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International audience; Highly unsaturated fatty acids of the omega-3 series (HUFA) are major constituents of cell membranes. yet are poorly synthesised de novo by consumers. Their production, mainly supported by aquatic microalgae, has been decreasing with global change. The consequences of such reductions may be profound for ectotherm consumers, as temperature tightly regulates the HUFA content in cell membranes, maintaining their functionality. Integrating individual, tissue and molecular approaches, we examined the consequences of the combined effects of temperature and HUFA depletion on the key cardio-respiratory functions of the golden grey mullet, an ectotherm grazer of high ecological importance. For 4 months, fish were exposed to two contrasting HUFA diets [4.8% eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) on dry matter (DM) versus 0.2% EPA+DHA on DM] at 12 and 20 degrees C. Ventricular force development coupled with gene expression profiles measured on cardiac muscle suggest that combining HUFA depletion with warmer temperatures leads to: (1) a proliferation of sarcolemmal and sarcoplasmic reticulum Ca2+ channels and (2) a higher force-generating ability by increasing extracellular Ca2+ influx via sarcolemmal channels when the heart has to sustain excessive effort due to stress and/or exercise. At the individual scale, these responses were associated with a greater aerobic scope, maximum metabolic rate and net cost of locomotion, suggesting the higher energy cost of this strategy. This impaired cardiac performance could have wider consequences for other physiological performance such as growth, reproduction or migration, all of which greatly depend on heart function.

WOS:000514971500002; International audience; The decrease in ocean pH that results from the increased concentration of dissolved carbon dioxide (CO2) is likely to influence many physiological functions in organisms. It has been shown in different fish species that ocean acidification (OA) mainly affects sensory systems, including olfaction. Impairment of olfactory function may be due to a dysfunction of the GABAergic system and to an alteration of neuronal plasticity in the whole brain and particularly in olfactory bulbs. Recent studies revealed that OA-driven effects on sensory systems are partly mediated by the regulation of the expression of genes involved in neurotransmission and neuronal development. However, these studies were performed in fish exposed to acidified waters for short periods, of only a few days. In the present paper, we investigated whether such effects could be observed in adult (4-years old) European sea bass (Dicentrarchus labrax) exposed to two hypercapnic and acidified conditions (PCO2 approximate to 980 mu atm; pH total = 7.7 and PCO2 approximate to 1520 mu atm; pH total = 7.5) from the larval stage. In a first approach, we analyzed by qPCR the expression of five genes involved in neurogenesis (DCX) or expressed in GABAergic (Gabra3), glutamatergic (Gria1) or dopaminergic (TH and DDC) neurons in the olfactory bulbs. The tested experimental conditions did not change the expression of any of the five genes. This result would indicate that a potential disruption of the olfactory function of sea bass exposed for a long term to near-future OA, either occurs at a level other than the transcriptional one or involves other actors of the sensory function.

International audience; Optimizing the metabolic use of these carbohydrate-rich plant products in fish feeds could be achieved through nutritional programming strategies of fish larvae. The present study aims at testing the long-term effect of an early-life high carbohydrate nutritional (34% of starch in the feed )stimulus in European sea bass(Dicentrarchus labrax), by examining several biochemical and molecular parameters involved in carbohydrate metabolism in sea bass larvae and juveniles, compared to a control group fed standard larval regime (phase 1). As awaited, specific activities of digestive enzymes, from pancreatic and intestinal segments, involved in glucide digestion (amylase, sucrase) were enhanced together with an up-regulation of glucokinase gene and down-regulation of glucose6-phosphatase gene assayed in whole-body arvae fed the high-carbohydrate diet.However, this regime also induced a significant lowering in growth and survival. Six months later,while all the fish were fed the same standard diet,a first hypoxia challenge test allowed to distinguish two groups of fish according their nutritional history at larval stage: fish had been fed high-carbo-hydrate regime performed much better than control.When fed again during 2 months with a high-carbohydrate diet (phase 2), such larval nutritional history did not confer any subsequent “advantage” or “disadvantage” in term of carbohydrate metabolism and growth.A second hypoxia challenge test performed just after phase2,did not confirm the first one. All together our results indicated that the larval conditioning may fade over time. It was suggested to apply regular nutritional stress pulses during thefirst months of fish life.

International audience; Maternal mRNA governs early embryonic development in fish and variation in abundance of maternal transcripts may contribute to variation in embryonic survival and hatch success in European eel, Anguilla anguilla. Previous studies have shown that quantities of the maternal gene products β-tubulin, insulin-like growth factor 2 (igf2), nucleoplasmin (npm2), prohibitin 2 (phb2), phosphatidylinositol glycan biosynthesis class F protein 5 (pigf5), and carnitine O-palmitoyltransferase liver isoform-like 1 (cpt1) are associated with embryonic developmental competence in other teleosts. Here, the relations between relative mRNA abundance of these genes in eggs and/or embryos and egg quality, was studied and analyzed. We compared egg quality of the two groups: i) batches with hatching and ii) batches with no hatching. Results showed no significant differences in relative mRNA abundance between the hatch and no hatching groups for any of the selected genes at 0, 2.5, and 5 HPF. However, at 30 HPF the hatch group showed significantly higher abundance of cpt1a, cpt1b, β-tubulin, phb2, and pigf5 transcripts than the no hatch group. Therefore, these results indicate that up-regulation of the transcription of these genes in European eel after the mid-blastula transition, may be needed to sustain embryonic development and hatching success.

