Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

Effects of dietary dehydrated lemon peel on some biochemical markers related to general metabolism,welfare and stress in gilthead seabream (Sparus aurata L.)

Dehydrated lemon peel (DLP) at two levels (1.5% and 3%) was included for 30 days in the diet of gilthead seabream (Sparus aurata L.) and after 15 and 30 days the serum and skin mucus of fish were analysed. In serum, both experimental diets led to an initial decrease in glucose and lactate after which the levels became comparable to the control. Dietary DLP modulated the activity of transaminases, alanine aminotransferase and aspartate aminotransferase. The total antioxidant status showed a progressive increase in relation to levels of inclusion of DLP and time of administration (p < .05). In skin mucus, some biomolecular markers related to general stress, oxidative stress and apoptosis exhibited variations that suggest an adaptive response of fish to the new metabolic situation, resulting from the experimental diet. The inclusion of DLP in the diet seems to positively modulate the central metabolism and welfare of gilthead seabream.     

Implication of mucus-secreting cells,acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ-carrageenin to gilthead seabream (Sparus aurata) specimens

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin–eosin, Alcian blue or periodic acid–Schiff. Furthermore, immunohistochemistry and expression analyses of several cells’ markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were up-regulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.     

Dietary lysine requirement of juvenile gilthead seabream Sparus aurata L.*

The dietary lysine requirement of juvenile gilthead seabream was determined by the growth response of duplicate groups of fish (3.5 g initial weight) fed on six isonitrogenous (427 g kg^?1) and isolipidic (135 g kg^?1) diets containing graded levels of crystalline l ‐lysine HCl, with dietary lysine content ranging from 36.3 to 79.7 g kg^?1 of protein. The final indispensable amino acid profile of the diets except for lysine was formulated so as to resemble that of wild seabream whole body. Except for the reduced growth performance of fish groups fed the lysine‐deficient diets no other deficiency signs were apparent. Survival observed throughout the feeding period of 6 weeks was excellent. Weight gain (in %), specific growth rate, feed efficiency and daily protein deposition (DPD) were significantly improved in response to the increasing levels of dietary lysine up to 52.7 g kg^?1 of protein and remained nearly constant thereafter. Whole‐body protein content followed a similar pattern as growth parameters in relation to dietary lysine level. Non‐linear regression analysis of DPD against dietary lysine level using the four‐parameter saturation kinetic model indicated a lysine requirement of 50.4 g kg^?1 of protein for this species to support growth.     

Effects of the dietary protein : lipid ratio on growth and nutrient utilization in gilthead seabream (Sparus aurata L.)

Gilthead seabream Sparus aurata L. (initial mean body weight: 42.5 g) were fed four experimental diets containing either 47 or 51% of dry matter (DM) as protein and either 15 or 21% as lipid for 12 weeks. Each diet was hand-distributed to triplicate groups of 60 fish, three times a day until satiation. The digestibility coefficients of the dietary components were determined using chromic oxide as a marker. The levels of protein or lipid in the diets did not affect the digestibility. Fish regulated their feed intake and attained the same weight at the end of the experiment. However, feed efficiency varied between diets, with best values obtained with both diets containing 21% lipid. When diets contained only 15% lipid, feed efficiency increased with dietary protein level. Nitrogen retention was significantly higher with high fat diets regardless of dietary protein level. Neutral lipid deposition was significantly higher in liver for diets rich in lipids. It was elevated in muscle only in fish fed the diet containing 47% protein and 21% lipid and this deposition in muscle contributed to a significant increase in body fat content. Phosphorus load to the environment, measured as percentage retention of ingested or digestible phosphorus, was significantly lower with both diets higher in lipids.     

