Comparative analysis of blood protein fractions in two mediterranean farmed fish: Dicentrarchus labrax and Sparus aurata.

Total protein levels in fish are widely used in health and welfare studies, providing a simple and accessible measure. However, the multifaceted role of blood proteins makes it sometimes challenging to link total protein content to specific health issues, while specific protein fractions may offer more precise insights into fish biology and health, particularly in farmed fish species where such data is often lacking. Data were gathered from two experiments involving Dicentrarchus labrax and Sparus aurata, key species in European marine aquaculture. The aim was (1) to assess how different globulin fractions contribute to total protein content in blood and (2) how these contributions vary across different sampling times in healthy animals. In D. labrax, the beta1 globulin fraction emerged as the major contributor (34.16%), followed by albumin and alpha2 globulins (18.24% and 16.41%, respectively). In contrast, pre-albumins and alpha1 fractions had the least contribution (5.49% and 7.71%). S. aurata exhibited albumin as the primary contributor (23.39%), followed by beta1 and alpha2 globulins (19.71% and 19.15%, respectively), with gamma and alpha1 fractions contributing the least (5.34% and 8.63%). Notably, the study revealed relatively stable contributions of globulin fractions to total proteins within both species, albeit with minor variations over time, potentially linked to environmental and individual factors. Furthermore, larger fish displayed higher total protein levels. This research underscores the need for further investigation into the diverse factors influencing globulin contributions to total proteins, ultimately enhancing health and welfare monitoring for farmed fish species.

Growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax) fed an environmentally and economically sustainable low marine protein diet in sea cages.

The large use of fish meal/fish oil in carnivorous fish feeds is the main concern regarding environmental sustainability of aquaculture. Here, we evaluated the effects of an innovative diet, designed to be (1) environmentally sustainable by lowering the marine protein content while being (2) cost effective by using sustainable alternative raw materials with acceptable cost and produced on an industrial scale, on growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax), a key species of the Mediterranean marine aquaculture, reared in sea cages. Results show that the specific growth rate of fish fed the low marine protein diet was significantly lower than those fed conventional diet (0.67% vs 0.69%). Fatty acid profile of fillets from fish fed a low marine protein diet presented significant lower n-6 and higher n-3 content when compared to conventional ones. Then, a significant increase in the abundance of Vibrio and reduction of Photobacterium were found in the gut of fish fed with the low marine protein diet but effects on sea bass health needs further investigation. Finally, no major health and welfare alterations for fish fed the low marine protein diet were observed, combined with a potential slight benefit related to humoral immunity. Overall, these results suggest that despite the low marine protein diet moderately affects growth performance, it nevertheless may enhance environmental and economic sustainability of the sea bass aquaculture.

Water temperature explains part of the variation in basal plasma cortisol level within and between fish species.

Within the thermal tolerance range of fish, metabolism is known to escalate with warming. Rapid thermic changes also trigger a series of physiological responses, including activation of the stress axis, producing cortisol. Fish have adapted to their environment by producing a low level of plasmatic cortisol when unstressed (basal), so that thriving in their natural temperature should not impact their basal cortisol levels. Yet, surprisingly, little is known on how temperature affects cortisol within and between fish species. Here, we conducted a phylogenetic meta-analysis to (1) test whether temperature can explain the differences in basal cortisol between species and (2) evaluate the role of temperature on differences in cortisol levels between individuals of a same species. To do this, we retrieved basal plasma cortisol data from 126 studies, investigating 33 marine and freshwater fish species, and correlated it to water temperature. Intra-species variability in basal plasma cortisol levels was further investigated in two species: the European sea bass Dicentrarchus labrax and the Nile tilapia Oreochromis niloticus. Factors such as life stage, sex and weight were also considered in the analyses. Overall, our phylogenetic analysis revealed a clear positive correlation between basal cortisol level and the temperature at which the fish live. The role of temperature has also been confirmed within D. labrax, while it failed to be significant in O. niloticus. In this paper, the influence of habitat, life stage, sex and weight on basal plasma cortisol levels is also discussed. Since some abiotic parameters were not included in the analysis, our study is a call to encourage scientists to systematically report other key factors such as dissolved oxygen or salinity to fully depict the temperature-cortisol relationship in fishes.

