Ocean warming combined with lower omega-3 nutritional availability impairs the cardio-respiratory function of a marine fish.

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°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.

The highly variable microbiota associated to intestinal mucosa correlates with growth and hypoxia resistance of sea bass, Dicentrarchus labrax, submitted to different nutritional histories.

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.

Lipid-free tuna muscle samples are suitable for total mercury analysis.

Tropical tunas are largely consumed worldwide, providing major nutritional benefits to humans, but also representing the main exposure to methylmercury, a potent neurotoxin that biomagnifies along food webs. The combination of ecological tracers (nitrogen and carbon stable isotopes, δ15N and δ13C) to mercury concentrations in tunas is scarce yet crucial to better characterize the influence of tuna foraging ecology on mercury exposure and bioaccumulation. Given the difficulties to get modern and historical tuna samples, analyses have to be done on available and unique samples. However, δ13C values are often analysed on lipid-free samples to avoid bias related to lipid content. While lipid extraction with non-polar solvents is known to have no effect on δ15N values, its impact on mercury concentrations is still unclear. We used white muscle tissues of three tropical tuna species to evaluate the efficiency and repeatability of different lipid extraction protocols commonly used in δ13C and δ15N analysis. Dichloromethane was more efficient than cyclohexane in extracting lipids in tuna muscle, while the automated method appeared more efficient but as repeatable as the manual method. Lipid extraction with dichloromethane had no effect on mercury concentrations. This may result from i) the affinity of methylmercury to proteins in tuna flesh, ii) the low lipid content in tropical tuna muscle samples, and iii) the non-polar nature of dichloromethane. Our study suggests that lipid-free samples, usually prepared for tropical tuna foraging ecology research, can be used equivalently to bulk samples to document in parallel mercury concentrations at a global scale.

Metabolic response to hypoxia in European sea bass (Dicentrarchus labrax) displays developmental plasticity.

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° and 20°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<1×10-6) under hypoxic conditions and were not improved by prior early-life exposure to hypoxia. Growth was 1.3 times higher (p<5×10-3) in juveniles reared at 15°C during the larval stage than those reared at 20°C, suggesting that compensatory growth had occurred. Early-life exposure to hypoxia induced higher (p<2×10-6) 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<1×10-3) lipid content in the liver of juveniles that had experienced 15°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.

Depletion of Essential Fatty Acids in the Food Source Affects Aerobic Capacities of the Golden Grey Mullet Liza aurata in a Warming Seawater Context.

The objective of this study was to evaluate the combined effects of thermal acclimation and n-3 highly unsaturated fatty acids (n-3 HUFA) content of the food source on the aerobic capacities of fish in a thermal changing environment. The model used was the golden grey mullet Liza aurata, a species of high ecological importance in temperate coastal areas. For four months, fish were exposed to two food sources with contrasting n-3 HUFA contents (4.8% ecosapentaenoic acid EPA + docosahexaenoic acid DHA on the dry matter DM basis vs. 0.2% EPA+DHA on DM) combined with two acclimation temperatures (12°C vs. 20°C). The four experimental conditions were LH12, LH20, HH12 and HH20. Each group was then submitted to a thermal challenge consisting of successive exposures to five temperatures (9°C, 12°C, 16°C, 20°C, 24°C). At each temperature, the maximal and minimal metabolic rates, metabolic scope, and the maximum swimming speed were measured. Results showed that the cost of maintenance of basal metabolic activities was particularly higher when n-3 HUFA food content was low. Moreover, fish exposed to high acclimation temperature combined with a low n-3 HUFA dietary level (LH20) exhibited a higher aerobic scope, as well as a greater expenditure of energy to reach the same maximum swimming speed as other groups. This suggested a reduction of the amount of energy available to perform other physiological functions. This study is the first to show that the impact of lowering n-3 HUFA food content is exacerbated for fish previously acclimated to a warmer environment. It raises the question of the consequences of longer and warmer summers that have already been recorded and are still expected in temperate areas, as well as the pertinence of the lowering n-3 HUFA availability in the food web expected with global change, as a factor affecting marine organisms and communities.

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.

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.