Smaller herring larval size-at-stage in response to environmental changes is associated with ontogenic processes and stress response.

Global change puts coastal systems under pressure, affecting the ecology and physiology of marine organisms. In particular, fish larvae are sensitive to environmental conditions, and their fitness is an important determinant of fish stock recruitment and fluctuations. To assess the combined effects of warming, acidification and change in food quality, herring larvae were reared in a control scenario (11°C*pH 8.0) and a scenario predicted for 2100 (14°C*pH 7.6) crossed with two feeding treatments (enriched in phosphorus and docosahexaenoic acid or not). The experiment lasted from hatching to the beginning of the post-flexion stage (i.e. all fins present) corresponding to 47 days post-hatch (dph) at 14°C and 60 dph at 11°C. Length and stage development were monitored throughout the experiment and the expression of genes involved in growth, metabolic pathways and stress responses were analysed for stage 3 larvae (flexion of the notochord). Although the growth rate was unaffected by acidification and temperature changes, the development was accelerated in the 2100 scenario, where larvae reached the last developmental stage at a smaller size (-8%). We observed no mortality related to treatments and no effect of food quality on the development of herring larvae. However, gene expression analyses revealed that heat shock transcripts expression was higher in the warmer and more acidic treatment. Our findings suggest that the predicted warming and acidification environment are stressful for herring larvae, inducing a decrease in size-at-stage at a precise period of ontogeny. This could either negatively affect survival and recruitment via the extension of the predation window or positively increase the survival by reducing the larval stage duration.

Effect of Early Peptide Diets on Zebrafish Skeletal Development.

Incorporation of dietary peptides has been correlated with decreased presence of skeletal abnormalities in marine larvae. In an attempt to clarify the effect of smaller protein fractions on fish larval and post-larval skeleton, we designed three isoenergetic diets with partial substitution of their protein content with 0% (C), 6% (P6) and 12% (P12) shrimp di- and tripeptides. Experimental diets were tested in zebrafish under two regimes, with inclusion (ADF-Artemia and dry feed) or lack (DF-dry feed only) of live food. Results at the end of metamorphosis highlight the beneficial effect of P12 on growth, survival and early skeletal quality when dry diets are provided from first feeding (DF). Exclusive feeding with P12 also increased the musculoskeletal resistance of the post-larval skeleton against the swimming challenge test (SCT). On the contrary, Artemia inclusion (ADF) overruled any peptide effect in total fish performance. Given the unknown species' larval nutrient requirements, a 12% dietary peptide incorporation is proposed for successful rearing without live food. A potential nutritional control of the larval and post-larval skeletal development even in aquaculture species is suggested. Limitations of the current molecular analysis are discussed to enable the future identification of the peptide-driven regulatory pathways.

The role of starter diets in the development of skeletal abnormalities in zebrafish Danio rerio (Hamilton, 1822).

Fish skeletal development has long been correlated with nutritional factors. Lack of zebrafish nutritional standardization, especially during the early stages, decreases the reproducibility of the conducted research. The present study represents an evaluation of four commercial (A, D, zebrafish specific; B, generic for freshwater larvae; C, specific for marine fish larvae) and one experimental (Ctrl) early diets on zebrafish skeletal development. Skeletal abnormalities rates in the different experimental groups were assessed at the end of the larval period (20 days post-fertilization, dpf) and after a swimming challenge test (SCT, 20-24 dpf). At 20 dpf, results revealed a significant effect of diet on the rate of caudal-peduncle scoliosis and gill-cover abnormalities, which were relatively elevated in B and C groups. SCT results focused on swimming-induced lordosis, which was comparatively elevated in diets C and D (83% ± 7% and 75% ± 10%, respectively, vs. 52% ± 18% in diet A). No significant effects of dry diets were observed on the survival and growth rate of zebrafish. Results are discussed with respect to the deferential diet composition between the groups and the species requirements. A potential nutritional control of haemal lordosis in finfish aquaculture is suggested.

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.

The relationships between growth rate and mitochondrial metabolism varies over time.