Pigmentation of gilthead seabream, Sparus aurata (L. 1875), using Chlorella vulgaris (Chlorophyta, Volvocales) microalga

A feeding experiment was conducted over 9 weeks with seven groups of 30 (fish per group) unpigmented gilthead seabream, Sparus aurata (L. 1875) (initial mean weight = 145.2 ± 12.3 g). Three experimental diets were prepared by adding to a basal diet free of carotenoid (final pigment content of around 40 mg per kg feed): (i) a biomass of the carotenogenic Chlorella vulgaris (Chlorophyta, Volvocales); (ii) a synthetic astaxanthin; and (iii) a mixture (1:1) of microalgal biomass and synthetic astaxanthin. At 3‐week intervals, five fish were sampled from each tank for total carotenoids analysis in skin and muscle. The carotenoid pigments (total amount = 0.4%) identified in the carotenogenic alga were lutein (0.3%), β‐carotene (1.2%), canthaxanthin (36.2%), astaxanthin, free and esterified forms (55.0%), and other pigments (7.3%). Carotenoid pigments were significantly deposited in the four skin zones studied during the feeding trial: the forefront between the eyes, the opercule, along the dorsal fin and in the abdominal area. In the muscle, regardless of the astaxanthin source, the amount of carotenoids measured was very low (less than 1 mg kg^?1) and differences not significant. Moreover, no muscle pigmentation was evident, and there was no variation in the amount of carotenoid analysed in skin tissue, through the trial, for each treatment. It was concluded that supplementing the feed with C. vulgaris would be an acceptable practice in aquaculture to improve the market appeal of the gilthead seabream.     

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.     

Development of a reference diet for use in indispensable amino acid requirement studies of gilthead seabream Sparus aurata L.

The development of a test diet supporting good fish growth to be used in gilthead seabream indispensable amino acid (IAA) requirement studies based on dose–response relationship was the objective of the present study. Four isonitrogenous diets, one serving as control (diet C) based on fish meal LT, and three others in which the fish meal component was reduced to 45% and the rest of the nitrogen was provided by mixtures of crystalline AA were formulated. Diet S resembled the whole body IAA profile of wild seabream. Diet D was made deficient in all IAA and a 10% excess of IAA compared with diet S was used in diet E. Triplicate groups of fish were fed the diets to satiation for 6 weeks. Growth parameters and nutrient utilization efficiencies among diets C and S were similar, while excessive supplementation of IAA negatively affected most of them, compared with the control. Whole body composition was differentiated in relation to the diet fed. No significant differences were observed among groups fed the control and diet S. As shown by the results, the presence of crystalline AA in diet S does not significantly affect growth, and could therefore serve as control diet in IAA requirement studies of gilthead seabream.     

Energy and protein requirements for maintenance and growth in gilthead seabream (Sparus aurata L.)

Factorial determinations of energy and protein requirements in growing Sparus aurata were carried out at 23–24°C. The energy content in the whole fish was dependent on fish weight and ranged from 5 to 11 MJ kg⁻¹ body mass for 1–250 g fish, whereas the protein content remained constant at 179 g kg⁻¹.
During starvation the fish lost 42.5 kJ body weight (BW) (kg)^−0.83 day⁻¹ and 0.42 g protein BW (kg)^−0.70 day⁻¹. The maintenance requirement for energy was calculated to be 55.8 kJ BW (kg)^−0.83 day⁻¹ and for protein 0.86 g BW (kg)^−0.70 day⁻¹. Utilization of digestible energy and digestible crude protein below and at maintenance was determined as 0.72 and 0.51, respectively. Utilization of digestible energy and digestible crude protein for growth above maintenance was determined as 0.46 and 0.28, respectively.
These values allow estimation of requirements for growing Sparus aurata .     

Interference of Listonella anguillarum with potential probiotic microorganisms isolated from farmed gilthead seabream (Sparus aurata, L.)

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 10⁸ CFU g^?1 of this isolate for 15 days. An other group of similar characteristics received the non‐supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non‐supplemented commercial diet.     

Alternative formulations for gilthead seabream diets: Towards a more sustainable production

To support the expected increase in aquaculture production during the next years, a wider range of alternative ingredients to fishmeal is needed, towards contributing to an increase in production sustainability. This study aimed to test diets formulated with non‐conventional feed ingredients on gilthead seabream (Sparus aurata) growth performance, feed utilization, apparent digestibility of nutrients and nutrient outputs to the environment. Four isonitrogenous and isoenergetic diets were formulated: a control diet (CTRL) similar to a commercial feed and three experimental diets containing, as main protein sources, plant by‐products, glutens and concentrates (PLANT); processed animal proteins (PAP); or micro/macroalgae, insect meals and yeast (EMERG). Diets were tested in triplicate during 80 days. The EMERG treatment resulted in lower fish growth performance, higher FCR and lower nutrient and energy retentions than the other treatments. The lowest protein digestibility was found for the EMERG diet, which caused increased nitrogen losses. The PLANT and PAP treatments resulted in better fish growth performance, higher nutrient and energy retentions, and lower FCR than the CTRL treatment. The significant improvement in FCR found for fish fed PLANT and PAP diets and the high protein digestibility of these diets contribute towards minimizing the environmental impacts of seabream production.     

