Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Circulating MicroRNAs Indicative of Sex and Stress in the European Seabass (Dicentrarchus labrax): Toward the Identification of New Biomarkers.

MicroRNAs (miRNAs) constitute a new category of biomarkers. Studies on miRNAs in non-mammalian species have drastically increased in the last few years. Here, we explored the use of miRNAs as potential, poorly invasive markers, to identify sex and characterize acute stress in fish. The European seabass (Dicentrarchus labrax) was chosen as a model because of its rapid response to stress and its specific sex determination system, devoid of sexual chromosomes. We performed a small RNA-sequencing analysis in the blood plasma of male and female European seabass (mature and immature) as well as in the blood plasma of juveniles submitted to an acute stress and sampled throughout the recovery period (at 0 h, 0.5 h, 1.5 h and 6 h). In immature individuals, both miR-1388-3p and miR-7132a-5p were up-regulated in females, while miR-499a-5p was more abundant in males. However, no miRNAs were found to be differentially expressed between sexes in the blood plasma of mature individuals. For the acute stress analysis, five miRNAs (miR-155-5p, miR-200a-3p, miR-205-1-5p, miR-143-3p, and miR-223-3p) followed cortisol production over time. All miRNAs identified were tested and validated by RT-qPCR on sequenced samples. A complementary analysis on the 3'UTR sequences of the European seabass allowed to predict potential mRNA targets, some of them being particularly relevant regarding stress regulation, e.g., the glucocorticoid receptor 1 and the mineralocorticoid receptor. The present study provides new avenues and recommendations on the use of miRNAs as biomarkers of sex or stress of the European seabass, with potential application on other fish species.

Temperature-dependent metabolic consequences of food deprivation in the European sardine.

Aquatic ecosystems can exhibit seasonal variation in resource availability and animals have evolved to cope with the associated caloric restriction. During winter in the NW Mediterranean Sea, the European sardine Sardina pilchardus naturally experiences caloric restriction owing to a decrease in the diversity and quantity of plankton. However, ongoing global warming has had deleterious effects on plankton communities such that food shortages may occur throughout the year, especially under warm conditions in the summer. We investigated the interactive effects of temperature and food availability on sardine metabolism by continuously monitoring whole-animal respiration of groups of control (fed) and food-deprived sardines over a 60-day experiment in winter (12°C) or summer (20°C) conditions under natural photoperiod. In addition, we measured mitochondrial respiration of red muscle fibres, biometric variables and energy reserves of individuals sampled at 30 and 60 days. This revealed that winter food deprivation elicits energy saving mechanisms at whole animal and cellular levels by maintaining a low metabolism to preserve energy reserves, allowing high levels of survival. By contrast, despite energy saving mechanisms at the mitochondrial level, whole animal metabolic rate was high during food deprivation in summer, causing increased consumption of energy reserves at the muscular level and high mortality after 60 days. Furthermore, a 5-day re-feeding did not improve survival, and mortalities continued, suggesting that long-term food deprivation at high temperatures causes profound stress in sardines that potentially impairs nutrient absorption.

Is starvation a cause of overmortality of the Mediterranean sardine?

Animal mortality is difficult to observe in marine systems, preventing a mechanistic understanding of major drivers of fish population dynamics. In particular, starvation is known to be a major cause of mortality at larval stages, but adult mortality is often unknown. In this study, we used a laboratory food-deprivation experiment, on wild caught sardine Sardina pilchardus from the Gulf of Lions. This population is interesting because mean individual phenotype shifted around 2008, becoming dominated by small, young individuals in poor body condition, a phenomenon that may result from declines in energy availability. Continuous monitoring of body mass loss and metabolic rate in 78 captive food-deprived individuals revealed that sardines could survive for up to 57 days on body reserves. Sardines submitted to long-term caloric restriction prior to food-deprivation displayed adaptive phenotypic plasticity, reducing metabolic energy expenditure and enduring starvation for longer than sardines that had not been calorie-restricted. Overall, entry into critical fasting phase 3 occurred at a body condition of 0.72. Such a degree of leanness has rarely been observed over 34 years of wild population monitoring. Still, the proportion of sardines below this threshold has doubled since 2008 and is maximal in January and February (the peak of the reproductive season), now reaching almost 10 % of the population at that time. These results indicate that the demographic changes observed in the wild may result in part from starvation-related adult mortality at the end of the winter reproductive period, despite adaptive plastic responses.