Exposure of zebrafish to an environmental mixture of persistent organic pollutants triggers an increase in anxiety-like syndrome but does not affect boldness in unexposed offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs) that are present as complex mixtures in all environmental compartments, including aquatic ecosystems. However, little is known about the effects of such complex mixtures on teleost behaviour. In this study, zebrafish (Danio rerio) were chronically exposed to an environmentally relevant mixture (MIX) containing 22 PCB and 7 PBDE congeners through diet from 5 days post fertilization onwards. MIX-exposed F0 fish produced offspring (F1 and F2 generations) that were fed using plain food and grown until adulthood. In each generation, five behavioural traits (i.e. boldness, activity, sociality, exploration and anxiety) were evaluated by the mean of different experimental set-ups. Two distinct behavioural syndromes were identified: boldness, positively correlated to activity and exploration; and anxiety, associated with low sociality. F0 fish did not display any behavioural disruption resulting from POP exposure whereas F1 MIX fish were bolder than fish from other generations but did not differ significantly from F1 controls. F2 MIX fish displayed a higher anxiety syndrome than F2 controls. This is of particular importance since such behavioural changes in offspring generations may have persistent ecological consequences, may affect fitness and hence cause detrimental effects on wild fish populations exposed to POP mixtures.

Transcriptomic profiles of consistent risk-taking behaviour across time and contexts in European sea bass.

Bolder individuals have greater access to food sources and reproductive partners but are also at increased risk of predation. Boldness is believed to be consistent across time and contexts, but few studies have investigated the stability of this trait across variable environments, such as varying stress loads or long periods of time. Moreover, the underlying molecular components of boldness are poorly studied. Here, we report that boldness of 1154 European sea bass, evaluated using group risk-taking tests, is consistent over seven months and for individuals subjected to multiple environments, including a chronically stressful environment. Differences in risk-taking behaviour were further supported by differences observed in the responses to a novel environment test: shy individuals displayed more group dispersion, more thigmotaxic behaviour and lower activity levels. Transcriptomic analyses performed on extreme phenotypes revealed that bold individuals display greater expression for genes involved in social and exploration behaviours, and memory in the pituitary, and genes involved in immunity and responses to stimuli in the head kidney. This study demonstrates that personality traits come with an underpinning molecular signature, especially in organs involved in the endocrine and immune systems. As such, our results help to depict state-behaviour feedback mechanisms, previously proposed as key in shaping animal personality.

Mapping the Energetic Costs of Free-Swimming Gilthead Sea Bream (Sparus aurata), a Key Species in European Marine Aquaculture.

Measurement of metabolic rates provides a valuable proxy for the energetic costs of different living activities. However, such measurements are not easy to perform in free-swimming fish. Therefore, mapping acceleration from accelerometer tags with oxygen consumption rates (MO2) is a promising method to counter these limitations and could represent a tool for remotely estimating MO2 in aquaculture environments. In this study, we monitored the swimming performance and MO2 of 79 gilthead sea bream (Sparus aurata; weight range, 219-971 g) during a critical swimming test. Among all the fish challenged, 27 were implanted with electromyography (EMG) electrodes, and 27 were implanted with accelerometer tags to monitor the activation pattern of the red/white muscles during swimming. Additionally, we correlated the acceleration recorded by the tag with the MO2. Overall, we found no significant differences in swimming performance, metabolic traits, and swimming efficiency between the tagged and untagged fish. The acceleration recorded by the tag was successfully correlated with MO2. Additionally, through EMG analyses, we determined the activities of the red and white muscles, which are indicative of the contributions of aerobic and anaerobic metabolisms until reaching critical swimming speed. By obtaining insights into both aerobic and anaerobic metabolisms, sensor mapping with physiological data may be useful for the purposes of aquaculture health/welfare remote monitoring of the gilthead sea bream, a key species in European marine aquaculture.

Calibrating Accelerometer Tags with Oxygen Consumption Rate of Rainbow Trout (Oncorhynchus mykiss) and Their Use in Aquaculture Facility: A Case Study.

