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Background: Efforts towards utilisation of diets without fish meal (FM) or fish oil (FO) in finfish aquaculture have been being made for more than two decades. Metabolic responses to substitution of fishery products have been shown to impact growth performance and immune system of fish as well as their subsequent nutritional value, particularly in marine fish species, which exhibit low capacity for biosynthesis of long-chain poly-unsaturated fatty acids (LC-PUFA). The main objective of the present study was to analyse the effects of a plant-based diet on the hepatic transcriptome of European sea bass (Dicentrarchus labrax)., Results: We report the first results obtained using a transcriptomic approach on the liver of two half-sibfamilies of the European sea bass that exhibit similar growth rates when fed a fish-based diet (FD), but significantly different growth rates when fed an all-plant diet (VD). Overall gene expression was analysed using oligo DNA microarrays (GPL9663). Statistical analysis identified 582 unique annotated genes differentially expressed between groups of fish fed the two diets, 199 genes regulated by genetic factors, and 72 genes that exhibited diet-family interactions. The expression of several genes involved in the LC-PUFA and cholesterol biosynthetic pathways was found to be up-regulated in fish fed VD, suggesting a stimulation of the lipogenic pathways. No significant diet-family interaction for the regulation of LC-PUFA biosynthesis pathways could be detected by microarray analysis. This result was in agreement with LC-PUFA profiles, which were found to be similar in the flesh of the two half-sibfamilies. In addition, the combination of our transcriptomic data with an analysis of plasmatic immune parameters revealed a stimulation of complement activity associated with an immunodeficiency in the fish fed VD, and different inflammatory status between the two half-sibfamilies. Biological processes related to protein catabolism, amino acid transaminations, RNA splicing and blood coagulation were also found to be regulated by diet, while the expression of genes involved in protein and ATP synthesis differed between the half-sibfamilies., Conclusions: Overall, the combined gene expression, compositional and biochemical studies demonstrated a large panel of metabolic and physiological effects induced by total substitution of both FM and FO in the diets of European sea bass and revealed physiological characteristics associated with the two half-sibfamilies.

The influence of dietary ascorbic acid (AA) on growth and morphogenesis during the larval development of European sea bass (Dicentrarchus labrax) was evaluated until 45days post hatching. Diets incorporated 0, 5, 15, 30, 50 or 400mg AA per kg diet to give AA-0, AA-5, AA-15, AA-30, AA-50 and AA-400 dietary treatments, respectively. Dietary AA levels lower than 15mg/kg reduced larval growth and survival was affected in specimens fed diets devoid of AA. Globally, disruption of the expression of genes involved in AA and calcium absorption in the intestine (SVCT-1, TRPV-6), skeletogenesis (BMP-4, IGF-1, RARγ) and bone mineralization (VDRβ, osteocalcin) were observed in groups fed doses lower and higher than 50mg AA/kg diet. Such disturbances detected at molecular level were associated with disruptions of the ossification process and the appearance of skeletal abnormalities., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Supplies of marine fish oils are limited, and continued growth in aquaculture production dictates that lipid substitutes in fish diets must be used without compromising fish health and product quality. In this study, the total substitution of a fish meal and fish oil by a blend of vegetable meals (corn, soybean, wheat and lupin) and linseed oil in the diet of European sea bass (Dicentrachus labrax) was investigated. Two groups of European sea bass were fed with fish diet (FD) or vegetable diet (VD) for 9months. VD, totally deprived of eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), revealed a nutritional deficiency and affected growth performance. Whilst VD induced a significant increase in fatty acid desaturase 2 (FADS2) and sterol binding regulatory element-binding protein 1 (SREBP-1) mRNA levels, the desaturation rate of [1-(14)C]18:3n-3 into [1-(14)C]18:4n-3, analysed in microsomal preparations using HPLC method, did not show an upregulation of FADS2 activities in liver and intestine of fish fed VD. Moreover Western-blot analysis did not revealed any significant difference of FADS2 protein amount between the two dietary groups. These data demonstrate that sea bass exhibits a desaturase (FADS2) activity whatever their diet, but a post-transcriptional regulation of fads2 RNA prevents an increase of enzyme in fish fed a HUFA-free diet. This led to a lower fish growth and poor muscle HUFA content., (2010 Elsevier Inc. All rights reserved.)