International audience; Marine plastic pollution is a major environmental issue. Given their ubiquitous nature and small dimensions, ingestion of microplastic (MP) and nanoplastic (NP) particles and their subsequent impact on marine life are a growing concern worldwide. Transfers along the trophic chain, including possible translocation, for which the hazards are less understood, are also a major preoccupation. Effects of MP ingestion have been studied on animals through laboratory exposure, showing impacts on feeding activity, reserve depletion and inflammatory responses, with consequences for fitness, notably reproduction. However, most experimental studies have used doses of manufactured virgin microspheres that may not be environmentally realistic. As for most ecotoxicological issues, the environmental relevance of laboratory exposure experiments has recently been debated. Here we review constraints and priorities for conducting experimental exposures of marine wildlife to microplastics based on the literature, feedback from peer reviewers and knowledge gained from our experience. Priorities are suggested taking into account the complexity of microplastics in terms of (i) aggregation status, surface properties and interactions with organic and inorganic materials, (ii) diversity of encountered particles types and concentrations, (iii) particle bioavailability and distribution in experimental tanks to achieve reproducibility and repeatability in estimating effects, and (iv) strict experimental procedures to verify the existence of genuine translocation. Relevant integrative approaches encompass a wide spectrum of methods from -omics to ecophysiological approaches, includingmodeling, are discussed to provide novel insights on the impacts of MP/NP on marine ecosystems from a long-term perspective. Knowledge obtained in this way would inform stakeholders in such a way as to help them mitigate impacts of the micro- and nano-plastic legacy.

WOS:000435090700050; International audience; European eel (Anguilla anguilla) is a euryhaline species, that has adapted to cope with both, hyper- and hypo-osmotic environments. This study investigates the effect of salinity, from a morphological and molecular point of view on European eel larvae reared from 0 to 12 days post hatch (dph). Offspring reared in 36 practical salinity units (psu; control), were compared with larvae reared in six scenarios, where salinity was decreased on 0 or 3 dph and in rates of 1, 2 or 4 psu/day, towards iso-osmotic conditions. Results showed that several genes relating to osmoregulation (nkcc2 alpha, nkcc2 beta, aqp1dup, aqpe), stress response (hsp70, hsp90), and thyroid metabolism (thr alpha A, thr alpha B, thr beta B, diol, dio2, dio3) were differentially expressed throughout larval development, while nkcc1 alpha, nkcc2 beta, aqp3, aqp 1dup, aqpe, hsp90, thr alpha A and dio3 showed lower expression in response to the salinity reduction. Moreover, larvae were able to keep energy metabolism related gene expression (atp6, cox1) at stable levels, irrespective of the salinity reduction. As such, when reducing salinity, an energy surplus associated to reduced osmoregulation demands and stress (lower nkcc, aqp and hsp expression), likely facilitated the observed increased survival, improved biometry and enhanced growth efficiency. Additionally, the salinity reduction decreased the amount of severe deformities such as spinal curvature and emaciation but also induced an edematous state of the larval heart, resulting in the most balanced mortality/deformity ratio when salinity was decreased on 3 dph and at 2 psu/day. However, the persistency of the pericardial edema and if or how it represents an obstacle in further larval development needs to be further clarified. In conclusion, this study clearly showed that salinity reduction regimes towards iso-osmotic conditions facilitated the European eel pre-leptocephalus development and revealed the existence of highly sensitive and regulated osmoregulation processes at such early life stage of this species.

WOS:000417657800001; International audience; Several physiological functions in fish are shaped by environmental stimuli received during early life. In particular, early-life hypoxia has been reported to have long-lasting effects on fish metabolism, with potential consequences for fish life history traits. In the present study, we examine whether the synergistic stressors hypoxia (40% and 100% air saturation) and temperature (15 degrees and 20 degrees C), encountered during early life, could condition later metabolic response in European sea bass (Dicentrarchus labrax) juveniles. Growth rate and metabolic parameters related to carbohydrate and lipid metabolism in the liver were investigated at the juvenile stage under normoxic and chronic hypoxic conditions. Juvenile growth rates were significantly lower (p \textless 1 x 10(-6)) under hypoxic conditions and were not improved by prior early-life exposure to hypoxia. Growth was 1.3 times higher (p \textless 5 x 10(-3)) in juveniles reared at 15 degrees C during the larval stage than those reared at 20 degrees C, suggesting that compensatory growth had occurred. Early-life exposure to hypoxia induced higher (p \textless 2 x 10(-6)) glycogen stores in juveniles even though there was no apparent regulation of their carbohydrate metabolism. In the liver of juveniles exposed to chronic hypoxia, lower glycogen content combined with stimulation of phosphoenolpyruvate carboxykinase gene expression and higher lactate concentration indicated a stimulation of the anaerobic glycolytic pathway. Furthermore, hypoxia only induced lower (p \textless 1 x 10(-3)) lipid content in the liver of juveniles that had experienced 15 degrees C at the larval stage. The present study provides evidence that environmental conditions experienced during early life shape the metabolic traits of D. labrax with potential consequences for juvenile physiological performance.