Metabolic rates are linked to the energetic costs of different activities of an animal's life. However, measuring the metabolic rate in free-swimming fish remains challenging due to the lack of possibilities to perform these direct measurements in the field. Thus, the calibration of acoustic transmitters with the oxygen consumption rate (MO2) could be promising to counter these limitations. In this study, rainbow trout (Oncorhynchus mykiss Walbaum, 1792; n = 40) were challenged in a critical swimming test (Ucrit) to (1) obtain insights about the aerobic and anaerobic metabolism throughout electromyograms; and (2) calibrate acoustic transmitters' signal with the MO2 to be later used as a proxy of energetic costs. After this calibration, the fish (n = 12) were implanted with the transmitter and were followed during ~50 days in an aquaculture facility, as a case study, to evaluate the potential of such calibration. Accelerometer data gathered from tags over a long time period were converted to estimate the MO2. The MO2 values indicated that all fish were reared under conditions that did not impact their health and welfare. In addition, a diurnal pattern with higher MO2 was observed for the majority of implanted trout. In conclusion, this study provides (1) biological information about the muscular activation pattern of both red and white muscle; and (2) useful tools to estimate the energetic costs in free-ranging rainbow trout. The use of acoustic transmitters calibrated with MO2, as a proxy of energy expenditure, could be promising for welfare assessment in the aquaculture industry.

Human protection drives the emergence of a new coping style in animals.

Wild animals face novel environmental threats from human activities that may occur along a gradient of interactions with humans. Recent work has shown that merely living close to humans has major implications for a variety of antipredator traits and physiological responses. Here, we hypothesize that when human presence protects prey from their genuine predators (as sometimes seen in urban areas and at some tourist sites), this predator shield, followed by a process of habituation to humans, decouples commonly associated traits related to coping styles, which results in a new range of phenotypes. Such individuals are characterized by low aggressiveness and physiological stress responses, but have enhanced behavioral plasticity, boldness, and cognitive abilities. We refer to these individuals as "preactive," because their physiological and behavioral coping style falls outside the classical proactive/reactive coping styles. While there is some support for this new coping style, formal multivariate studies are required to investigate behavioral and physiological responses to anthropogenic activities.

An environmentally relevant mixture of polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) disrupts mitochondrial function, lipid metabolism and neurotransmission in the brain of exposed zebrafish and their unexposed F2 offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants still present in aquatic environments despite their total or partial ban. Previously, we observed that an environmentally realistic mixture of these compounds affects energy balance, growth, and reproduction in exposed zebrafish (F0), and behavior in their unexposed offspring (F1-F4). In the present work, we performed lipidomic and transcriptomic analyses on brains of zebrafish (F0-F2) from exposed and control lineages to identify molecular changes that could explain the observed phenotypes. The use of both technologies highlighted that F0 zebrafish displayed impaired mitochondrial function and lipid metabolism regulation (depletion in triacylglycerols and phospholipids) which can explain disruption of energy homeostasis. A subset of the regulated biological pathways related to energetic metabolism and neurotransmission were inherited in F2. In addition, there were increasing effects on epigenetic pathways from the F0 to the F2 generation. Altogether, we show that the effects of an environmental exposure to PCBs and PBDEs on energetic metabolism as well as neurotransmission extend over 2 generations of zebrafish, possibly due to transgenerational epigenetic inheritance.

Temperature increase and its effects on fish stress physiology in the context of global warming.

The capacity of fishes to cope with environmental variation is considered to be a main determinant of their fitness and is partly determined by their stress physiology. By 2100, global ocean temperature is expected to rise by 1-4°C, with potential consequences for stress physiology. Global warming is affecting animal populations worldwide through chronic temperature increases and an increase in the frequency of extreme heatwave events. As ectotherms, fishes are expected to be particularly vulnerable to global warming. Although little information is available about the effects of global warming on stress physiology in nature, multiple studies describe the consequences of temperature increases on stress physiology in controlled laboratory conditions, providing insight into what can be expected in the wild. Chronic temperature increase constitutes a physiological load that can alter the ability of fishes to cope with additional stressors, which might compromise their fitness. In addition, rapid temperature increases are known to induce acute stress responses in fishes and might be of ecological relevance in particular situations. This review summarizes knowledge about effects of temperature increases on the stress physiology of fishes and discusses these in the context of global warming.

