Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet.

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.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

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.

Dietary phospholipids are more efficient than neutral lipids for long-chain polyunsaturated fatty acid supply in European sea bass Dicentrarchus labrax larval 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.

Ontogeny of the gastrointestinal tract of marine fish larvae.

Marine fish larvae undergo major morphological and cellular changes during the first month of life. The ontogeny of the gastrointestinal tract combines these two aspects of the larval development and is very interesting in that the timing of functional changes appears genetically hard-wired. The goal of this paper is to give an overview of the gastrointestinal development process in marine fish larvae, with particular attention to three species: sea bass; red drum; and sole, since the description of gut maturation in fish larvae was initiated during the last decade with these species. During the early stages, marine fish larvae exhibit particular digestive features. Concerning the exocrine pancreas, amylase expression decreases with age from the third week post-hatching in sea bass and red drum (approximately 400 degree days), whereas expression of other enzymes (trypsin, lipase, phospholipase A2...) increases until the end of the larva period. Moreover, secretory function of the exocrine pancreas progressively develops and becomes efficient after the third week of life. Concerning the intestine, enzymes of the enterocyte cytosol (in particular peptidase) have higher activity in young larvae than in older. Approximately in the fourth week of post-hatching development in sea bass, red drum and sole larvae, the cytosolic activities dramatically decline concurrently with a sharp increase in membranous enzyme activities of the brush border, such as alkaline phosphatase, aminopeptidase N, maltase. This process characterises the normal maturation of enterocytes in developing fish larvae and also in other vertebrates' species. The establishment of an efficient brush border membrane digestion represents the adult mode of digestion of enterocytes. This paper also describes the role of diet on the development of the gastrointestinal tract. Indeed, the maturational process of digestive enzyme can be enhanced, stopped, or delayed depending on the composition of the diet.

High dietary lipid levels enhance digestive tract maturation and improve dicentrarchus labrax larval development.

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.

Partial substitution of di- and tripeptides for native proteins in sea bass diet improves Dicentrarchus labrax larval development.

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.

Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae.

Sea bass (Dicentrarchus labrax) larvae were fed from day 15 to day 35 with 4 isoenergetic formulated diets that varied in protein (30, 40, 50, 60%) and carbohydrate (37, 27, 17, 7%) content. The diets were designated as P30, P40, P50 and P60, respectively. Best growth and survival were noted for P50 larvae. Poor growth and survival were observed for P30 larvae. The specific activity of amylase increased in direct relation to the dietary carbohydrate level from day 18 onwards. This increase was the result of extensive amylase synthesis. Trypsin activity was positively correlated with dietary protein level only at day 35. This study shows that the mechanisms involved in amylase regulation are efficient in very young sea bass larvae, while those related to trypsin appear later in ontogeny and thus suggests an age-dependent regulation of enzyme synthesis.

Effect of the molecular form of dietary nitrogen supply in sea bass larvae: Response of pancreatic enzymes and intestinal peptidases.

Sea bass (Dicentrarchus labrax) larvae were weaned at day 25 with microparticulated diets in which 10% of the nitrogen supply had different molecular forms: amino acid mixture (SLAA), casein hydrolysate (SLH) or fish meal (SLP). The control group (LP) was fed live prey. No difference was observed in larval growth between the weaned groups, but the survival was significantly higher in the SLH group. Trypsin secretion was stimulated in the SLAA group, whereas the SLH diet reduced the secretion from the exocrine pancreas. The activity of the leucine-alanine peptidase, located in the cytosol of enterocytes, remained high in all weaned groups. However, the activity of the peptidases of the brush border membrane increased during the development phase in the control group. These results suggest that weaning with a classic compound diet delays enterocyte differentiation by maintaining the larval features of digestion. A compound diet containing protein hydrolysate can attenuate the delay of intestinal maturation.

Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus labrax): effect of weaning with different protein sources.

The maturation of the digestive functions in sea bass (Dicentrarchus labrax) larvae was evaluated by the enzymatic profile of pancreas and intestine brush border membranes. Sea bass larvae were weaned at day 25 with three simplified diets different by their protein nature: 100% fish meal (FP), 100% casein mixture (CP) and 50% fish meal-50% casein mixture (CFP). The casein mixture contained 35% of hydrolysate. The control group was fed live preys. The specific activity of amylase decreased with age irrespectively of the diets whereas the specific activity of trypsin was enhanced. The casein mixture reduced pancreatic secretion in amylase and trypsin. The CFP group differed from the other groups fed on compound diets, exhibiting as soon as day 32 high activities of brush border enzymes, similar to controls. This sharp increase between day 25 and 32 appeared to be crucial for larval survival. The addition of a protein hydrolysate in a weaning diet seems to facilitate this maturation process.