José L. Zambonino-Infante

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 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.

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.

BackgroundEfforts 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).ResultsWe 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.ConclusionsOverall, 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.

Ontogenic effects of early feeding of sea bass (Dicentrarchus labrax) larvae with a range of dietary n-3 highly unsaturated fatty acid levels on the functioning of polyunsaturated fatty acid desaturation pathways.

Four replicated groups of sea bass (Dicentrarchus labrax) larvae were fed diets containing an extra-high level of highly unsaturated fatty acids (HUFA) (XH; 3.7 % EPA+DHA), a high level of HUFA (HH; 1.7 %), a low level of HUFA (LH; 0.7 %) or an extra-low level of HUFA (XLH; 0.5 %) from day 6 to day 45 (experiment 1; XH1, HH1, LH1, XLH1). After a subsequent 1-month period feeding a commercial diet (2.7 % EPA+DHA), the capacity of the four initial groups to adapt to an n-3 HUFA-restricted diet (0.3 % EPA+DHA; R-groups: XH2R, HH2R, LH2R, XLH2R) was tested for 35 d. Larval dietary treatments had no effect on larval and juvenile survival rates. The wet weight of day 45 larvae was higher in XH1 and HH1 (P < 0.001), but the R-juvenile mass gains were similar in all treatments. Delta-6-desaturase (Delta6D) mRNA level was higher in LH1 and XLH1 at day 45 (P < 0.001), and higher in LH2R and XLH2R, with a significant increase at day 118.Concomitantly, PPARalpha and PPARbeta mRNA levels were higher in XLH1 at day 45, and PPARbeta and gamma mRNA levels were higher in XLH2R at day 118, suggesting possible involvement of PPAR in stimulation of Delta6D expression, when drastic dietary larval conditioning occurred. The low DHA content in the polar lipids (PL) of LH1 and XLH1 revealed an n-3-HUFA deficiency in these groups. Larval conditioning did not affect DHA content in the PL of R-juveniles. The present study showed (i) a persistent Delta6D mRNA enhancement in juveniles pre-conditioned with an n-3 HUFA-deficient larval diet, over the 1-month intermediate period, and (ii) brought new findings suggesting the involvement of PPAR in the Delta6D mRNA level stimulation. However, such nutritional conditioning had no significant effect on juvenile growth and lipid composition.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

BackgroundVitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream.ResultsDuring gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp.ConclusionsPresent data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

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 (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.

Hypoxia tolerance of common sole juveniles depends on dietary regime and temperature at the larval stage: evidence for environmental conditioning

An individuals environmental history may have delayed effects on its physiology and life history at later stages in life because of irreversible plastic responses of early ontogenesis to environmental conditions. We chose a marine fish, the common sole, as a model species to study these effects, because it inhabits shallow marine areas highly exposed to environmental changes. We tested whether temperature and trophic conditions experienced during the larval stage had delayed effects on life-history traits and resistance to hypoxia at the juvenile stage. We thus examined the combined effect of global warming and hypoxia in coastal waters, which are potential stressors to many estuarine and coastal marine fishes. Elevated temperature and better trophic conditions had a positive effect on larval growth and developmental rates; warmer larval temperature had a delayed positive effect on body mass and resistance to hypoxia at the juvenile stage. The latter suggests a lower oxygen demand of individuals that had experienced elevated temperatures during larval stages. We hypothesize that an irreversible plastic response to temperature occurred during early ontogeny that allowed adaptive regulation of metabolic rates and/or oxygen demand with long-lasting effects. These results could deeply affect predictions about impacts of global warming and eutrophication on marine organisms.

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.

Dietary Cholecalciferol Regulates the Recruitment and Growth of Skeletal Muscle Fibers and the Expressions of Myogenic Regulatory Factors and the Myosin Heavy Chain in European Sea Bass Larvae

The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

Effects of the total replacement of fish-based diet with plant-based diet on the hepatic transcriptome of two European sea bass (Dicentrarchus labrax

BackgroundEfforts 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).ResultsWe 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.ConclusionsOverall, 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.