WOS:000426336600006; International audience; Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3'-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 degrees C) from hatch until first-feeding. We identified 2 subtypes of thr (thr alpha and thr beta) with 2 isoforms each (thr alpha A, thr alpha B, thr beta A, thr beta beta) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel.

International audience; Six candidate gene SNP assays and 12 new microsatellites were developed in the European flounder (Platichthys flesus), in order to provide new tools to (1) further investigate signatures of selection suspected on candidate genes, and (2) better depict the neutral evolutionary background shaping the differentiation of populations. All markers were successfully tested in two populations from contrasted estuaries, and should be helpful to better understand the possible adaptive responses of flounder populations to anthropogenic pressures (e.g. global warming, eutrophication, pollutions), over the whole distribution range of the species.

International audience; In the context of global change, ectotherms are increasingly impacted by abiotic perturbations. Along the distribution area of a species, the populations at low latitudes are particularly exposed to temperature increase and hypoxic events. In this study, we have compared the proteomic responses in the liver of European flounder populations, by using 2-D electrophoresis. One southern peripheral population from Portugal vs two northern core populations from France, were reared in a common garden experiment. Most of the proteomic differences were observed between the two experimental conditions, a cold vs a warm and hypoxic conditions. Consistent differentiations between populations were observed in accumulation of proteins involved in the bioenergetics- and methionine-metabolisms, fatty acids transport, and amino-acid catabolism. The specific regulation of crucial enzymes like ATP-synthase and G6PDH, in the liver of the southern population, could be related to a possible local adaptation. This southern peripheral population is spatially distant from northern core populations and has experienced dissimilar ecological conditions; thus it may contain genotypes that confer resilience to climate changes.

International audience; Hemoglobins (Hb) play a critical role in satisfying the oxygen demand of vertebrate aerobic metabolism. The present study reports the characterization of the European sea bass (Dicentrarchus labrax) Hb genes, including genomic organization, phylogeny, and spatio-temporal gene expression. These Hb genes are divided into two unlinked clusters, the “MN” cluster containing eleven genes (five Hbα genes named MN-Hbα1-5 and six Hbβ genes named MN-Hbβ1–6) and the “LA” cluster consisting of three genes (two Hbα genes named LA-Hbα1-2 and one Hbβ gene named LA-Hbβ1). Comparative analysis of Hb amino acid sequences indicates that most of the important amino acid residues involved in hemoglobin-oxygen binding, particularly in the Bohr and Root effects, are generally well conserved, except in MN-Hbβ3. Six genes were mainly expressed during early life (MN-Hbα3-5, MN-Hbβ4–6), while the others were predominantly expressed at juvenile–adult stages. Adult fish expressed Hb genes at high levels in the head kidney and spleen; the main organs involved in blood formation. The Hb genes expressed in non-hematopoietic organs (intestine, gills, heart, brain, and liver) may facilitate oxygen homeostasis or be involved in antimicrobial defense. Stage- and tissue-specific gene expression patterns, together with the sequence features of the different Hb proteins, suggest a broad range of roles in European sea bass.

WOS:000408631800021; International audience; European sea bass (Dicentrarchus labrax) inhabits coastal waters and may be exposed to hypoxia at different life stages, requiring physiological and behavioral adaptation. In the present study, we attempted to determine whether regulation of hemoglobin (Hb) gene expression plays a role in the physiological response to chronic moderate hypoxia in whole larvae and hematopoietic tissues (head kidney and spleen) of juveniles. We also tested the hypothesis that hypoxia exposure at the larval stage could induce a long-term effect on the regulation of Hb gene expression. For this purpose, D. labrax were exposed to a non-lethal hypoxic condition (40% air saturation) at the larval stage from 28 to 50 days post-hatching (dph) and/or at the juvenile stage from 196 to 296 dph. Data obtained from larvae indicate that hypoxia induced a subtype-specific regulation of Hb gene expression, with a significant decrease of MN-Hba3, MN-Hb beta 4 and MN-Hb beta 5 and increase of MN-Hb alpha 2, LA-Hb alpha 1 and LA-Hb beta 1 transcript levels. Hypoxia did not induce regulation of Hb gene expression in juveniles, except in the head kidney for those that experienced hypoxia at the larval stage. The latter exhibited a significant hypoxia-induced stimulation of MN-Hba2, LA-Hba1 and LA-Hb beta 1 gene expression, associated with stimulation of the PHD-3 gene involved in the hypoxia-inducible factor oxygen-sensing pathway. We conclude that subtype-and stage-specific regulation of Hb gene expression plays a role in the physiological response of D. labrax to cope with hypoxia and that early exposure to low oxygen concentration has a long-term effect on this response.