Cardiac and behavioural responses to hypoxia and warming in free-swimming gilthead seabream, Sparus aurata.

Gilthead seabream were equipped with intraperitoneal biologging tags to investigate cardiac responses to hypoxia and warming, comparing when fish were either swimming freely in a tank with conspecifics or confined to individual respirometers. After tag implantation under anaesthesia, heart rate (fH) required 60 h to recover to a stable value in a holding tank. Subsequently, when undisturbed under control conditions (normoxia, 21°C), mean fH was always significantly lower in the tank than in the respirometers. In progressive hypoxia (100% to 15% oxygen saturation), mean fH in the tank was significantly lower than in the respirometers at oxygen levels down to 40%, with significant bradycardia in both holding conditions below this level. Simultaneous logging of tri-axial body acceleration revealed that spontaneous activity, inferred as the variance of external acceleration (VARm), was low and invariant in hypoxia. Warming (21 to 31°C) caused progressive tachycardia with no differences in fH between holding conditions. Mean VARm was, however, significantly higher in the tank during warming, with a positive relationship between VARm and fH across all temperatures. Therefore, spontaneous activity contributed to raising fH of fish in the tank during warming. Mean fH in respirometers had a highly significant linear relationship with mean rates of oxygen uptake, considering data from hypoxia and warming together. The high fH of confined seabream indicates that respirometry techniques may bias estimates of metabolic traits in some fishes, and that biologging on free-swimming fish will provide more reliable insight into cardiac and behavioural responses to environmental stressors by fish in their natural environment.

Potential application and beneficial effects of a marine microalgal biomass produced in a high-rate algal pond (HRAP) in diets of European sea bass, Dicentrarchus labrax.

Microalgae have been used as live food in aquatic species. In recent years, the interest in microalgae has considerably increased, thanks to the evolution of production techniques that have identified them as an ecologically attractive aquafeed ingredient. The present study provides the first data about the effects of dietary inclusion of a microalgae consortium grown in a high-rate algal pond system on zootechnical performance, morphometric indices, and dietary nutrient digestibility as well as morphology and functionality of the digestive system of European sea bass, Dicentrarchus labrax. A dietary treatment including a commercial mono-cultured microalgae (Nannochloropsis sp.) biomass was used for comparison. Six hundred and thirty-six European sea bass juveniles (18 ± 0.28 g) were randomly allotted into 12 experimental groups and fed 4 different diets for 10 weeks: a control diet based on fish meal, fish oil, and plant protein sources; a diet including 10% of Nannochloropsis spp. biomass (100 g/kg diet); and two diets including two levels (10% and 20%) of the microalgal consortium (100 and 200 g/kg diet). Even at the highest dietary inclusion level, the microalgal consortium (200 g/kg diet) did not affect feed palatability and fish growth performance. A significant decrease in the apparent digestibility of dry matter, protein, and energy was observed in diets including 10 and 20% of the microalgal consortium, but all fish exhibited a well-preserved intestinal histomorphology. Moreover, dietary inclusion with the microalgal consortium significantly increased the enzymatic activity of maltase, sucrase-isomaltase, and ɤ-glutamil transpeptidase in the distal intestine of the treated European sea bass. Algal consortium grown using fish farm effluents represents an attempt to enhance the utilization of natural biomasses in aquafeeds when used at 10 % as substitute of vegetable ingredients in diet for European sea bass.

Changes in foraging mode caused by a decline in prey size have major bioenergetic consequences for a small pelagic fish.