Zebrafish Danio rerio shows behavioural cross-context consistency at larval and juvenile stages but no consistency between stages.

Coping style is defined as a set of individual physiological and behavioural characteristics that are consistent across time and context. In the zebrafish Danio rerio, as well as in many other animals, several covariations have been established among behavioural, physiological and molecular responses. Nonetheless, not many studies have addressed the consistency in behavioural responses over time starting at the larval stage. Therefore, this study aimed to improve the understanding of behavioural consistency across contexts and over time in zebrafish from the larval to juvenile stages. Two distinct experiments were conducted: a larval stage experiment (from 8 to 21 days post fertilization, dpf) and a juvenile stage experiment (from 21 to 60 dpf). On one hand, the larval experiment allows to focus on the transition between 8 and 21 dpf, marked by significant morphological changes related to the end of larval stage and initiation of metamorphosis. On the other hand, the juvenile experiment allows to properly cover the period extending from the end of larval stage to the juvenile stage (60 dpf), including metamorphosis which is itself completed around 45 dpf. Within each experiment, boldness was determined using a group risk-taking test to identify bold and shy individuals. A novel environment test was then performed at the same age to evaluate consistency across contexts. Groups of fish (either bold or shy) were bathed in an alizarin red S solution for later identification of their initially determined coping style to evaluate behavioural consistency over time. Fish were then reared under common garden conditions and challenged again with the same behavioural tests at a later age (21 and 60 dpf in the larval and juvenile experiments, respectively). Behavioural consistency was observed across contexts, with bold fish being more active and expressing higher thigmotaxis regardless of age. There was, however, little behavioural consistency over age, suggesting behavioural plasticity during development. Moreover, the use of alizarin red S to conduct this experiment provides new perspectives for the further study of the longitudinal evolution of various traits, including behaviour, over life stages in fish.

Behavioral and physiological responses to stocking density in sea bream (Sparus aurata): Do coping styles matter?

Stocking density is considered a stress factor for fish and is therefore one of the numerous concerns about fish welfare in an aquaculture context. Stress coping styles (SCS) are defined as a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context and appear to be promising for improving fish welfare in aquaculture. The aim of the present study was to describe the physiological and zootechnical performances of gilthead sea bream (Sparus aurata) at different stocking densities (low density, LD: 15 kg/m3 and high density, HD: 30 kg/m3), depending on individual SCS. To do so, the fish SCS were first screened by measuring boldness (prior to the experiment). Three consecutive samplings were performed over the experiment to measure several blood parameters, including hematocrit (Hct), red blood cell count (RBCC), hemoglobin (Hb), cortisol, adrenalin, noradrenalin, glucose, lactate, and lysozyme, to infer the consequence of the SCS profile on the welfare condition in response to stocking density. Finally, swimming activity was recorded in a subsample of individuals (9 BOLD and 9 SHY individuals per density), and BOLD individuals displayed higher swimming activity than SHY ones at HD, while the opposite pattern was observed at LD. According to principal component analysis, physiological parameters are linked to the SCS profile, mostly at the beginning of the experiment, while density effects on physiology remain during the entire experiment duration. In conclusion, regarding all the variables observed, fish SCS appeared to be promising criteria to select the most adaptive individuals relating to rearing conditions and therefore improve welfare.

Coping styles in European sea bass: The link between boldness, stress response and neurogenesis.