Mitochondrial metabolism varies significantly between individuals of the same species and can influence animal performance, such as growth. However, growth rate is usually determined before the mitochondrial assay. The hypothesis that natural variation in mitochondrial metabolic traits is linked to differences in both previous and upcoming growth remains untested. Using biopsies to collect tissue in a non-lethal manner, we tested this hypothesis in a fish model (Dicentrarchus labrax) by monitoring individual growth rate, measuring mitochondrial metabolic traits in the red muscle, and monitoring the growth of the same individuals after the mitochondrial assay. Individual variation in growth rate was consistent before and after the mitochondrial assay; however, the mitochondrial traits that explained growth variation differed between the growth rates determined before and after the mitochondrial assay. While past growth was correlated with the activity of the cytochrome c oxidase, a measure of mitochondrial density, future growth was linked to mitochondrial proton leak respiration. This is the first report of temporal shift in the relationship between growth rate and mitochondrial metabolic traits, suggesting an among-individual variation in temporal changes in mitochondrial traits. Our results emphasize the need to evaluate whether mitochondrial metabolic traits of individuals can change over time.

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.

Balancing between Artemia and microdiet usage for normal skeletal development in zebrafish (Danio rerio).

Targeting in zebrafish fast growth, high survival rates and improved reproductive performance has led over the last years in variable feeding regimes between different facilities. Despite its significance on fish function and welfare, normal skeletal development has rarely been evaluated in establishing the best feeding practices for zebrafish. The aim of this study was to establish a protocol for normal skeletal development, growth and survival of zebrafish larvae through live feed-to-microdiet transition at an appropriate rate. Four feeding regimes including feeding exclusively on Artemia nauplii (A) or dry microdiet (D), and feeding on both Artemia and microdiet at two different transition rates (slow (B) or fast (C)) were applied from 5 to 24 dpf (days post-fertilization). Results demonstrated a significant effect of feeding regimes on the incidence of skeletal abnormalities (gill cover, fins and vertebral column, p < .05) in zebrafish larvae. The A and B experimental groups presented the highest (88 ± 3 and 84 ± 17%, respectively), but the C and D the lowest (18 ± 14 and 11 ± 2%, respectively), rates of normal fish (fish without any abnormality). Similarly, growth rate was comparatively elevated in A and B groups. No significant differences were observed in fish survival between A, B and C groups. However, D group presented a significantly lower survival rate. To our knowledge, this is the first study to show that the live feed-to-microdiet transition rate influences larval growth, survival and abnormality rates in a non-homogenous pattern.

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.

Food availability modulates the combined effects of ocean acidification and warming on fish growth.

When organisms are unable to feed ad libitum they may be more susceptible to negative effects of environmental stressors such as ocean acidification and warming (OAW). We reared sea bass (Dicentrarchus labrax) at 15 or 20 °C and at ambient or high PCO2 (650 versus 1750 µatm PCO2; pH = 8.1 or 7.6) at ad libitum feeding and observed no discernible effect of PCO2 on the size-at-age of juveniles after 277 (20 °C) and 367 (15 °C) days. Feeding trials were then conducted including a restricted ration (25% ad libitum). At 15 °C, growth rate increased with ration but was unaffected by PCO2. At 20 °C, acidification and warming acted antagonistically and low feeding level enhanced PCO2 effects. Differences in growth were not merely a consequence of lower food intake but also linked to changes in digestive efficiency. The specific activity of digestive enzymes (amylase, trypsin, phosphatase alkaline and aminopeptidase N) at 20 °C was lower at the higher PCO2 level. Our study highlights the importance of incorporating restricted feeding into experimental designs examining OAW and suggests that ad libitum feeding used in the majority of the studies to date may not have been suitable to detect impacts of ecological significance.

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.

Combined effects of ocean acidification and temperature on larval and juvenile growth, development and swimming performance of European sea bass (Dicentrarchus labrax).

Ocean acidification and ocean warming (OAW) are simultaneously occurring and could pose ecological challenges to marine life, particularly early life stages of fish that, although they are internal calcifiers, may have poorly developed acid-base regulation. This study assessed the effect of projected OAW on key fitness traits (growth, development and swimming ability) in European sea bass (Dicentrarchus labrax) larvae and juveniles. Starting at 2 days post-hatch (dph), larvae were exposed to one of three levels of PCO2 (650, 1150, 1700 µatm; pH 8.0, 7.8, 7.6) at either a cold (15°C) or warm (20°C) temperature. Growth rate, development stage and critical swimming speed (Ucrit) were repeatedly measured as sea bass grew from 0.6 to ~10.0 (cold) or ~14.0 (warm) cm body length. Exposure to different levels of PCO2 had no significant effect on growth, development or Ucrit of larvae and juveniles. At the warmer temperature, larvae displayed faster growth and deeper bodies. Notochord flexion occurred at 0.8 and 1.2 cm and metamorphosis was completed at an age of ~45 and ~60 days post-hatch for sea bass in the warm and cold treatments, respectively. Swimming performance increased rapidly with larval development but better swimmers were observed in the cold treatment, reflecting a potential trade-off between fast grow and swimming ability. A comparison of the results of this and other studies on marine fish indicates that the effects of OAW on the growth, development and swimming ability of early life stages are species-specific and that generalizing the impacts of climate-driven warming or ocean acidification is not warranted.

