The Digestive Function of Pseudoplatystoma punctifer Early Juveniles Is Differentially Modulated by Dietary Protein, Lipid and Carbohydrate Content and Their Ratios.

Pseudoplatystoma punctifer is an Amazonian catfish highly appreciated for its high flesh quality, size, and commercial value. Its aquaculture is pursued to satisfy the demands of an increasing population in the region. However, knowledge of the nutritional needs during the early life stages is necessary for improving growth and reducing the incidence of cannibalism, factors that limit the success of its commercial farming. This study aimed at evaluating the influence of four diets containing different protein and lipid levels (30:15, 30:10, 45:15, or 45:10 in %) in the digestive physiology and performance of early juveniles. The results showed that the dietary protein:lipid as well as carbohydrate levels and ratios influenced differently the whole-body proximate composition, the digestive physiology and development, and hence growth and survival. The 45:15 diet promoted the best growth, survival, and the most rapid development of the digestive system, as shown at histological (higher number of hepatocytes, goblet cells in the anterior intestine and enterocytes in all intestinal portions, and longer folds in the posterior intestine), molecular (highest amylase, lipoprotein lipase, phospholipase, trypsinogen, and pepsinogen gene expression), and biochemical (highest lipase and pepsin activities and higher alkaline phosphatase:leucine alanine peptidase activity ratio) levels. Lipids were favored over carbohydrates as source of energy, with lipids promoting a protein-sparing effect at adequate energy:protein ratio. Carbohydrate content higher than 25% was excessive for this species, leading to unbalanced lipid metabolism and fat deposition in the liver.

An early life hypoxia event has a long-term impact on protein digestion and growth in juvenile European sea bass.

Ocean warming, eutrophication and the consequent decrease in oxygen lead to smaller average fish size. Although such responses are well known in an evolutionary context, involving multiple generations, this appears to be incompatible with current rapid environmental change. Instead, phenotypic plasticity could provide a means for marine fish to cope with rapid environmental changes. However, little is known about the mechanisms underlying plastic responses to environmental conditions that favour small phenotypes. Our aim was to investigate how and why European sea bass that had experienced a short episode of moderate hypoxia during their larval stage subsequently exhibited a growth depression at the juvenile stage compared with the control group. We examined whether energy was used to cover higher costs for maintenance, digestion or activity metabolisms, as a result of differing metabolic rate. The lower growth was not a consequence of lower food intake. We measured several respirometry parameters and we only found a higher specific dynamic action (SDA) duration and lower SDA amplitude in a fish phenotype with lower growth; this phenotype was also associated with a lower protein digestive capacity in the intestine. Our results contribute to the understanding of the observed decrease in growth in response to climate change. They demonstrate that the reduced growth of juvenile fishes as a consequence of an early life hypoxia event was not due to a change of fish aerobic scope but to a specific change in the efficiency of protein digestive functions. The question remains of whether this effect is epigenetic and could be reversible in the offspring.

Does the chronic chemical contamination of a European flounder population decrease its thermal tolerance?

Juvenile flounders (Platichthys flesus), collected in two estuaries with similar temperature regimes (the heavily polluted Seine and the moderately contaminated Vilaine), were submitted to a common garden experiment. After an acclimation period, both populations were challenged by a thermal stress (9-24°C for 15days, then maintenance at 24°C for 19days). The condition factor of the Vilaine fish increased in both conditions, while it decreased for the heated Seine flounders after 34days. The expression of genes related to the energetic metabolism was measured in the liver. The expression levels for ATP-F0 and COII were significantly reduced for heated vs. standard fish from both estuaries, while a decrease of the 12S expression was detected only in heated vs. standard fish from the Seine estuary. Thus, it is suggested that highly contaminated fish from Seine could display a lower tolerance to thermal stress, compared to moderately contaminated fish from Vilaine.

Characteristics of fads2 gene expression and putative promoter in European sea bass (Dicentrarchus labrax): comparison with salmonid species and analysis of CpG methylation.

Marine fish species exhibit low capacity to biosynthesise highly unsaturated fatty acid (HUFA) in comparison to strict freshwater and anadromous species. It is admitted that the Delta(6) desaturase (FADS2) is a key enzyme in the HUFA biosynthetic pathway. We investigated by quantitative PCR the relative amounts of FADS2 mRNA in European sea bass (Dicentrarchus labrax) in comparison with a salmonid species, the rainbow trout (Oncorhynchus mykiss L.). The analysis of the expression data was performed regarding the difference of the characteristics of a critical fragment of the fads2 gene promoter between sea bass and Atlantic salmon. The lower level of fads2 gene expression observed in sea bass suggested that fads2 gene putative promoter, which exhibited an E-box like Sterol Regulatory Element (SRE) site but lacked a Sp1 site, is less active in this marine species. The cytosine methylation of CpG sites in the putative promoter region including E-box like SRE and NF-Y binding sites of sea bass fads2 gene was also investigated following a nutritional conditioning of larvae. However, no significant difference of CpG methylation could be found for any of the 28 CpGs analysed between larvae fed diet with high or low HUFA contents. In conclusion, the present data revealed lower constitutive expression of the fads2 gene possibly related to different characteristics of gene promoter in sea bass in comparison with salmonid species, and indicated that long-term conditioning of fads2 gene expression did not influence the methylation of the gene promoter at potential SRE binding site.

