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.     

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.     

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass,Dicentrarchus labrax

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues—eye, oesophagus, gills and skin—as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.     

New perspective to control of tenacibaculosis in sea bass Dicentrarchus labrax L

This work was carried out to investigate the effects of injection of Tenacibaculum maritimum formalin‐killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) as well as 1% feed supplements of oil extracts of Echinacea purpurea and Origanum vulgare on sea bass immunity improvement to the favour of T. maritimum experimental infection control after 4 weeks of the experiment. Tenacibaculum maritimum isolated from naturally infected sea bass showed brown to yellowish‐brown lesions (sores) on gills, skin and/or fins and identified by different biochemical methods and polymerase chain reaction technique. Immune parameters namely, total protein, globulin and lysozyme activity, as well as the relative level of protection were improved by T. maritimum (FKC), (LPS), (ECPs), O. vulgare and E. purpurea, respectively compared with control. Histopathological examination of T. maritimum naturally infected sea bass indicated many pathological changes in gill, skin and musculatures. Present study could be concluded that application of T. maritimum (FKC), (LPS), (ECPs), O. vulgare or E. purpurea improved sea bass immunity to the favour of disease resistance against T. maritimum. Further investigations on the combination between the previous control methods and the vaccine application methods will be needed.     

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.     

Incidence and prevalence of Diplectanum aequans and its influence on the fitness of juvenile sea bass (Dicentrarchus labrax) in the Black Sea

High rates of unexplained mortalities (up to 70%) are anticipated in the cultured juvenile sea bass (Dicentrarchus labrax) in farms in the eastern Black Sea at least for the last 8 years. Diplectanum aequans, often blamed for the observed mortalities, is the only prevalent parasite impacting the health of sea bass in the brackish water of the Black Sea. To investigate the seasonal fluctuation of D. aequans prevalence, mean intensities and the potential effects of D. aequans on the fitness of sea bass, eight cages from one farm (Farm A) and four cages at another distinct site (Farm B) were surveyed monthly from May to October of 2008. Six hundred and sixty‐one juvenile sea bass from Farm A and 236 from Farm B were individually examined for the presence and intensity of the parasite. The prevalence of D. aequans in cultured sea bass (96.3 ± 11.11; Mean ± SD) ranged from 66.7% to 100% in June, the beginning of 6‐month growing season. The mean intensity was 6.08 ± 2.19, significantly higher than that (2.74 ± 0.87; P < 0.05) in December (P < 0.05), the end of the growing season. Fish with low fitness had significantly higher number of parasite than the fish with higher fitness (P < 0.005). Although D. aequans negatively influenced host's condition factor, the impact was not enough to lead mortalities in the cultured sea bass in the Black Sea.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Tolerance of juvenile black sea bass Centropristis striata to acute ammonia and nitrite exposure at various salinities

ABSTRACT:   Experiments were conducted to determine the acute tolerance of juvenile (mean weight ± standard error, 9.9 ± 0.9 g) black sea bass Centropristis striata to environmental un-ionized ammonia-nitrogen (NH₃-N) and nitrite-nitrogen (NO₂-N) exposure at various salinities. Specifically, median lethal concentrations (LC50 values) of NH₃-N and NO₂-N at 24, 48 and 96 h of exposure were determined at salinities of 10, 20 and 30 g/L at 22°C. With the exception of LC50 values determined at 48 h, median lethal concentrations of NH₃-N to black sea bass were not influenced by environmental salinity; 24, 48, and 96 h LC50 values ranged from 0.81 to 0.85, 0.65–0.77, and 0.46–0.54 mg-NH₃-N/L, respectively. In contrast, tolerance of black sea bass to environmental NO₂-N was compromised at reduced salinities. Median lethal concentrations of NO₂-N to fish at 24, 48 and 96 h of exposure ranged from 288.3 to 429.0, 258.4 to 358.8 and 190.0 to 241.9 mg-NO₂-N/L, respectively. Results indicate that while juvenile black sea bass are relatively sensitive to acute NH₃-N exposure, they are highly resistant to NO₂-N exposure.     

Peptide molecular weight distribution of soluble protein fraction affects growth performance and quality in European sea bass (Dicentrarchus labrax) larvae

Dicentrarchus labrax were fed from 10 to 40 days posthatching with six microdiets differing in the inclusion level (60 g kg^‐1 and 120 g kg^‐1) and type of protein hydrolysate (PH; yeast, YPH; pig blood, PBPH; pig red blood cells, PRBCPH). A microdiet containing 120 g kg^‐1 fish PH (FPH) was used as a control. PH differed in their amino acid (AA) profile and molecular weight distribution and therefore the tested microdiets too. The estimated content in FAA and di‐ and tripeptides in the FPH microdiet was 2 g kg^‐1 and 44 g kg^‐1, respectively. FAA estimated levels in YPH and PBPH microdiets were 26 g kg^‐1 and 53 g kg^‐1, whereas levels of di‐ and tripeptides were 30 g kg^‐1 and 60 g kg^‐1, respectively. The estimated levels of FAA in PRBCPH microdiets were 8 g kg^‐1 and 17 g kg^‐1, whereas estimated levels of di‐ and tripeptides were 11 g kg^‐1 and 22 g kg^‐1, respectively. Results revealed that FPH may be replaced by alternative PH from yeast and pig blood products, as fish fed those diets performed, in terms of growth, survival, digestive function and incidence of skeletal deformities, as well as those fed the FPH microdiet. Using YPH, PBPH and PRBCPH, the inclusion level of PH in microdiets might be reduced to a half with respect to current practices using FPH.     

Replacement of fishmeal with rice protein concentrate in practical diets for European sea bass Dicentrarchus labrax reared at winter temperatures

As no information is available regarding the efficacy of using rice protein concentrate (RPC) to replace fishmeal (FM) in diets for European sea bass (Dicentrarchus labrax), a trial was conducted to determine the optimum inclusion for European sea bass juveniles. Diets were formulated to replace 25%, 50% and 75% of fishmeal with RPC. Additional high level inclusion diets (50% and 75%) were supplemented with lysine and methionine to determine if these were limiting factors. Fish (18.0 ± 0.06 g) were fed the experimental diets for 12 weeks. The final mean weight and specific growth rate (SGR) were significantly higher in fish fed the control diet (diet FM) and low level RPC (diet RPC25) than in fish fed the other diets. The supplementation of the amino acids had a positive effect on growth, elevating the final weight of the high level RPC (RPC75 + AA) group above that of the non‐supplemented group (RPC75). However, this was still not comparable to fish fed FM and RPC25 diets. No significant differences in body composition were observed. A digestibility study demonstrated an inverse relationship between elevating inclusion levels of RPC and apparent digestibility coefficients (ADCs) for dry matter and lipid, with significantly lower values than the control being observed with RPC inclusions above 25%. The mesenteric fat index followed this trend. Circulating leucocyte levels, leucocyte ratios and serum lysozyme activity remained unaffected by dietary treatment. However, compared with the control group, fish fed RPC75, RPC50 + AA RPC75 + AA displayed significantly lower haematocrit values. The present study demonstrates that the inclusion of RPC at 140 g kg^?1 (effectively replacing 25% of FM content) does not compromise European sea bass growth performance, body quality or basic haematological parameters. It is suggested that future studies including higher levels test the efficacy of additional amino acid supplementation (e.g. tryptophan).     

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.