Global warming is causing profound modifications of aquatic ecosystems and one major outcome appears to be a decline in adult size of many fish species. Over the last decade, sardine populations in the Gulf of Lions (NW Mediterranean Sea) have shown severe declines in body size and condition as well as disappearance of the oldest individuals, which could not be related to overfishing, predation pressure or epizootic diseases. In this study, we investigated whether this situation reflects a bottom-up phenomenon caused by reduced size and availability of prey that could lead to energetic constraints. We fed captive sardines with food items of two different sizes eliciting a change in feeding mode (filter-feeding on small items and directly capturing larger ones) at two different rations for several months, and then assessed their muscle bioenergetics to test for changes in cellular function. Feeding on smaller items was associated with a decline in body condition, even at high ration, and almost completely inhibited growth by comparison to sardines fed large items at high ration. Sardines fed on small items presented specific mitochondrial adjustments for energy sparing, indicating a major bioenergetic challenge. Moreover, mitochondria from sardines in poor condition had low basal oxidative activity but high efficiency of ATP production. Notably, when body condition was below a threshold value of 1.07, close to the mean observed in the wild, it was directly correlated with basal mitochondrial activity in muscle. The results show a link between whole-animal condition and cellular bioenergetics in the sardine, and reveal physiological consequences of a shift in feeding mode. They demonstrate that filter-feeding on small prey leads to poor growth, even under abundant food and an increase in the efficiency of ATP production. These findings may partially explain the declines in sardine size and condition observed in the wild.

Le changement global entraîne de profondes modifications des écosystèmes aquatiques, l'une des principales étant le déclin de la taille des adultes chez de nombreuses espèces de poissons. Au cours de la dernière décennie, les populations de sardines du Golfe du Lion (Nord-Ouest de la Méditerranée) ont montré une importante diminution de leur taille et de leur condition corporelle ainsi qu'une disparition des individus les plus âgés, qui n'ont pas pu être liées à la surpêche, à la pression de prédation ou aux épizooties. Dans cette étude, nous avons cherché à savoir si cette situation reflète un phénomène ascendant causé par la réduction de la taille et de la disponibilité des proies qui pourrait entraîner des contraintes énergétiques chez la sardine. Nous avons ainsi nourri des sardines captives avec des granulés de deux tailles différentes provoquant un changement de mode d'alimentation (filtration des petits granulés et capture directe des plus gros) et à deux rations différentes pendant plusieurs mois, puis nous avons évalué leur bioénergétique musculaire pour tester les changements au niveau de leur fonction cellulaire. L'alimentation à base de petits granulés a été associée à un déclin de la condition corporelle, même à une ration élevée, et à une croissance quasiment inhibée par rapport aux sardines nourries avec des plus gros granulés à une ration élevée. Les sardines nourries avec des petits granulés ont également présenté des ajustements mitochondriaux spécifiques pour économiser de l'énergie, indiquant un défi bioénergétique majeur. De plus, les mitochondries des sardines en mauvaise condition présentaient une faible activité oxydative basale, mais une efficacité élevée de production d'ATP. Notamment, lorsque la condition corporelle était inférieure à une valeur seuil de 1,07, proche de la moyenne observée dans la nature, elle était directement corrélée à l'activité mitochondriale basale dans le muscle. Ces résultats montrent un lien entre la condition de l'animal entier et la bioénergétique cellulaire chez la sardine, et révèlent les conséquences physiologiques d'un changement de mode d'alimentation. Ils démontrent que le nourrissage via la filtration de petites proies entraîne une faible croissance, même en cas de nourriture abondante, et une augmentation de l'efficacité de la production d'ATP. Ces résultats peuvent expliquer en partie le déclin de la taille et de la condition des sardines observé dans la nature.

Oxygen uptake, heart rate and activities of locomotor muscles during a critical swimming speed protocol in the gilthead sea bream Sparus aurata.

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue.