Ocean warming, eutrophication and consequent decrease in oxygen lead to smaller average fish size. Although such responses are well-known in an evolutionary context, involving multiple generations, it appears to be incompatible with current rapid environmental change. Rather, 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 to 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 feed intake. We measured several respirometry parameters and we only found a higher SDA (Specific Dynamic Action) 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.

WOS:000407548800031; International audience; Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24 degrees C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22 degrees C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16 degrees C) or accelerated at warm temperatures (20-22 degrees C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16 degrees C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22 degrees C, suggesting thermal stress. Furthermore, 24 degrees C was shown to be deleterious (resulting in 100% mortality), while 16 degrees C and 22 degrees C (similar to 50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18 degrees C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment has increased very substantially over the last decades leading to high concentrations in sediments of contaminated areas. To evaluate the consequences of long-term chronic exposure to PAHs, zebrafish were exposed, from their first meal at 5 days post fertilisation until they became reproducing adults, to diets spiked with three PAH fractions at three environmentally relevant concentrations with the medium concentration being in the range of 4.6-6.7 μg g(-1) for total quantified PAHs including the 16 US-EPA indicator PAHs and alkylated derivatives. The fractions used were representative of PAHs of pyrolytic (PY) origin or of two different oils of differing compositions, a heavy fuel (HO) and a light crude oil (LO). Fish growth was inhibited by all PAH fractions and the effects were sex specific: as determined with 9-month-old adults, exposure to the highest PY inhibited growth of females; exposure to the highest HO and LO inhibited growth of males; also, the highest HO dramatically reduced survival. Morphological analysis indicated a disruption of jaw growth in larvae and malformations in adults. Intestinal and pancreatic enzyme activities were abnormal in 2-month-old exposed fish. These effects may contribute to poor growth. Finally, our results indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can promote lethal and sublethal effects which are likely to be detrimental for fish recruitment.

Background The better understanding of how intestinal microbiota interacts with fish health is one of the key to sustainable aquaculture development. The present experiment aimed at correlating active microbiota associated to intestinal mucosa with Specific Growth Rate (SGR) and Hypoxia Resistance Time (HRT) in European sea bass individuals submitted to different nutritional histories: the fish were fed either standard or unbalanced diets at first feeding, and then mixed before repeating the dietary challenge in a common garden approach at the juvenile stage. Results A diet deficient in essential fatty acids (LH) lowered both SGR and HRT in sea bass, especially when the deficiency was already applied at first feeding. A protein-deficient diet with high starch supply (HG) reduced SGR to a lesser extent than LH, but it did not affect HRT. In overall average, 94 % of pyrosequencing reads corresponded to Proteobacteria, and the differences in Operational Taxonomy Units (OTUs) composition were mildly significant between experimental groups, mainly due to high individual variability. The highest and the lowest Bray-Curtis indices of intra-group similarity were observed in the two groups fed standard starter diet, and then mixed before the final dietary challenge with fish already exposed to the nutritional deficiency at first feeding (0.60 and 0.42 with diets HG and LH, respectively). Most noticeably, the median percentage of Escherichia-Shigella OTU_1 was less in the group LH with standard starter diet. Disregarding the nutritional history of each individual, strong correlation appeared between (1) OTU richness and SGR, and (2) dominance index and HRT. The two physiological traits correlated also with the relative abundance of distinct OTUs (positive correlations: Pseudomonas sp. OTU_3 and Herbaspirillum sp. OTU_10 with SGR, Paracoccus sp. OTU_4 and Vibrio sp. OTU_7 with HRT; negative correlation: Rhizobium sp. OTU_9 with HRT). Conclusions In sea bass, gut microbiota characteristics and physiological traits of individuals are linked together, interfering with nutritional history, and resulting in high variability among individual microbiota. Many samples and tank replicates seem necessary to further investigate the effect of experimental treatments on gut microbiota composition, and to test the hypothesis whether microbiotypes may be delineated in fish. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0885-2) contains supplementary material, which is available to authorized users.

00000 ăWOS:000382802100022; International audience; Human alteration of marine ecosystems is substantial and growing. Yet, no adequate methodology exists that provides reliable predictions of how environmental degradation will affect these ecosystems at a relevant level of biological organization. The primary objective of this study was to develop a methodology to evaluate a fish's capacity to face a well-established environmental challenge, an exposure to chemically dispersed oil, and characterize the long-term consequences. Therefore, we applied high-throughput, non-lethal challenge tests to assess hypoxia tolerance, temperature susceptibility and maximal swimming speed as proxies for a fish's functional integrity. These whole animal challenge tests were implemented before (1 month) and after (1 month) juvenile European sea bass (Dicentrarchus labrax) had been acutely exposed (48 h) to a mixture containing 0.08 g L-1 of weathered Arabian light crude oil plus 4% dispersant (Corexit(C) EC9500A), a realistic exposure concentration during an oil spill. In addition, experimental populations were then transferred into semi-natural tidal mesocosm ponds and correlates of Darwinian fitness (growth and survival) were monitored over a period of 4 months. Our results revealed that fish acutely exposed to chemically dispersed oil remained impaired in terms of their hypoxia tolerance and swimming performance, but not in temperature susceptibility for 1 month post-exposure. Nevertheless, these functional impairments had no subsequent ecological consequences under mildly selective environmental conditions since growth and survival were not impacted during the mesocosm pond study. Furthermore, the earlier effects on fish performance were presumably temporary because re-testing the fish 10 months post-exposure revealed no significant residual effects on hypoxia tolerance, temperature susceptibility and maximal swimming speed. We propose that the functional proxies and correlates of Darwinian fitness used here provide a useful assessment tool for fish health in the marine environment.