Fish facing global change: are early stages the lifeline?

The role of phenotypic plasticity in the acclimation and adaptive potential of an organism to global change is not currently accounted for in prediction models. The high plasticity of marine fishes is mainly attributed to their early stages, during which morphological, structural and behavioural functions are particularly sensitive to environmental constraints. This developmental plasticity can determine later physiological performances and fitness, and may further affect population dynamics and ecosystem functioning. This review asks the essential question of what role early stages play in the ability of fish to later cope with the effects of global change, considering three key environmental factors (temperature, hypoxia and acidification). After having identified the carry-over effects of early exposure reported in the literature, we propose areas that we believe warrant the most urgent attention for further research to better understand the role of developmental plasticity in the responses of marine organisms to global change.

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.

Assessing the long-term effect of exposure to dispersant-treated oil on fish health using hypoxia tolerance and temperature susceptibility as ecologically relevant biomarkers.

The ecological and economic importance of fish act as a brake on the development of chemical dispersants as operational instruments following oil spills. Although a valuable and consistent body of knowledge exists, its use in spill response is limited. The objective of the present study was to increase current knowledge base to facilitate the translation of published data into information of operational value. Thus we investigated the dose-response relationship between dispersant-treated oil exposure and ecologically relevant consequences by combining laboratory and field experiments. Effects were examined over almost a year using juveniles of the slowly growing, commercially important European sea bass (Dicentrarchus labrax). A reliable interpretation of biomarker responses requires a complete knowledge of the factors likely to affect them. Interpopulational variability is of particular importance in environmental impact assessment because biomarker responses from a population collected in an impacted area are classically compared with those collected in a clean site. Our study revealed no effect of the exposure to dispersant-treated oil on fish hypoxia tolerance and temperature susceptibility at 1 and 11 mo post exposure. Similarly, no effect of the exposure was observed on the ability of the fish to cope with environmental contingencies in the field, regardless of the dose tested. Thus we feel confident to suggest that a 48-h exposure to chemically treated oil does not affect the ability of sea bass to cope with mild environmental contingencies. Finally, investigation of interpopulation variability revealed large differences in both hypoxia tolerance and temperature susceptibility among the 2 populations tested, suggesting that this variability may blur the interpretation of population comparisons as classically practiced in impact assessment. Environ Toxicol Chem 2019;38:210-221. © 2018 SETAC.

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.

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.

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.

Effects of warming rate, acclimation temperature and ontogeny on the critical thermal maximum of temperate marine fish larvae.

Most of the thermal tolerance studies on fish have been performed on juveniles and adults, whereas limited information is available for larvae, a stage which may have a particularly narrow range in tolerable temperatures. Moreover, previous studies on thermal limits for marine and freshwater fish larvae (53 studies reviewed here) applied a wide range of methodologies (e.g. the static or dynamic method, different exposure times), making it challenging to compare across taxa. We measured the Critical Thermal Maximum (CTmax) of Atlantic herring (Clupea harengus) and European seabass (Dicentrarchus labrax) larvae using the dynamic method (ramping assay) and assessed the effect of warming rate (0.5 to 9°C h-1) and acclimation temperature. The larvae of herring had a lower CTmax (lowest and highest values among 222 individual larvae, 13.1-27.0°C) than seabass (lowest and highest values among 90 individual larvae, 24.2-34.3°C). At faster rates of warming, larval CTmax significantly increased in herring, whereas no effect was observed in seabass. Higher acclimation temperatures led to higher CTmax in herring larvae (2.7 ± 0.9°C increase) with increases more pronounced at lower warming rates. Pre-trials testing the effects of warming rate are recommended. Our results for these two temperate marine fishes suggest using a warming rate of 3-6°C h-1: CTmax is highest in trials of relatively short duration, as has been suggested for larger fish. Additionally, time-dependent thermal tolerance was observed in herring larvae, where a difference of up to 8°C was observed in the upper thermal limit between a 0.5- or 24-h exposure to temperatures >18°C. The present study constitutes a first step towards a standard protocol for measuring thermal tolerance in larval fish.