Transgenerational exposure to ocean acidification impacts the hepatic transcriptome of European sea bass (Dicentrarchus labrax).

Physiological effects of ocean acidification associated with elevated CO2 concentrations in seawater is the subject of numerous studies in teleost fish. While the short time within-generation impact of ocean acidification (OA) on acid-base exchange and energy metabolism is relatively well described, the effects associated with transgenerational exposure to OA are much less known. Yet, the impacts of OA can vary in time with the potential for acclimation or adaptation of a species. Previous studies in our lab demonstrated that transgenerational exposure to OA had extensive effects on the transcriptome of the olfactory epithelium of European sea bass (Dicentrarchus labrax), especially on genes related to ion balance, energy metabolism, immune system, synaptic plasticity, neuron excitability and wiring. In the present study, we complete the previous work by investigating the effect of transgenerational exposure to OA on the hepatic transcriptome of European sea bass. Differential gene expression analysis was performed by RNAseq technology on RNA extracted from the liver of two groups of 18 months F2 juveniles that had been exposed since spawning to the same AO conditions as their parents (F1) to either actual pH or end-of-century predicted pH levels (IPCC RCP8.5), respectively. Here we show that transgenerational exposure to OA significantly impacts the expression of 236 hepatic transcripts including genes mainly involved in inflammatory/immune responses but also in carbohydrate metabolism and cellular homeostasis. Even if this transcriptomic impact is relatively limited compared to what was shown in the olfactory system, this work confirmed that fish transgenerationally exposed to OA exhibit molecular regulation of processes related to metabolism and inflammation. Also, our data expand the up-regulation of a key gene involved in different physiological pathways including calcium homeostasis (i.e. pthr1), which we already observed in the olfactory epithelium, to the liver. Even if our experimental design does not allow to discriminate direct within F2 generation effects from transgenerational plasticity, these results offer the perspective of more functional analyses to determine the potential physiological impact of OA exposure on fish physiology with ecological relevance.

Ocean acidification alters the acute stress response of a marine fish.

The absorption of anthropogenic carbon dioxide from the atmosphere by oceans generates rapid changes in seawater carbonate system and pH, a process termed ocean acidification. Exposure to acidified water can impact the allostatic load of marine organism as the acclimation to suboptimal environments requires physiological adaptive responses that are energetically costly. As a consequence, fish facing ocean acidification may experience alterations of their stress response and a compromised ability to cope with additional stress, which may impact individuals' life traits and ultimately their fitness. In this context, we carried out an integrative study investigating the impact of ocean acidification on the physiological and behavioral stress responses to an acute stress in juvenile European sea bass. Fish were long term (11 months) exposed to present day pH/CO2 condition or acidified water as predicted by IPCC "business as usual" (RCP8.5) scenario for 2100 and subjected to netting stress (fish transfer and confinement test). Fish acclimated to acidified condition showed slower post stress return to plasma basal concentrations of cortisol and glucose. We found no clear indication of regulation in the central and interrenal tissues of the expression levels of gluco- and mineralocorticoid receptors and corticoid releasing factor. At 120 min post stress, sea bass acclimated to acidified water had divergent neurotransmitters concentrations pattern in the hypothalamus (higher serotonin levels and lower GABA and dopamine levels) and a reduction in motor activity. Our experimental data indicate that ocean acidification alters the physiological response to acute stress in European sea bass via the neuroendocrine regulation of the corticotropic axis, a response associated to an alteration of the motor behavioral profile. Overall, this study suggests that behavioral and physiological adaptive response to climate changes related constraints may impact fish resilience to further stressful events.

Temporal variations in scale cortisol indicate consistent local-and broad-scale constraints in a wild marine teleost fish.

Environmental changes can alter the nursery function of coastal areas through their impact on juveniles' growth and survival rates, an effect mediated by individuals' chronic stress response. Fish chronic stress can be quantified using scale cortisol but no study has yet been quantified the spatio-temporal variations in scale cortisol and its relationship with growth in wild nurseries. We collected wild sea bass juveniles (Dicentrarchus labrax, four years, three nurseries) and found that scale cortisol levels increased consistently with age and across cohorts in 2019 and 2020 probably due to greater stress history in older fish and/or heatwaves that occurred in summers of 2018 and 2019. Growth was impaired in fish with high scale cortisol in 2019 and 2020, confirming the usefulness of scale cortisol as a biomarker of broad and local constraints in wild fish; longer time series will enable us to identify environmental factors underpinning these temporal variations.