The present study investigates the effects of dietary phospholipids (PL) on intestinal lipid absorption in fry and early juvenile stages of rainbow trout ( Oncorhynchus mykiss ) fed fresh or oxidized lipids. Six semi-purified casein-based diets were formulated with 12% fresh or oxidized fish oil and 6% PL supplied as soybean lecithin or egg lecithin or 6% soybean oil for PL-free diets. Each diet was distributed to 3 replicate tanks of 700 swim-up fry (initial mean body weight: 66 mg) or 200 early juvenile fish (initial mean body weight: 455 mg) over a 3-week or 17-week growth trial at a water temperature of 17 °C. Feeding oxidized compared to fresh lipids significantly reduced body weight of both fry and juvenile trout (0.15 ± 0.02 vs. 0.28 ± 0.07 g and 10 ± 2 vs. 22 ± 2 g, respectively). At fry stages, within groups fed fresh lipids, trout fry fed the PL-supplemented diets had a significantly higher final body weight than fry fed the PL-free diet (0.31 ± 0.06 vs. 0.21 ± 0.02 g). Histological examination revealed a higher occurrence of intestinal steatosis (accumulation of large lipid droplets in the enterocytes of the anterior intestine) in fish fed the PL-free diet compared to fish fed PL-supplemented diets at fry stages (13/16 vs. 1/32), whereas no significant differences were recorded at juvenile stages (1/18 vs. 0/18). The levels of circulating total cholesterol, triacylglycerol (TAG) and PL in the juvenile fish were not significantly modified by dietary PL supplementation whereas the plasma TAG/PL ratio was lower in juvenile fish fed oxidized compared to fresh lipids (0.31 ± 0.06 vs. 0.45 ± 0.08). At the fry stages, within groups fed fresh lipids, expression of the gene coding for apolipoprotein B (apoB) was 2.3-fold reduced in the intestine of fish fed the PL-free diet compared to the PL-supplemented diets whereas no significant difference was recorded at juvenile stages. The expression of other apolipoproteins or key-enzymes involved in intestinal lipoprotein synthesis was not significantly modified by dietary PL supplementation. In conclusion, the results indicate that the beneficial effect of dietary PL on growth and intestinal lipid absorption is restricted to the fry stages and support previous opinions that PL are needed for intestinal TAG absorption. The limiting step seems to be the synthesis of apoB by the enterocytes in rainbow trout fry fed fresh lipids but this requires further investigation in fish fed oxidized lipids.

P>The alcian blue-alizarin red technique was successfully adjusted to stain developing European sea bass (Dicentrarchus labrax) larvae. For an optimal staining protocol design both larval size and their morphological characteristics at each developmental stage were considered, since such parameters notably influence the staining of tissues. The incubation times of the different solutions were adjusted to allow the stain penetration for revealing the integrity of cartilaginous and bony tissues without significant tissue degradation. Three developmental windows were determined for an optimal staining procedure: (i) 4.5-6.4 mm, (ii) 6.7-8.7 mm, and (iii) 12.8-15.5 mm total length (TL). In order to validate the continuity of staining along the larval development, quantification of bone mineralization and osteocalcin gene expression were also monitored. Quantitative analysis revealed that ossification followed an exponential kinetic that was positively correlated with the osteocalcin gene expression pattern (Rs = 0.9762, P < 0.05). The mineralized tissue increased from 6.4 mm TL onwards, corresponding with the detection of the first ossified structures. The quantity of bony tissue increased gradually until 7.6 mm TL, since mineralization remained limited to the skull. From 8.3 to 15.5 mm TL, the mineralized bone was notable and nearly concerned the whole larval skeleton (skull, vertebral column and caudal complex). Since it was possible to detect the first cartilaginous and mineralized structures in specimens as small as 4.5 and 6.4 mm TL, respectively, this procedure is a useful tool to study the European sea bass skeletal ontogenesis, to precociously diagnose skeletal malformations in small larvae and eventually to better characterize the effect of different environmental and/or nutritional factors on the ossification status of specific skeletal components.