Enhancement of gilthead seabream (Sparus aurata) larval growth by dietary vitamin E in relation to two different levels of essential fatty acids

The objective of this study was to determine the effect of dietary vitamin E on gilthead seabream (Sparus aurata) growth and survival, at two different highly unsaturated fatty acids (HUFAs) levels. Eighteen days old gilthead seabream larvae were fed four formulated experimental diets combining two different dietary levels of HUFAs (M: medium 2.5 + 1.5, DHA + EPA, H: high 5 + 2.5 DHA + EPA g per 100 g) with two different levels of vitamin E (M: medium 540 mg kg^?1, H: high 2900 mg kg^?1): MM, MH, HM, HH (HUFA/vitamin E). After 2‐week feeding trial, the average survival rate was 52.6% and there were no significant differences found among treatments. Increase in vitamin E up to high level markedly improved larval growth, particularly when dietary HUFA levels were lower, suggesting a higher protection value when these fatty acids are more limiting. At medium dietary HUFA levels, increase in vitamin E from medium to high level enhanced larval growth performance in terms of total length. Moreover, increase in vit E enhanced HUFAs content in the larval polar lipids denoting the anti‐oxidative effect of vitamin E.     

Debaryomyces hansenii L2‐enriched diet enhances the immunity status,gene expression and intestine functionality in gilthead seabream (Sparus aurata L.)

This study evaluates the effects of the dietary administration of the live yeast Debaryomyces hansenii strain L2 on the immune responses of gilthead seabream for 4 weeks. Cellular immune parameters were measured from serum and head‐kidney leucocytes respectively. The expression levels of immune‐associated genes were quantified by real‐time polymerase chain reaction. In addition, the profile of intestinal microbiota was studied by denaturing gradient gel electrophoresis. The results showed that seabream fed a diet containing D. hansenii had significantly increased cellular immune parameters. The yeast‐supplemented diet up‐regulated the expression of most seabream genes at week 2 and down‐regulated all of them at week 4, except in the head‐kidney. Finally, a reduction in the diversity of the intestinal microbiota was detected in those specimens receiving the yeast‐supplemented diet. These results support the idea that the live yeast D. hansenii strain L2 stimulates the immune system of gilthead seabream.     

Comparison of the swimming performance of farmed and wild gilthead sea bream, Sparus aurata

Farmed gilthead sea bream, Sparus aurata, frequently escape from the sea cages and interact with wild populations. The impact of these interactions on the wild populations will depend, in part, on differences in performance of the bream. This study compared the swimming performance of the wild and farmed fish in a current channel. The absolute critical swimming speed (U_crit) increased with increasing size while the relative U_crit decreased. Even at the same length there were noticeable performance differences between the individuals. The wild sea bream have significantly higher (P<0.05) absolute U_crit performance (0.86±0.01 m s⁻¹) than the farmed fish (0.79±0.01 m s⁻¹) and significantly higher (P<0.05) relative U_crit performance (4.52±0.05 BL s⁻¹) than the farmed fish (4.21±0.05 BL s⁻¹). The present study suggests that cultured sea bream may not have the ability to compete with wild sea bream in native seawaters.     

Influence of lupin and rapeseed meals on the integrity of digestive tract and organs in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles

Two rearing trials were conducted to evaluate the effects of dietary lupin (LM) and rapeseed (RM) meals in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles. Each plant meal was incorporated at the rate of 200 g kg^?1 in two distinct diets, which were compared with a fishmeal‐based diet as control. After 1 month, the two plant diets did not influence the whole body growth, but the digestive systems were affected. The splenosomatic index was reduced with two plant meals in goldfish and with RM in seabream. The hepatosomatic index was only reduced in LM‐fed seabream. Cellular characteristics were also affected. The largest liver cells were observed in RM‐fed goldfish suggesting changes in metabolic function. The LM and RM diets stimulated in seabream, especially the reaction in haematopoietic tissues with the proliferation of melano‐macrophages centres, and a tendency for elongated villus height in the anterior intestine thus that possibly compensated for a reduction in digestive function. Such adaptive structural modifications and the absence of degenerative signs allowed concluding that the integrity of the digestive system was maintained in fish fed plant meals.     