Responses of early life stages of European abalone (Haliotis tuberculata) to ocean acidification after parental conditioning: Insights from a transgenerational experiment.

CO2 absorption is leading to ocean acidification (OA), which is a matter of major concern for marine calcifying species. This study investigated the effects of simulated OA on the reproduction of European abalone Haliotis tuberculata and the survival of its offspring. Four-year-old abalone were exposed during reproductive season to two relevant OA scenarios, ambient pH (8.0) and low pH (7.7). After five months of exposure, abalone were induced to spawn. The gametes, larvae and juveniles were then exposed for five months to the same pH conditions as their parents. Several biological parameters involved in adult reproduction as well as in larval, post-larval and juvenile fitness were measured. No effects on gametes, fertilisation or larval oxidative stress response were detected. However, developmental abnormalities and significant decreases in shell length and calcification were observed at veliger stages. The expression profile of a GABA A receptor-like gene appeared to be regulated by pH, depending on larval stage. Larval and post-larval survival was not affected by low pH. However, a lower survival and a reduction of growth were recorded in juveniles at pH 7.7. Our results confirm that OA negatively impacts larval and juvenile fitness and suggest the absence of carry-over effects on abalone offspring. This may compromise the survival of abalone populations in the near future.

Natural cortisol production is not linked to the sexual fate of European sea bass.

In this study, we aimed to investigate the relationship between cortisol and the determination of sexual fate in the commercially important European sea bass (Dicentrarchus labrax). To test our hypothesis, we designed two temperature-based experiments (19 ℃, 21 ℃ and 23 ℃, experiment 1; 16 ℃ and 21 ℃, experiment 2) to assess the effects of these thermal treatments on European sea bass sex determination and differentiation. In the fish from the first experiment, we evaluated whether blood cortisol levels and expression of stress key regulatory genes were different between differentiating (149 to 183 dph) males and females. In the second experiment, we assessed whether cortisol accumulated in scales over time during the labile period for sex determination as well as the neuroanatomical localisation of brain cells expressing brain aromatase (cyp19a1b) and corticotropin-releasing factor (crf) differed between males and females undergoing molecular sex differentiation (117 to 124 dph). None of the gathered results allowed to detect differences between males and females regarding cortisol production and regulatory mechanisms. Altogether, our data provide strong physiological, molecular and histochemical evidence, indicating that in vivo cortisol regulation has no major effects on the sex of European sea bass.

CO2 induced seawater acidification impacts survival and development of European eel embryos.

Fish embryos may be vulnerable to seawater acidification resulting from anthropogenic carbon dioxide (CO2) emissions or from excessive biological CO2 production in aquaculture systems. This study investigated CO2 effects on embryos of the European eel (Anguilla anguilla), a catadromous fish that is considered at risk from climate change and that is targeted for hatchery production to sustain aquaculture of the species. Eel embryos were reared in three independent recirculation systems with different pH/CO2 levels representing "control" (pH 8.1, 300 µatm CO2), end-of-century climate change ("intermediate", pH 7.6, 900 µatm CO2) and "extreme" aquaculture conditions (pH 7.1, 3000 µatm CO2). Sensitivity analyses were conducted at 4, 24, and 48 hours post-fertilization (hpf) by focusing on development, survival, and expression of genes related to acute stress response (crhr1, crfr2), stress/repair response (hsp70, hsp90), water and solute transport (aqp1, aqp3), acid-base regulation (nkcc1a, ncc, car15), and inhibitory neurotransmission (GABAAα6b, Gabra1). Results revealed that embryos developing at intermediate pH showed similar survival rates to the control, but egg swelling was impaired, resulting in a reduction in egg size with decreasing pH. Embryos exposed to extreme pH had 0.6-fold decrease in survival at 24 hpf and a 0.3-fold change at 48 compared to the control. These observed effects of acidification were not reflected by changes in expression of any of the here studied genes. On the contrary, differential expression was observed along embryonic development independent of treatment, indicating that the underlying regulating systems are under development and that embryos are limited in their ability to regulate molecular responses to acidification. In conclusion, exposure to predicted end-of-century ocean pCO2 conditions may affect normal development of this species in nature during sensitive early life history stages with limited physiological response capacities, while extreme acidification will negatively influence embryonic survival and development under hatchery conditions.

Effect of long-term intergenerational exposure to ocean acidification on ompa and ompb transcripts expression in European seabass (Dicentrarchus labrax).