International audience; The influence of dietary vitamins on growth, survival, and morphogenesis was evaluated until day 38 of posthatching life in European sea bass larvae (Dicentrarchus labrax). A standard vitamin mix (VM), at double the concentration of the U. S. National Research Council's recommendations, was incorporated into larval feeds at 0.5%, 1.5%, 2.5%, 4.0%, and 8.0% to give treatments VM 0.5, VM 1.5, VM 2.5, VM 4.0, and VM 8.0, respectively. The group fed the VM 0.5 diet all died before day 30. At day 38, the larvae group fed VM 1.5 had 33% survival, while the other groups, with higher vitamin levels, showed at least 50% survival. The higher the percentage VM in the diet, the lower the percentage of column deformities. High dietary vitamin levels positively influenced the formation of mineralized bone in larvae: the higher the dietary vitamin level, the higher the ossification status. In the larvae group fed at the highest vitamin levels, we observed a temporal sequence of coordinated growth factor expression, in which the expression of bone morphometric protein (BMP-4) preceded the expression of IGF-1, which stimulated the maturation of osteoblasts (revealed by high osteocalcin expression levels). In groups fed lower proportions of vitamins, elevated proliferator peroxisome-activated receptors (PPAR-gamma) expression coincided with low BMP-4 expression. Our results suggest that high levels of PPAR-gamma transcripts in larvae-fed diets with a low VM content converted some osteoblasts into adipocytes during the first two weeks of life. This loss of osteoblasts is likely to have caused skeletal deformities.

International audience

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.

International audience; The main goal of this work was to determine the effect of dietary live yeast Debaryomyces hansenii on the enzymatic antioxidative status of sea bass Dicentrarchus labrax larvae. Growth, activity and expression of the main antioxidative enzymes: catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD), and heat shock protein (HSP70) were measured in sea bass larvae at 23 and 48 days after hatching. Larvae were fed on two microdiets: group one, fed microdiet containing live yeast and the control group fed microdiet without yeast. Heat shock protein 70 showed the same expression levels in both fish larvae fed yeast and the control diet. The group fed D. hansenii showed highest growth and lower activity and expression levels of GPX and SOD compared to fish fed control diet. In our work the differences in activity and gene expression patterns could only be attributed to the presence of yeast, assuming a possible involvement of superoxide anion retention in fish larvae, which could represent importance to the host to increase cell or tissue responsiveness to growth- and/or differentiation-enhancing factors. (c) 2009 Elsevier B.V. All rights reserved.

Chantier qualité GA; 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 9 months. 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. (C) 2010 Elsevier Inc. All rights reserved.

International audience; The objective of the present study was to investigate the effects of dietary vitamin A supplementation on reproduction and fry quality of rainbow trout (Oncorhynchus mykiss). Two feeding trials were conducted with rainbow trout broodstock at a water temperature of 7 degrees C over a 6-month period prior to spawning. Three practical diets based on fish meal and fish oil (trial 1) and on plant ingredients (trial 2) were used. Diets 1A0, 1A20 and 1A700 were supplemented with 0,20 and 700 IU/g vitamin A supplied as retinyl acetate resulting in dietary vitamin A contents of 45, 59 and 627 Wig in trial 1; whereas, diets 2A0, 2A20 and 2A200 supplemented with 0,20 and 200 IU/g vitamin A displayed total vitamin A contents of 21,39 and 194 IU/g diet in trial 2. In both trials, gonadosomatic indices were not significantly different among dietary groups. Egg size remained unaffected by the levels of vitamin A in the broodstock diets. In both feeding trials, the levels of retinyl palmitate and retinol, the two main storage forms of vitamin A, were significantly higher in oocytes from 1A700 and 2A200 groups compared to other dietary groups; whereas, the level of retinoic acid remained unaffected. In trial 2, broodstock females fed diet 2A200 showed better fecundity than females fed diets 2A0 and 2A20. A significant decline in survival from the eyed stage onwards was noticeable in group 1A700 in trial 1 but no differences were found in trial 2. At the swim-up stage, trout fry from broodstock fed diet 1A20 displayed significantly improved growth rates compared to other dietary groups in trial 1; whereas, in trial 2, trout fry from 2A200 group exhibited the best growth performance. The expression of genes coding for retinoid receptors and other associated nuclear receptors was not significantly different among dietary groups at any developmental stage considered and no skeletal malformation was recorded in offspring. The results indicate that feeding rainbow trout broodstock a high level of vitamin A induced mortality at embryonic stages but was not teratogenic as no skeletal malformation was recorded at late developmental stages. However, quite high levels of vitamin A (60-200 IU/g) were necessary to sustain fecundity of broodstock and early growth of offspring. (C) 2010 Elsevier BM. All rights reserved.

Vitamins D and C are essential in many physiological functions. Vitamin D, a fat soluble vitamin, is crucial to preserve calcium and phosphate homeostasis and to protect the skeletal integrity. This hormone functions through the vitamin D receptor (VDR) inducing the expression of various calcium binding and transport proteins in the intestine to stimulate active calcium uptake, thus preserving normocalcemia and, indirectly, maintaining bone mineralization. Besides, vitamin D also acts directly on osteoblasts, the resident bone-forming cells of the skeleton, to inhibit proliferation, modulate differentiation, and regulate mineralization of the extracellular matrix. Vitamin C, a water soluble vitamin, acts as a co-substrate for hydroxylase and oxygenase enzymes involved in the biosynthesis of pro-collagen, carnitine and neurotransmitters, among other numerous physiological functions such as antioxidant or pro-oxidant. Both vitamins should be supplied by the diet because fish are unable to synthesize them. However, their wide range of action makes it difficult to adjust the adequate amount of these vitamins to achieve an optimal fish performance. Besides, the dietary vitamin needs of fish depend on several factors such as developmental stage, physiological, environmental/ecological and genetic conditions. In this sense, vitamin requirements of flatfish do not necessarily meet those of pelagic fish and depends also on their feeding habits (carnivorous, planktivorous or detritivorous); the dietary vitamin demands of an adult fish differ from those of a larva; and even within the same fish species and developmental stage, the environmental conditions would also influence the vitamin needs (i.e., under stress conditions, high vitamin C levels have been demonstrated to improve stress resistance and, consequently, growth). The present paper gives a general overview about the requirements of vitamins D and C in fish and specifically reviews the role of these vitamins in fish skeletogenesis and their influence in the development of skeletal deformities. In addition, new insights on the molecular pathways involving these vitamins in the skeletal ossification process are provided.