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

The effect of the feeding period on larval development was investigated in European sea bass larvae by considering the expression level of some genes involved in morphogenesis. Larvae were fed a control diet except during three different periods (period A: from 8 to 13 d post-hatching (dph); period B: from 13 to 18 dph; period C: from 18 to 23 dph) with two compound diets containing high levels of vitamin A or PUFA. European sea bass morphogenesis was affected by these two dietary nutrients during the early stages of development. The genes involved in morphogenesis could be modulated between 8 and 13 dph, and our results indicated that retinoids and fatty acids influenced two different molecular pathways that in turn implicated two different gene cascades, resulting in two different kinds of malformation. Hypervitaminosis A delayed development, reducing the number of vertebral segments and disturbing bone formation in the cephalic region. These malformations were correlated to an upregulation of retinoic acid receptor gamma, retinoid X receptor (RXR) alpha and bone morphogenetic protein (BMP)4. An excess of PUFA accelerated the osteoblast differentiation process through the upregulation of RXRalpha and BMP4, leading to a supernumerary vertebra. These results suggest that the composition of diets devoted to marine fish larvae has a particularly determining effect before 13 dph on the subsequent development of larvae and juvenile fish.

The effect of the nature and form of supply of dietary lipids on larval development was investigated in European sea bass larvae, by considering the expression of several genes involved in morphogenesis. Fish were fed from 7 to 37 d post-hatch with five isoproteic and isolipidic compound diets incorporating different levels of EPA and DHA provided by phospholipid or neutral lipid. Phospholipid fraction containing 1.1 % (PL1 diet) to 2.3 % (PL3 diet) of EPA and DHA sustained good larval growth and survival, with low vertebral and cephalic deformities. Similar levels of EPA and DHA provided by the neutral lipid fraction were teratogenic and lethal. Nevertheless, dietary phospholipids containing high levels of DHA and EPA (PL5 diet) induced cephalic (8.5 %) and vertebral column deformities (35.3 %) adversely affecting fish growth and survival; moreover, a down-regulation of retinoid X receptor alpha (RXRalpha), retinoic acid receptor alpha, retinoic acid receptor gamma and bone morphogenetic protein-4 genes was also noted in PL5 dietary group at day 16. High levels of dietary PUFA in neutral lipid (NL3 diet) first up-regulated the expression of RXRalpha at day 16 and then down-regulated most of the studied genes at day 23, leading to skeletal abnormalities and death of the larvae. A moderate level of PUFA in neutral lipids up-regulated genes only at day 16, inducing a lesser negative effect on growth, survival and malformation rate than the NL3 group. These results showed that retinoid pathways can be influenced by dietary lipids leading to skeletal malformation during sea bass larvae development.

We evaluated the effects of dietary lipid class (phospholipid vs. neutral lipid) and level of n-3 long-chain PUFA (LC-PUFA) on the growth, digestive enzymatic activity, and histological organization of the intestine and liver in European sea bass larvae. Fish were fed from the onset of exogenous feeding at 7 to 37 d post-hatch with five isoproteic and isolipidic compound diets with different levels of EPA and DHA. Diet names indicated the percentage of EPA and DHA contained in the phospholipids (PL) and neutral lipids (NL), as follows: PL5, PL3, PL1, NL1, and NL3. Histological observations showed different patterns of lipid absorption and accumulation in the intestinal mucosa depending on the level and nature of the dietary lipid fraction. Fish fed high levels of neutral lipids (11%, NL3 diet: 2.6% of EPA + DHA in the NL fraction) showed large intracellular and intercellular lipid deposits in the anterior intestine, but no such lipid accumulation was detected when larvae were fed with low and moderate levels of EPA and DHA in the phospholipid and neutral lipid fractions of the diet (PL and NL1 diets). PL were preferentially absorbed in the postvalvular intestine, and the accumulation of marine PL was inversely correlated to their dietary level. The postvalvular intestinal mucosa and liver showed signs of steatosis; large lipid vacuoles were observed in this region of the intestine and in the liver and were inversely correlated with the level of dietary neutral lipids. The best results in terms of growth, survival, and development (maturation of the digestive system and histological organization of the liver and intestinal mucosa) were obtained in the group fed with 2.3% of EPA and DHA in the PL fraction of the diet (PL3 diet), revealing that European sea bass larvae use the LC-PUFA contained in the PL fraction more efficiently than those from the NL fraction of the diet.