Coping styles consist of a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context. Such consistent inter-individual differences in behavior have already been shown in European sea bass (Dicentrarchus labrax), but the associated mechanisms are still poorly understood. Here, we combine physiological measurements with individual behavioral responses in order to characterize coping styles in fish. Fish were tagged and placed in a tank for group risk-taking tests (GRT) at 8 months of age to evaluate boldness using the proxy latency of leaving a sheltered area towards an open area. A subsample of these fish were individually challenged 16 months later using an open field test (OFT), in which the boldness was assessed after being placed in a shelter within an open arena. Latency to exit the shelter, time spent in the shelter, and distance travelled were recorded for this purpose. The blood and brain were then collected to evaluate plasma cortisol concentration and neurotransmitter levels (dopamine, norepinephrine, serotonin, and related metabolites), as well as brain transcription of key genes involved in stress axis regulation (gr1, gr2, mr, crf), neurogenesis (neurod1, neurod2, pcna), and neuronal development (egr1). Fish acting bolder in the GRT were not necessarily those acting bolder in the OFT, highlighting the relatively low consistency across different types of tests performed with a 16-months interval. There was, however, a significant correlation between stress markers and boldness. Indeed, mRNA levels of mr, crf, gr2, egr1, and neurod2, as well as norepinephrine levels were higher in shy than bold fish, whereas brain serotonergic activity was lower in shy fish. Overall, our study highlights the fact that boldness was not consistent over time when testing context differed (group vs. alone). This is in agreement with previous literature suggesting that social context play a key role in boldness measurement and that the particular life history of each individual may account in shaping the personality fate of a fish.

Examining multi- and transgenerational behavioral and molecular alterations resulting from parental exposure to an environmental PCB and PBDE mixture.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants extensively used during the 20th century and still present in aquatic environments despite their ban. Effects of exposure to these compounds over generations are poorly documented. Therefore, our aims were to characterize behavioral responses and underlying molecular mechanisms in zebrafish exposed to an environmentally relevant mixture of PCBs and PBDEs as well as in four unexposed offspring generations. Zebrafish (F0) were chronically exposed from the first meal onward to a diet spiked with a mixture containing 22 PCB and 7 PBDE congeners in proportions and concentrations reflecting environmental situations (ΣPCBs = 1991 and ΣPBDEs = 411 ng/g). Four offspring generations (F1 to F4) were obtained from this F0 and were not further exposed. Behavior was assessed at both larval and adult stages. Mechanisms related to behavioral defects (habenula maturation and c-fos transcription) and methylation (dnmts transcription) were monitored in larvae. Exposed adult F0 as well as F1 and F3 adults displayed no behavioral change while F2 expressed anxiety-like behavior. Larval behavior was also disrupted, i.e. hyperactive after light to dark transition in F1 or hypoactive in F2, F3 and F4. Behavioral disruptions may be related to defect in habenula maturation (observed in F1) and change in c-fos transcription (observed in F1 and F2). Transcription of the gene encoding DNA methyltransferase (dnmt3ba) was also modified in all generations. Our results lead us to hypothesize that chronic dietary exposure to an environmentally relevant mixture of PCB and PBDE triggers multigenerational and transgenerational molecular and behavioral disruptions in a vertebrate model.

Fish life-history traits are affected after chronic dietary exposure to an environmentally realistic marine mixture of PCBs and PBDEs.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants that have been shown to affect fish life-history traits such as reproductive success, growth and survival. At the individual level, their toxicity and underlying mechanisms of action have been studied through experimental exposure. However, the number of experimental studies approaching marine environmental situations is scarce, i.e., in most cases, individuals are exposed to either single congeners, or single types of molecules, or high concentrations, so that results can hardly be transposed to natural populations. In the present study, we evaluated the effect of chronic dietary exposure to an environmentally realistic marine mixture of PCB and PBDE congeners on zebrafish life-history traits from larval to adult stage. Exposure was conducted through diet from the first meal and throughout the life cycle of the fish. The mixture was composed so as to approach environmentally relevant marine conditions in terms of both congener composition and concentrations. Life-history traits of exposed fish were compared to those of control individuals using several replicate populations in each treatment. We found evidence of slower body growth, but to a larger asymptotic length, and delayed spawning probability in exposed fish. In addition, offspring issued from early spawning events of exposed fish exhibited a lower larval survival under starvation condition. Given their strong dependency on life-history traits, marine fish population dynamics and associated fisheries productivity for commercial species could be affected by such individual-level effects of PCBs and PBDEs on somatic growth, spawning probability and larval survival.