniles each. The results showed that in both hatchery‐reared and wild juveniles, stimulus exposure elicited a significant decrease in the mean shoal distance from the bottom. Similarly, individuals from both groups engaged a freezing reaction, but the mean freezing duration was significantly higher in wild‐ than in hatchery‐reared juveniles. Results are discussed in the light of their relevance for the enhancement of restocking programmes.     

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass,Dicentrarchus labrax (L.)

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red‐spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV‐type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.     

Influence of tank volume on vitellogenesis and spawning performances in sea bass Dicentrarchus labrax L.

Sea bass, Dicentrarchus labrax (mean weight: 748±13 g), were maintained before and during vitellogenesis in 1, 3, 8, 16 and 32 m³ tanks, and then they were transferred to 2 m³ tanks, for the spawning season. During the first 2 months of the experiment, the growth rates were significantly lower in smaller tanks (1 m³). In August, the oocyte diameters were significantly lower in smaller tanks (1, 3 and 8 m³) than in larger (16–32 m³) tanks. At the end of the experiment, the fish mean weight in the 1 m³ tanks was significantly lower than in the 3 m³ tanks, but oocyte diameters and plasma oestradiol concentrations were not significantly different between the volumes. This shows a longer acclimation requirement in smaller volume rearing. Although all the females had not spawned, one spawn at least was collected in each volume. The variation in conditioning volume has not blocked the spawning process. The qualitative and quantitative characteristics of spawns were not significantly different between volumes. The conditioning volume of 3 m³ seems to be a minimal volume required to obtain good reproduction of sea bass.     

Compensatory growth response of European sea bass (Dicentrarchus labrax L.) under cycled starvation and restricted feeding rate

The compensatory growth response of the European sea bass (Dicentrarchus labrax) that faced cycling starvation and restricted ration was assessed. Juveniles (10.5 g) were stocked into 15 tanks at a density of 25 fish per tank. Five different feeding regimes were tested on triplicate groups of fish: C_Satiation: control fed for 60 days without deprivation, C_Restricted: 25% restricted feeding, S‐R: 1 day starvation then 4 days C_Restricted feeding, R‐F: 1 day C_Restricted feeding then 4 days C_Satiation feeding, and, finally, S‐F: 1 day starvation then 4 days C_Satiation feeding. The specific growth rate of fish in the C_Satiation (2.5 ± 0.06% day^?1), S‐F (2.5 ± 0.11% day^?1) and R‐F (2.4 ± 0.18% day^?1) were significantly higher than that of C_Restricted (2.2 ± 0.05% day^?1) or S‐R (2.0 ± 0.01% day^?1). Fish in S‐F group were able to achieve catching up with the C_Satiation. There was no significant difference in feed conversion rates but R‐F and S‐F consumed approximately 34% more feed than C_Satiation following the first re‐feeding day. Although, the highest lipid content was observed in C_Satiation (14.4%), S‐R (33.3% dry matter) had the highest water content compared with the C_Satiation and C_Restricted (37.8% and 36.9% dry matter respectively). In conclusion, it may be concluded that sea bass has rapid response to cycling starvation/re‐feeding and that a 25% restricted feeding ratio is insufficient to invoke a compensatory growth response in sea bass.     

Growth,development and survival of European sea bass (Dicentrarchus labrax) larvae cultured under different light spectra and intensities

We assessed the effects of light intensity and spectrum on the growth, development and survival of Dicentrarchus labrax larvae from 30 to 96 days after hatching. Twelve lighting regimes were applied using 0.3, 1 and 2 W/m² full spectrum white, blue, red and green light. By day 66 of exposure, growth performance was significantly better in larvae reared under green or red light compared with white or blue light (p < 0.05), while it was significantly better at 2.0 W/m² compared with 1.0 or 0.3 W/m² (p < 0.05). Larvae reared under white or blue light had significantly more deformities (p < 0.05) than did larvae reared under light of other spectra, while larvae reared under 0.3 W/m² light intensity had a significantly higher proportion of jaw malformations than reared under 1.0 and 2.0 W/m² (p ? 0.05). Dicentrarchus labrax larvae subjected to green light showed significantly lower survival compared with the other light spectra (p < 0.05). Moreover, light significantly affected brain aanat1a, aanatt1b, mellc, mt1, mt2, gh, tsh and crf mRNA expression. Therefore, the red and green light groups (or under 2.0 W/m²) could improve the growth performance and enhance secretion of melatonin, gh and tsh, and reduce the proportion of jaw malformations.     