Since sensory system allows organisms to perceive and interact with their external environment, any disruption in their functioning may have detrimental consequences on their survival. Ocean acidification has been shown to potentially impair olfactory system in fish and it is therefore essential to develop biological tools contributing to better characterize such effects. The olfactory marker protein (omp) gene is involved in the maturation and the activity of olfactory sensory neurons in vertebrates. In teleosts, two omp genes (ompa and ompb) originating from whole genome duplication have been identified. In this study, bioinformatic analysis allowed characterization of the ompa and ompb genes from the European seabass (Dicentrarchus labrax) genome. The European seabass ompa and ompb genes differ in deduced amino acid sequences and in their expression pattern throughout the tissues. While both ompa and ompb mRNA are strongly expressed in the olfactory epithelium, ompb expression was further observable in different brain areas while ompa expression was also detected in the eyes and in other peripheral tissues. Expression levels of ompa and ompb mRNA were investigated in adult seabass (4 years-old, F0) and in their offspring (F1) exposed to pH of 8 (control) or 7.6 (ocean acidification, OA). Under OA ompb mRNA was down-regulated while ompa mRNA was up-regulated in the olfactory epithelium of F0 adults, suggesting a long-term intragenerational OA-induced regulation of the olfactory sensory system. A shift in the expression profiles of both ompa and ompb mRNA was observed at early larval stages in F1 under OA, suggesting a disruption in the developmental process. Contrary to the F0, the expression of ompa and ompb mRNA was not anymore significantly regulated under OA in the olfactory epithelium of juvenile F1 fish. This work provides evidence for long-term impact of OA on sensorial system of European seabass as well as potential intergenerational acclimation of omp genes expression to OA in European seabass.

Transgenerational regulation of cbln11 gene expression in the olfactory rosette of the European sea bass (Dicentrarchus labrax) exposed to ocean acidification.

Elevated amounts of atmospheric CO2 are causing ocean acidification (OA) that may affect marine organisms including fish species. While several studies carried out in fish revealed that OA induces short term dysfunction in sensory systems including regulation of neurons activity in olfactory epithelium, information on the effects of OA on other physiological processes and actors is scarcer. In the present study we focused our attention on a European sea bass (Dicentrarchus labrax) sghC1q gene, a member of the C1q-domain-containing (C1qDC) protein family. In vertebrates, C1qDC family includes actors involved in different physiological processes including immune response and synaptic organization. Our microsynteny analysis revealed that this sghC1q gene is the orthologous gene in European sea bass to zebrafish (Danio rerio) cbln11 gene. We cloned the full length cbln11 mRNA and identified the different domains (the signal peptide, the coiled coil region and the globular C1q domain) of the deduced protein sequence. Investigation of mRNA expression by qPCR and in situ hybridization revealed that cbln11gene is especially expressed in the non-sensory epithelium of the olfactory rosette at larval and adult stages. The expression of cbln11 mRNA was analysed by qPCR in the first generation (F0) of European sea bass broodstock exposed since larval stages to water pH of 8.0 (control) or 7.6 (predicted for year 2100) and in their offspring (F1) maintained in the environmental conditions of their parents. Our results showed that cbln11 mRNA expression level was lower in larvae exposed to OA then up-regulated at adult stage in the olfactory rosette of F0 and that this up-regulation is maintained under OA at larval and juvenile stages in F1. Overall, this work provides evidence of a transgenerational inheritance of OA-induced up-regulation of cbln11 gene expression in European sea bass. Further studies will investigate the potential immune function of cbln11 gene and the consequences of these regulations, as well as the possible implications in terms of fitness and adaptation to OA in European sea bass.

Climate change impacts on fish reproduction are mediated at multiple levels of the brain-pituitary-gonad axis.

Anthropogenic emissions of carbon dioxide in the atmosphere have generated rapid variations in atmospheric composition which drives major climate changes. Climate change related effects include changes in physico-chemical proprieties of sea and freshwater, such as variations in water temperature, salinity, pH/pCO2 and oxygen content, which can impact fish critical physiological functions including reproduction. In this context, the main aim of the present review is to discuss how climate change related effects (variation in water temperature and salinity, increases in duration and frequency of hypoxia events, water acidification) would impact reproduction by affecting the neuroendocrine axis (brain-pituitary-gonad axis). Variations in temperature and photoperiod regimes are known to strongly affect sex differentiation and the timing and phenology of spawning period in several fish species. Temperature mainly acts at the level of gonad by interfering with steroidogenesis, (notably on gonadal aromatase activity) and gametogenesis. Temperature is also directly involved in the quality of released gametes and embryos development. Changes in salinity or water acidification are especially associated with reduction of sperm quality and reproductive output. Hypoxia events are able to interact with gonad steroidogenesis by acting on the steroids precursor cholesterol availability or directly on aromatase action, with an impact on the quality of gametes and reproductive success. Climate change related effects on water parameters likely influence also the reproductive behavior of fish. Although the precise mechanisms underlying the regulation of these effects are not always understood, in this review we discuss different hypothesis and propose future research perspectives.