The purpose of this study was to examine the impact of graded levels of dietary vitamin A on sea bass larval performances and to determine optimal retinol levels at different larval stages to avoid specific skeletal malformations. Retinol was incorporated into larval feeds at 0, 5,10,15, 25, 35 and 70 mg kg(-1) dry matter (giving rise to RET0, RET5, RET10, RET15, RET25, RET35, RET70 groups, respectively). Analysis of the several types of deformities affecting the skull, vertebral column or fins of the fish were observed depending on experimental groups. On one hand, the incidence of skull malformations affecting the maxillary and premaxillary bones. dentaries, operculum, branchiostegal rays and glossohyal was lower for the RET0 and RET5 groups. On the other hand, the frequency of vertebral (slight fusions and kyphosis of the anterior five vertebrae, over-mineralization and lordosis of the haemal vertebrae, the transformation of the last pre-haemal vertebra into haemal) and fin (deformations of the dorsal and anal pterygiophores, deviations of the related rays, modifications of the anatomy of the caudal supporting elements, partial to complete lack of the pelvic fins) deformities were lower for the RET5-70 groups. In the RET0 group, lower level of Hoxd-9 expression coincided with partial or complete lack of pelvic fin. Our results suggest that the optimal level of retinol for harmonious ontogenesis fluctuate along sea bass larvae development and that inadequate dietary retinol levels alters morphogenesis through the modulation of Hox gene expression, at least for the pelvic fin. (c) 2009 Elsevier B.V. All rights reserved.

16 páginas, 12 figuras, 4 tablas., The effects of different levels of vitamin A (VA) in Senegalese sole larval performance and development were evaluated by means of a dietary dose–response experiment using enriched Artemia metanauplii as a carrier of this micronutrient. Larvae were fed from 6 to 27 days post hatch (dph) with enriched Artemia containing graded levels of total VA (1.3, 2.1, 4.5 and 12.9 µg VA mg− 1 DW). The content of VA in live prey directly affected its accumulation in larvae and early juveniles. Retinyl palmitate accumulated during larval ontogeny, whereas retinol showed the opposite trend, decreasing from hatching until 41 dph and then remaining constant until the end of the study. In metamorphic larvae (10 and 15 dph), VA did not affect the number of thyroid follicles or the intensity of the immunoreactive staining of T3 and T4. However, at older stages of development (post-metamorphic larvae: 20, 30, 41 and 48 dph), VA decreased the number of thyroid follicles but increased their mean size and enhanced T3 and T4 immunoreactive staining. A dietary excess of VA did not affect either larval performance in terms of growth and survival or the maturation of the digestive system. However, the most remarkable impact of this morphogenetic nutrient was detected during skeletal morphogenesis. Dietary VA accelerated the intramembranous ossification of vertebral centrums, which led to the formation of a supranumerary haemal vertebra and a high incidence of fused and compressed vertebrae in fish fed 2.1, 4.5 and 12.9 mg VA mg− 1 DW. In addition, VA also affected those structures from vertebrae and caudal fin formed by chondral ossification, leading to defects in their shape and fusions with adjacent skeletal elements. In particular, the caudal fin was the region most affected by the dietary treatments. In order of importance, the bones with more developmental anomalies were the modified neural and haemal spines, epural, hypurals and parahypural. The impact of systemic factors such as thyroidal hormones in skeletogenesis should not be neglected since present results revealed that an excess of dietary VA affected the levels of T3 and T4, which might have affected bone formation and remodelling, leading to skeletal deformities., This work was funded by the Ministry of Education and Culture (MEC) of the Spanish Government (project AGL2005-02478).