Behaviour of European sea bass,Dicentrarchus labrax L., in cages – impact of early life rearing conditions and management

European sea bass, Dicentrarchus labrax L., groups reared under different conditions during larval stages (mesocosm and intensive rearing) were monitored during on‐growing in sea cages until marketable size (350–400 g). Four caged groups were followed for a period of 17 months each at a pilot scale farm, where vertical distribution behaviour was monitored. This was performed while fish were under calm conditions, during feeding and when stress events occurred. Also during two specific periods: (i) spawning and (ii) high water temperature. Clear differences in the behavioural pattern of swimming depth, displacement and used space between the groups were observed. The individuals from the mesocosm rearing were more sensitive to human presence, showing stronger reactions (speed of displacement and vertical distribution). Most pronounced differences were observed during the ‘extreme’ warm period and the reproductive season. Within the seasons, European sea bass responded during feeding and stress showing a tendency to move deeper compared to calm conditions. The increased displacements were longer during feeding and stress.     

Effect of lordosis on the swimming performance of juvenile sea bass (Dicentrarchus labrax L.)

The critical swimming speed (U_crit, cm s⁻¹) value of normal juvenile sea bass (Dicentrarchus labrax) (64.7±6.33) was significantly higher than that of fish with lordosis (60.3±6.66) (P<0.05). Both normal fish and those with lordosis showed increased endurance with increasing length during exercise at a fixed water velocity of 50 cm s⁻¹. This speed was used to investigate the separation possibility of deformed specimen from the normal developed fish based on their swimming performance, and 28% separation was achieved without losing any normal fish by the end of the 10th minute. To achieve a better separation of the deformities from the normal, first grading of the juveniles and then a swimming endurance test is advised.     

Effects of conventional and organic feed on the mineral composition of cultured European sea bass (Dicentrarchus labrax)

European sea bass (Dicentrarchus labrax) is a widely consumed marine fish in Mediterranean areas, and different farming techniques are applied for fish culturing to satisfy the growing demand for seafood. The aim of this study was to investigate the effects of conventional and organic feed on the quality of cultured European sea bass (Dicentrarchus labrax) collected during the growth period. The concentrations of ash, moisture, essential macro‐elements (Ca, K, Mg, Na and P), micro‐elements (Co, Cr, Cu, Fe, Mn, Ni and Zn) and toxic elements (As, Cd, and Pb) were determined in feeds and in fillets of cultured fish. The results were compared to those obtained from wild sea bass. Results showed that the differences between organic diet‐fed and conventional diet‐fed sea basses varied in relation to the specific element measured and the growth period. The former showed higher concentration of Fe, Mg and Cr, and lower Na content. The amount of P, Na, Fe, Cu and Cr in wild sea bass was significantly (P ≤ 0.05) higher than that found in farmed fish. The levels of toxic elements in cultured sea bass were always within the allowed limit for fishery products. Wild samples had significantly (P ≤ 0.05) higher content of arsenic and lead than farmed sea bass. Cultured fish represent a valuable dietary source of essential macro‐ and micro‐elements. Controlled rearing systems and feedings were related to a decrease in the presence of some toxic metals in cultured fish compared with wild fish.     

Effects of successive size grading on the growth of juvenile European sea bass (Dicentrarchus labrax L.) at three temperatures

Juvenile European sea bass from the same fish stock were selected by successive size grading processes using 2, 3 and 4 mm bar graders at 79, 96 and 99 days post hatching, thus forming three groups (n=300) consisting of similar‐sized fish that differed by time of each group formation. The growth patterns of fish groups were studied at three temperatures during 5 weeks of rearing. Three‐way anova followed by the Tukey multiple comparison test (P<0.05) showed a high dependence of growth on the temperature applied. The smallest size and weight of fish were detected in all groups reared at 19 °C compared with fish held at 21 and 23 °C respectively. Differences in coefficients of variation of lengths were small and insignificant between groups and temperatures. Growth in the length of sea bass juveniles during the test period was a linear function of time and no differences were observed in growth rate among groups at a particular temperature. Growth rates of fish were 0.71 ± 0.02, 0.62 ± 0.01 and 0.52 ± 0.02 mm day^?1 at 23, 21 and 19 °C respectively. These results indicated that the variations in body size of juveniles in the test period were not the result of differences in the growth potential of individuals.     