Effects of the total replacement of fish-based diet with plant-based diet on the hepatic transcriptome of two European sea bass (Dicentrarchus labrax) half-sibfamilies showing different growth rates with the plant-based diet.

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.

Imbalanced dietary ascorbic acid alters molecular pathways involved in skeletogenesis of developing European sea bass (Dicentrarchus labrax).

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 (VDRß, 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.

Cloning, tissue expression analysis, and functional characterization of two Δ6-desaturase variants of sea bass (Dicentrarchus labrax L.).

Fish are the main source of the n-3 highly unsaturated fatty acids, which are crucial for human health. Their synthesis from C(18) precursors is mediated by desaturases and elongases, but the activity of these enzymes has not been conclusively established in marine fish species. This study reports the cloning, tissue expression, and functional characterization of a sea bass (Dicentrarchus labrax L.) Δ6-desaturase and one of its splicing variants. Two cDNAs with open reading frames of 1,346 and 1,354 bp were cloned and named D6D and D6D-V, respectively. Both deduced protein sequences (445 and 387 amino acids, respectively) contained two transmembrane regions and the N-terminal cytochrome b(5) domain with the HPGG motif characteristic of microsomal desaturases. D6D presents three histidine-rich regions, whereas in D6D-V, an insertion of eight nucleotides in the boundaries of exons 10 and 11 modified the third histidine-rich domain and led to insertion of a premature STOP codon, resulting in a shorter predicted protein. Quantitative real-time polymerase chain reaction assay of gene expression showed that D6D was highly expressed in the brain and intestine, and to a lesser extent, in muscle and liver; meanwhile, D6D-V was expressed in all tissues tested, but at level at least 200-fold lower than D6D. Functional analysis in yeast showed that sea bass D6D encodes a fully functional Δ6-desaturase with no residual Δ5-desaturase activity. This desaturase does not exhibit a clear preference for n-3 versus n-6 C(18) substrates. Interestingly, D6D-V is a nonfunctional protein, suggesting that the C-terminal end is indispensable for protein activity.

Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids.

A comparative proteomic approach was used to assess the protein expression profile in the liver of 34days old pikeperch larvae fed from day 10 post hatching, with three isoproteic and isolipidic formulated diets varying by their phospholipid (PL) contents (% dry diet weight): 1.4% (PL1), 4.7% (PL5) and 9.5% (PL9). Using 2D-DIGE minimal labelling of liver extracts, we were able to show 56 protein spots with a differential intensity (p<0.05) depending on the dietary PL content. Among these spots, 11 proteins were unambiguously identified using nanoLC-MS/MS tandem mass spectrometry. In the PL9 larvae, our results indicate that the glycolytic pathway could be down-regulated due to the under-expression of the fructose biphosphate aldolase B and the phosphoglucomutase 1. Meanwhile, propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was under-expressed. In addition, another gluconeogenic and lipogenic enzyme, pyruvate carboxylase, was identified in 3 different spots as being under-expressed in fish fed with the intermediate PL level (PL5). A high PL content increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. Moreover, several stress proteins (glutathione S-transferase M, glucose regulated protein 75 and peroxiredoxin-1) were modulated in response to the dietary PL level and fatty acid composition. In the larvae fed with the lowest dietary PL content (PL1), over-expression of both GSTM and GRP75 might indicate a cellular stress in this experimental treatment, while the under-expression of Prx1 might indicate a lower defence against oxidative stress. In conclusion, this nutriproteomic approach showed significant modifications of protein expression in the liver of pikeperch larvae fed different PL contents, highlighting the importance of these nutrients and their influence on metabolism processes and on stress response.

Intake of high levels of vitamin A and polyunsaturated fatty acids during different developmental periods modifies the expression of morphogenesis genes in European sea bass (Dicentrarchus labrax).

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.

Effect of nature of dietary lipids on European sea bass morphogenesis: implication of retinoid receptors.

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.

Dietary levels of all-trans retinol affect retinoid nuclear receptor expression and skeletal development in European sea bass larvae.

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.

Effect of dietary phospholipid level and phospholipid:neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet.

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.