Long-term exposure to near-future ocean acidification does not affect the expression of neurogenesis- and synaptic transmission-related genes in the olfactory bulb of European sea bass (Dicentrarchus labrax).

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 ≈ 980 µatm; pH total = 7.7 and PCO2 ≈ 1520 µatm; 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.

Molecular Ontogeny of First-Feeding European Eel Larvae.

Digestive system functionality of fish larvae relies on the onset of genetically pre-programmed and extrinsically influenced digestive functions. This study explored how algal supplementation (green-water) until 14 days post hatch (dph) and the ingestion of food [enriched rotifer (Brachionus plicatilis) paste] from 15 dph onward affects molecular maturation and functionality of European eel larval ingestion and digestion mechanisms. For this, we linked larval biometrics to expression of genes relating to appetite [ghrelin (ghrl), cholecystokinin (cck)], food intake [proopiomelanocortin (pomc)], digestion [trypsin (try), triglyceride lipase (tgl), amylase (amyl)], energy metabolism [ATP synthase F0 subunit 6 (atp6), cytochrome-c-oxidase 1 (cox1)], growth [insulin-like growth factor (igf1)] and thyroid metabolism [thyroid hormone receptors (thrαA, thrßB)]. Additionally, we estimated larval nutritional status via nucleic acid analysis during transition from endogenous and throughout the exogenous feeding stage. Results showed increased expression of ghrl and cck on 12 dph, marking the beginning of the first-feeding window, but no benefit of larviculture in green-water was observed. Moreover, expression of genes relating to protein (try) and lipid (tgl) hydrolysis revealed essential digestive processes occurring from 14 to 20 dph. On 16 dph, a molecular response to initiation of exogenous feeding was observed in the expression patterns of pomc, atp6, cox1, igf1, thrαA and thrßB. Additionally, we detected increased DNA contents, which coincided with increased RNA contents and greater body area, reflecting growth in feeding compared to non-feeding larvae. Thus, the here applied nutritional regime facilitated a short-term benefit, where feeding larvae were able to sustain growth and better condition than their non-feeding conspecifics. However, RNA:DNA ratios decreased from 12 dph onward, indicating a generally low larval nutritional condition, probably leading to the point-of-no-return and subsequent irreversible mortality due to unsuccessful utilization of exogenous feeding. In conclusion, this study molecularly identified the first-feeding window in European eel and revealed that exogenous feeding success occurs concurrently with the onset of a broad array of enzymes and hormones, which are known to regulate molecular processes in feeding physiology. This knowledge constitutes essential information to develop efficient larval feeding strategies and will hopefully provide a promising step toward sustainable aquaculture of European eel.

Salinity reduction benefits European eel larvae: Insights at the morphological and molecular level.

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 (nkcc2α, nkcc2ß, aqp1dup, aqpe), stress response (hsp70, hsp90), and thyroid metabolism (thrαA, thrαB, thrßB, dio1, dio2, dio3) were differentially expressed throughout larval development, while nkcc1α, nkcc2ß, aqp3, aqp1dup, aqpe, hsp90, thrαA 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.

Moderate hypoxia but not warming conditions at larval stage induces adverse carry-over effects on hypoxia tolerance of European sea bass (Dicentrarchus labrax) juveniles.

Environmental conditions, to which organisms are exposed during all their life, may cause possible adaptive responses with consequences in their subsequent life-history trajectory. The objective of this study was to investigate whether ecologically relevant combinations of hypoxia (40% and 100% air saturation) and temperature (15° and 20 °C), occurring during the larval period of European sea bass larvae (Dicentrarchus labrax), could have long-lasting impacts on the physiology of resulting juveniles. Hypoxic challenge tests were performed over one year to give an integrative evaluation of physiological performance. We revealed that juvenile performance was negatively impacted by hypoxia but not by the thermal conditions experienced at larval stage. This impact was related to the prevalence of opercular abnormalities. The present study indicates that exposure to a moderate hypoxia event during larval stage may have adverse carry-over effects, which could compromise fitness and population recruitment success.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol.