Growth Performance and Adaptability of European Sea Bass (Dicentrarchus labrax) Gut Microbiota to Alternative Diets Free of Fish Products.

Innovative fish diets made of terrestrial plants supplemented with sustainable protein sources free of fish-derived proteins could contribute to reducing the environmental impact of the farmed fish industry. However, such alternative diets may influence fish gut microbial community, health, and, ultimately, growth performance. Here, we developed five fish feed formulas composed of terrestrial plant-based nutrients, in which fish-derived proteins were substituted with sustainable protein sources, including insect larvae, cyanobacteria, yeast, or recycled processed poultry protein. We then analyzed the growth performance of European sea bass (Dicentrarchus labrax L.) and the evolution of gut microbiota of fish fed the five formulations. We showed that replacement of 15% protein of a vegetal formulation by insect or yeast proteins led to a significantly higher fish growth performance and feed intake when compared with the full vegetal formulation, with feed conversion ratio similar to a commercial diet. 16S rRNA gene sequencing monitoring of the sea bass gut microbial community showed a predominance of Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes phyla. The partial replacement of protein source in fish diets was not associated with significant differences on gut microbial richness. Overall, our study highlights the adaptability of European sea bass gut microbiota composition to changes in fish diet and identifies promising alternative protein sources for sustainable aquafeeds with terrestrial vegetal complements.

Muscle bioenergetics of two emblematic Mediterranean fish species: Sardina pilchardus and Sparus aurata.

We investigated links between swimming behavior and muscle bioenergetics in two emblematic Mediterranean fish species that have very different ecologies and activity levels. European sardines Sardina pilchardus are pelagic, they swim aerobically, school constantly and have high muscle fat content. Gilthead seabream Sparus aurata are bentho-pelagic, they show discontinuous spontaneous swimming patterns and store less fat in their muscle. Estimating the proportion of red and white muscle phenotypes, sardine exhibited a larger proportion of red muscle (~10% of the body mass) compared to gilthead seabream (~5% of the body mass). We firstly studied red and white muscle fiber bioenergetics, using high-resolution respirometers, showing a 4-fold higher oxidation capacity for red compared to white muscle. Secondly, we aimed to compare the red muscle ability to oxidize either lipids or carbohydrates. Sardine red muscle had a 3-fold higher oxidative capacity than gilthead seabream and a greater capacity to oxidize lipids. This study provides novel insights into physiological mechanisms underlying the different lifestyles of these highly-prized species.

Transcriptomic Profiling of Egg Quality in Sea Bass (Dicentrarchus labrax) Sheds Light on Genes Involved in Ubiquitination and Translation.

Variable and low egg quality is a major limiting factor for the development of efficient aquaculture production. This stems from limited knowledge on the mechanisms underlying egg quality in cultured fish. Molecular analyses, such as transcriptomic studies, are valuable tools to identify the most important processes modulating egg quality. However, very few studies have been devoted to this aspect so far. Within this study, the microarray-based transcriptomic analysis of eggs (of different quality) of sea bass (Dicentrarchus labrax) was performed. An Agilent oligo microarray experiment was performed on labelled mRNA extracted from 16 batches of eggs (each batch obtained from a different female) of sea bass, in which over 24,000 published probe arrays were used. We identified 39 differentially expressed genes exhibiting a differential expression between the groups of low (fertilization rate < 60 %) and high (fertilization rate > 60 %) quality. The mRNA levels of eight genes were further analyzed by quantitative PCR. Seven genes were confirmed by qPCR to be differentially expressed in eggs of low and high quality. This study confirmed the importance of some of the genes already reported to be potential molecular quality indicators (mainly rnf213 and irf7), but we also found new genes (mainly usp5, mem-prot, plec, cenpf), which had not yet been reported to be quality-dependent in fish. These results suggest the importance of genes involved in several important processes, such as protein ubiquitination, translation, DNA repair, and cell structure and architecture; these probably being the mechanisms that contribute to egg developmental competence in sea bass.