Dietary saponins and phytosterols do not affect growth,intestinal morphology and immune response of on‐growing European sea bass (Dicentrarchus labrax)

This study was the first to investigate the effect of soy antinutrients, saponins and phytosterols, in on‐growing sea bass. Seven diets were formulated: a control diet (fishmeal and fish oil based) and six diets containing 1 or 2 g kg^?1 levels of soya saponins, 5 or 10 g kg^?1 levels of phytosterols or a combination of 1 g kg^?1 saponins + 5 g kg^?1 phytosterols or 2 g kg^?1 saponins + 10 g kg^?1 phytosterols. After a 59 days feeding trial fish were weighed, faeces collected for digestibility studies and plasma sampled for cholesterol quantification. At day 15 and 59, distal intestine (DI) samples were collected for histological evaluation and quantification of maltase (Malt), alkaline phosphatase (AlP) and leucine aminopeptidase (LAP) activities. At day 59, DI was collected for gene expression of AlP, Malt and immune related genes. Antinutrients had no effect on fish performance, digestibility, plasma cholesterol levels and digestive enzymes activities and gene expression. Only mild morphological changes were present in groups fed high levels of saponins + phytosterols. Expression of immune related genes showed no differences between groups. Overall, on‐growing sea bass showed high tolerance to dietary antinutrients up to the levels tested.     

Dietary spermine supplementation induces intestinal maturation in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were fed microparticulated compound diet containing 0 (FP0), 0.10 (FP10) and 0.33% (FP33) of a polyamine, spermine, from day 20 to day 38. LP group was fed live prey. This group exhibited the highest growth and survival. The addition of spermine did not lead to growth enhancement. A 33% survival improvement was obtained in FP33 group compared to FP0 group. The spermine addition affected the activity of pancreatic enzymes, trypsin, chymotrypsin and amylase, during larvae development. This non specific effect suggested that the action of spermine would be mediated by hormones. In the intestine, the FP33 group exhibited from day 31 higher activities of brush border membrane enzymes (leucine aminopeptidase and alkaline phosphatase) and lower level in a cytosolic enzyme (leucine-alanine peptidase) compared to FP10 and FP0 group. The diet containing the highest spermine level induced an enzymatic profile similar to that obtained in LP group and characteristic of a mature enterocyte. The initiation of enterocyte maturation at a proper development stage was associated to the survival improvement observed in FP33 group.     

Acid–base balance in sea bass (Dicentrarchus labrax L.) in relation to water oxygen concentration

The influence of water oxygen concentration on the acid–base balance of sea bass was evaluated. Fish weighing 200–250 g were cultured under different dissolved oxygen concentrations of 64%, 97%, 150% and 250% saturation (92.7, 140.5, 217.5 and 362.7 mmHg respectively) under mild hypoxia, normoxia, mild hyperoxia and high hyperoxia conditions. The results showed that high hyperoxia and mild hypoxia conditions modified some blood parameters significantly when compared with fish held under the normoxia condition, while no differences were shown with respect to the acid–base balance of fish cultured under normoxia and mild hyperoxia conditions. This testifies that the mild hyperoxia condition does not produce physiological disturbances in the acid–base status of sea bass and it could be considered a favourable condition in sea bass land‐based farming, mainly in comparison with the mild hypoxia condition, responsible for other physiological problems.     

Antibody response in sea bass (Dicentrarchus labrax L.) in relation to water temperature and oxygenation

The influence of water temperature and water oxygenation on the specific antibody response was evaluated by indirect ELISA in sea bass (Dicentrarchus labrax L.) immunized against human‐γ‐ globulins emulsified in Freund's complete adjuvant. A higher antibody response was observed in fish reared at 24 and 30 °C, than at 12 and 18 °C. For the fish group reared at 24 °C, the immunological observation was carried out until 300 days after immunization. At the end of this period, fish kept at 24 °C still showed immunological competence, although the antibody response began to decrease significantly from 120 days after the immunization. Fish reared under mild hyperoxia and normoxia conditions had higher antibody responses than fish reared under mild hypoxia conditions. Moreover, at the end of the experimental period of 56 days the antibody response of fish reared under hyperoxia conditions was significantly higher than the immune response detected in fish reared under normoxia and hypoxia conditions.