European sea bass larvae were fed different dietary vitamin A levels. Growth, skeletal development and the expression of genes involved in larval morphogenesis were evaluated. From 7 to 42 d post-hatching, larvae were fed five isoproteic and isolipidic compound diets with graded levels of retinyl acetate (RA; RA0, RA10, RA50, RA250 and RA1000, containing 0, 10, 50, 250 and 1000 mg RA/kg DM, respectively), resulting in an incorporation of 12, 13, 31, 62 and 196 mg all-trans retinol/kg DM. Larvae fed extreme levels of RA had weights 19 % and 27 % lower than those of the RA50 group. The RA1000 diet induced a fall in growth with an increase of circulating and storage retinol forms in larvae, revealing hypervitaminosis. High levels of RA affected maturation of the pancreas and intestine. These data indicated that the optimal RA level was close to 31 mg/kg DM. Inappropriate levels of dietary RA resulted in an alteration of head organisation characterised by the abnormal development of the splanchnocranium and neurocranium, and scoliotic fish. Of the larvae fed RA1000, 78.8 % exhibited skeletal abnormalities, whereas the RA50 group presented with 25 % malformations. A linear correlation between vitamin A level and malformation percentage was observed and mainly associated with an upregulation of retinoic acid receptor-gamma expression in the RA1000 group during the 2 first weeks after hatching. The expression of retinoid X receptor-alpha decreased during normal larval development when that of the retinoic acid receptors increased. This work highlights the involvement of retinoid pathways in the appearance of dietary-induced skeletal malformations during post-hatching development in sea bass.

The aim of the study was to determine the influence of dietary phospholipid concentration on survival and development in sea bass (Dicentrarchus labrax) larvae. Larvae were fed from day 9 to day 40 post-hatch with an isoproteic and isolipidic formulated diet with graded phospholipid levels from 27 to 116 g/kg DM and different phospholipid:neutral lipid values. The best growth (32 mg at the end of the experiment) survival (73 %) and larval quality (only 2 % of malformed larvae) were obtained in the larvae fed the diet containing 116 g phospholipid/kg DM (P<0.05). These results were related to the amount of phosphatidylcholine and phosphatidylinositol included in this diet (35 and 16 g/kg respectively). Amylase, alkaline phosphatase and aminopeptidase N activities revealed a proper maturation of the digestive tract in the two groups fed the highest phospholipid levels. Regulation of lipase and phospholipase A2 by the relative amount of their substrate in the diet occurred mainly at the transcriptional level. The response of pancreatic lipase to dietary neutral lipid was not linear. As in mammals 200 g triacylglycerol/kg diet seems to represent a threshold level above which the response of pancreatic lipase is maximal. The response of phospholipase A2 to dietary phospholipid content was gradual and showed a great modulation range in expression. Sea bass larvae have more efficient capacity to utilize dietary phospholipid than neutral lipids. For the first time a compound diet sustaining good growth, survival and skeletal development has been formulated and can be used in total replacement of live prey in the feeding sequence of marine fish larvae.

This study was designed to determine the nutritional lipid requirement of seabass larvae and to understand the effects of dietary fat concentration on their digestive tract maturation. Seabass (Dicentrarchus labrax) larvae were fed, from d 15 to 38 of life, one of five isonitrogenous compound diets with different lipid levels, ranging from 10 to 30 g/100 g. The higher the lipid level, the greater the growth and survival of the larvae (P < 0.05). The lipolytic enzymes assayed, lipase and phospholipase A2, were stimulated by the increase in their respective dietary substrates, triglycerides and phospholipids, in 38-d-old larvae (P < 0.05). Nevertheless, a plateau in the activity of these two lipolytic enzymes was observed from 20% dietary lipids onwards. The similar mRNA levels of phospholipase A2 in the three groups fed the highest lipid levels suggested that the maximal synthesis level of lipolytic enzyme was reached at 20% dietary fat. Pancreatic secretion of trypsin and amylase were positively affected by the dietary lipid level; a possible involvement of a cholecystokinin-releasing factor is discussed. Diets containing >20% lipids led to the increase in activities of brush border membrane enzymes to the detriment of a cytosolic enzyme in enterocytes, leucine-alanine (Leu-Ala) peptidase. This enzymatic change reveals the earlier maturation of enterocytes in larva groups fed high lipid levels.

To determine whether incorporation of peptides into diets can improve larval development, sea bass (Dicentrarchus labrax) larvae were fed for 21 d one of three isonitrogenous, isoenergetic semipurified diets in which enzymatic hydrolysate (75% di- and tripeptides) of fish meal proteins was substituted for 0, 20 or 40% of native fish meal proteins. Growth and survival were significantly greater (P < 0.05) in larvae fed peptide diets compared to those fed only native protein, with the best performance exhibited by those fed the 20% level of peptides. Chymotrypsin activity was much higher in groups fed peptide diets compared to that fed all native protein (P < 0.001), indicating a greater proteolytic capacity of the pancreas. At the intestinal level, activities of the brush border enzymes, aminopeptidase, maltase and gamma-glutamyl transpeptidase, increased with age while the cytosolic enzyme, leu-ala peptidase, decreased with age (P < 0.001). These changes in enzymatic activities correspond to the normal development of intestinal digestion. This development occurred earlier in the group fed 20% peptide-substituted diet than in the two other groups. The better larval performances observed in groups fed diets containing peptides can be related to the enhanced proteolytic capacity of the pancreas and the earlier development of intestinal digestion.