Several nutritional studies have found a direct effect of several vitamins in chondrogenic and osteogenic development during early life stages of marine fish species. In the present study, the effect of vitamin A (VA) in gilthead sea bream skeletogenesis was evaluated by means of four different dietary regimes (enriched rotifers) containing increasing levels of total VA (75, 109, 188 and 723 ng total VA mg− 1 DW). Dietary treatments were offered to larvae during the rotifer-feeding phase (4–20 days after hatching), while later all groups were fed with Artemia nauplii and weaned onto the same inert diet. Different dietary doses of VA affected gilthead sea bream larval growth, survival, performance (maturation of the digestive system) and quality (incidence of skeletal deformities). Higher levels of dietary VA than those included in the commercial emulsion for rotifer enrichment led to different levels and typologies of skeletal deformities, indicating that gilthead sea bream larvae were very sensitive to small increases in dietary VA. The degree of ossification was affected by the level of VA in enriched rotifers: the higher amount of VA in the diet, the higher number of skeletal pieces ossified (R = 0.585, P = 0.04). Dietary VA affected the normal process of bone formation and skeletogenesis, the skeletal structures mostly affected by high amounts of dietary VA were those from the cranial skeleton (splanchnocranium), vertebral centrums and caudal fin complex. The premaxilla, maxilla and dentary bones were the cranial structures affected by dietary VA levels, resulting in a large incidence of animals with compressed snout. Dietary VA also affected the normal development of the opercular complex, and a dose–response dependant effect was observed in relation to the incidence of specimens with incomplete operculum. Body shape was also affected by the level of dietary VA, increasing the incidence of specimens with lordosis, kyphosis and/or scoliosis with the dose of VA, being the prehaemal and caudal vertebrae the most affected regions of the vertebral column with this kind of abnormalities. The caudal fin complex was the most affected region of the skeleton affected by dietary treatments as seen by the high incidence of skeletal deformities in fish fed different doses of dietary VA. Deformities affected all skeletal elements composing the caudal fin, although the most affected ones were, in order of importance, the epurals, hypurals, parahypural, neural arch and uroneurals. Differences in sensitivity to dietary VA amongst caudal fin skeletal elements might be due to their differential ontogenetic development and differences in the exposure time to VA. An excess of dietary VA also accelerated the intramembranous ossification process of vertebral centrums leading to one or two supranumerary vertebrae, and a high incidence of fused and compressed vertebral centrums. The sensibility of the developing skeletal structures to dietary VA levels should incline us to test lower doses of VA in live preys enrichments during early larval stages and higher doses afterwards., This work was funded by the Ministry of Education and Culture (MEC) of the Spanish Government (project AGL2005-02478). The collaboration with Y.K. (HMCR, Greece) was established thanks to the HG2004-0018 grant (MEC, Spain). I.F was supported by a predoctoral MEC fellowship and E.G. by the Programa Ramón y Cajal (MEC, Spain).

International audience; 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.

International audience; For sustainable aquaculture, the removal of marine resource ingredients in fish diets is an important objective. While most studies focus on the replacement of fish oil by vegetable oil, little is known on the nutritional effects of presence (which corresponds to the control diet) or absence of dietary fish oil. We studied fatty acid composition of brush-border membranes and digestive enzyme activities of the intestine and measured the expression and activities of several enzymes involved in the hepatic intermediary metabolism of rainbow trout (Oncorhynchus mykiss) fed for 7 weeks with or without fish oil. The diets were pair-fed to ensure that fish fed either diet had comparable carbohydrate and protein intakes. Absence of fish oil significantly reduced growth rate, protein efficiency and plasma lipid components. Activities of intestinal digestive enzymes were significantly decreased in the anterior intestine in fish fed without fish oil. In liver, dietary fish oil removal did not affect the transcript levels or activities of the main enzymes involved in lipogenesis (fatty acid synthase) and fatty acid b-oxidation (3-hydroxyacyl-CoA dehydrogenase), glycolysis or amino acid oxidation. It lowered the expression of the genes coding for gluconeogenic enzymes (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase), but their enzyme activities were not affected. The activities, but not gene expression of lipogenic enzymes, involved in NADPH and malonyl-CoA formation were also modified after fish oil removal as reflected by higher activities of isocitrate dehydrogenase/glucose-6-phosphate dehydrogenase and acetyl-CoA carboxylase enzymes. Overall, our results indicate that the intestinal digestive capacity was strongly modified by dietary fish oil removal, while hepatic intermediary metabolism was only marginally affected, in fed rainbow trout

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.

Microbiota was studied in 20-day old seabass larvae after 10 days of feeding compound diets, in comparison with a control group kept live food. This early weaning caused bacterial overload in the larvae fed the normal compound diet (109 CFU per g of body weight). The bacterial load was significantly limited with a diet lacking ferrous salt in the mineral premix (3 × 108 CFU g−1). A similar range of associated flora was observed before weaning at day 10, but only 107 CFU g−1 were counted in the larvae fed Artemia till day 20. The bacterial surge in weaned larvae was due to one strain of Vibrio, especially dominant in the iron-supplemented diet. In spite of its opportunism, the strain did not seem pathogenic to starving seabass larvae challenged from day 5 to day 10. This Vibrio produced siderophore, protease, phospholipase and amylase. These extracellular products were also produced by 25 to 60% of the diversified flora of the Artemia-fed larvae. Lipase was the main extracellular product observed in the latter flora (80% of the isolates), whereas it was seldom produced in the weaned groups (less than 10%). The omission of dietary ferrous salt had no detrimental effect on survival and growth rates of the larvae, but limited bacterial load. It is concluded that diet formulation should take into account the effects on microbiota, based on this first example with iron limitation.