Effect of diet composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparus aurata L.)

An experiment was conducted to evaluate the effects of dietary composition on apparent digestibility coefficients of protein, fat and carbohydrate of gilthead sea bream. In addition α‐amylase and proteolytic activities were measured in the digestive tract of fish, held at 20±1°C. Six experimental diets were formulated containing approximately 40%, 45% and 50% protein, 11% and 21% fat and a starch level which fluctuated from 14% to 36%. Fish having an average weight of 100–130 g were used, fed at 1.5% of their body weight daily, and digestibility was measured using an indirect method. Enzyme activities were measured in the digestive tract of fish, fed diets containing a combination of 40%, 50% protein with 11%, 21% fat at 0.5, 5, 10, 24 h after a single meal. Starch digestibility was reduced with its level in the diet. It also negatively affected fat digestibility as well as protein digestibility, the last only at the high fat level. Dietary fat level had a negative effect on starch and protein digestibility. Fat affected also strongly α‐amylase levels in the digestive tract, while its effect on protease activity was of smaller magnitude. These results indicate that digestive enzyme activities and nutrient digestibility values are affected by dietary composition, carbohydrates and fat indicating the strongest effect.     

Mono- and duoculture of juvenile sharpsnout seabream Diplodus puntazzo (Cetti) and gilthead seabream Sparus aurata L. in a recirculated water system

Although the growth of intensively reared sharpsnout seabream Diplodus puntazzo (DP) has been suggested to improve in duoculture with gilthead seabream Sparus aurata (SA), the stocking ratio for most effective duoculture rearing has not been investigated. For this purpose, juvenile D. puntazzo (1.68±0.011 g) and S. aurata (1.43±0.007 g) were reared in duplicated groups of 165 specimens each, in two monoculture (100% DP, 100% SA) and four duoculture combinations (80% DP–20% SA, 60% DP–40% SA, 40% DP–60% SA, 20% DP–80% SA) for 96 days under recirculated water system conditions. Both species exhibited the highest growth when stocked at 20% and the poorest growth when stocked at 80%. For each species, no major differences for coefficient of weight variation and carcass proximate composition were detected. As the percentage of stocked S. aurata increased, D. puntazzo aggressiveness and weight variability of whole population were reduced, while total biomass, food consumption and food conversion ratio were increased. The results obtained are discussed in relation to fish behaviour and social interactions. From the fish farming point of view, it is indicated that under the present experimental conditions, juvenile S. aurata should be reared in monoculture and D. puntazzo in duoculture 40% DP–60% SA.     

Tolerance response to water pH in larvae of two marine fish species, gilthead seabream, Sparus aurata (L.) and Senegal sole, Solea senegalensis (Kaup), during development

Gilthead seabream, Sparus aurata (L.) and Senegal sole, Solea senegalensis (Kaup) are two fish of primary importance in Mediterranean aquaculture. In the present study, the larvae of these species were exposed to different rearing‐water pH during 24 h to examine their tolerance. The 24‐h pHL50 values were calculated with low and high pH values at 7, 20 and 32 days after hatching (DAH) in S. senegalensis larvae, and 12, 20 and 52 DAH in S. aurata larvae. Low 24‐h pHL50 values ranged between 4.88 and 5.76, whereas high 24‐h pHL50 values ranged between 8.94 and 9.57 in S. senegalensis larvae. S. aurata larvae showed values of low 24‐h pHL50 that ranged between 4.82 and 5.55, whereas values of high 24‐h pHL50 ranged between 8.66 and 9.26. Both species showed similar tolerance response at all the tested ages. The high 24‐h pHL50 values found were close to pH values that eventually can be reached in the rearing tanks. The pH should be carefully controlled in rearing water during the first development stages of both species.