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae.

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°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°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20-22°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°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~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°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 development of contemporary European sea bass larvae (Dicentrarchus labrax) is not affected by projected ocean acidification scenarios.

Ocean acidification is a recognized consequence of anthropogenic carbon dioxide (CO2) emission in the atmosphere. Despite its threat to marine ecosystems, little is presently known about the capacity for fish to respond efficiently to this acidification. In adult fish, acid-base regulatory capacities are believed to be relatively competent to respond to hypercapnic conditions. However, fish in early life stage could be particularly sensitive to environmental factors as organs and important physiological functions become progressively operational during this period. In this study, the response of European sea bass (Dicentrarchus labrax) larvae reared under three ocean acidification scenarios, i.e., control (present condition, [Formula: see text] = 590 µatm, pH total = 7.9), low acidification (intermediate IPCC scenario, [Formula: see text] = 980 µatm, pH total = 7.7), and high acidification (most severe IPCC scenario, [Formula: see text] = 1520 µatm, pH total = 7.5) were compared across multiple levels of biological organizations. From 2 to 45 days-post-hatching, the chronic exposure to the different scenarios had limited influence on the survival and growth of the larvae (in the low acidification condition only) and had no apparent effect on the digestive developmental processes. The high acidification condition induced both faster mineralization and reduction in skeletal deformities. Global (microarray) and targeted (qPCR) analysis of transcript levels in whole larvae did not reveal any significant changes in gene expression across tested acidification conditions. Overall, this study suggests that contemporary sea bass larvae are already capable of coping with projected acidification conditions without having to mobilize specific defense mechanisms.

Early exposure to chronic hypoxia induces short- and long-term regulation of hemoglobin gene expression in European sea bass (Dicentrarchus labrax).

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-Hbα3, MN-Hbß4 and MN-Hbß5 and increase of MN-Hbα2, LA-Hbα1 and LA-Hbß1 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-Hbα2, LA-Hbα1 and LA-Hbß1 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.

An early life hypoxia event has a long-term impact on protein digestion and growth in juvenile European sea bass.

Ocean warming, eutrophication and the consequent decrease in oxygen lead to smaller average fish size. Although such responses are well known in an evolutionary context, involving multiple generations, this appears to be incompatible with current rapid environmental change. Instead, 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 with 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 food intake. We measured several respirometry parameters and we only found a higher specific dynamic action (SDA) 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.

Leptin receptor gene in the European sea bass (Dicentrarchus labrax): Cloning, phylogeny, tissue distribution and neuroanatomical organization.

In this study, we report the cloning of three transcripts for leptin receptor in the European sea bass, a marine teleost of economic interest. The two shortest variants, generated by different splice sites, encode all functional extracellular and intracellular domains but missed the transmembrane domain. The resulting proteins are therefore potential soluble binding proteins for leptin. The longest transcript (3605bp), termed sblepr, includes all the essential domains for binding and transduction of the signal. Thus, it is proposed as the ortholog for the human LEPR gene, the main responsible for leptin signaling. Phylogenetic analysis shows the sblepr clustered within the teleost leptin receptor group in 100% of the bootstrap replicates. The neuroanatomical localization of sblepr expressing cells has been assessed by in situ hybridization in brains of sea bass of both sexes during their first sexual maturation. At histological level, the distribution pattern of sblepr expressing cells in the brain shows no clear differences regarding sex or reproductive season. Transcripts of the sblepr have a widespread distribution throughout the forebrain and midbrain until the caudal portion of the hypothalamus. A high hybridization signal is detected in the telencephalon, preoptic area, medial basal and caudal hypothalamus and in the pituitary gland. In a more caudal region, sblepr expressing cells are identified in the longitudinal torus. The expression pattern observed for sblepr suggests that in sea bass, leptin is very likely to be involved in the control of food intake, energy reserves and reproduction.

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance.

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca(2+) response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 °C and changes in intracellular calcium concentration ([Ca(2+)]i) following KCl stimulation were measured using Fura-2, at 12 or 22 °C-test. The increase in [Ca(2+)]i resulted primarily from extracellular Ca(2+) entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca(2+) response was observed between 12- and 22 °C-acclimated fish. In particular, a greater increase in [Ca(2+)]i at a high level of adrenaline was observed in 22 °C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.