Heat hardening enhances metabolite-driven thermoprotection in the Mediterranean mussel Mytilus galloprovincialis.

Introduction: Temperature affects organisms' metabolism and ecological performance. Owing to climate change, sea warming constituting a severe source of environmental stress for marine organisms, since it increases at alarming rates. Rapid warming can exceed resilience of marine organisms leading to fitness loss and mortality. However, organisms can improve their thermal tolerance when briefly exposed to sublethal thermal stress (heat hardening), thus generating heat tolerant phenotypes. Methods: We investigated the "stress memory" effect caused by heat hardening on M. galloprovincialis metabolite profile of in order to identify the underlying biochemical mechanisms, which enhance mussels' thermal tolerance. Results: The heat hardening led to accumulation of amino acids (e.g., leucine, isoleucine and valine), including osmolytes and cytoprotective agents with antioxidant and anti-inflammatory properties that can contribute to thermal protection of the mussels. Moreover, proteolysis was inhibited and protein turnover regulated by the heat hardening. Heat stress alters the metabolic profile of heat stressed mussels, benefiting the heat-hardened individuals in increasing their heat tolerance compared to the non-heat-hardened ones. Discussion: These findings provide new insights in the metabolic mechanisms that may reinforce mussels' tolerance against thermal stress providing both natural protection and potential manipulative tools (e.g., in aquaculture) against the devastating climate change effects on marine organisms.

Metabolic remodeling caused by heat hardening in the Mediterranean mussel Mytilus galloprovincialis.

Organisms can modify and increase their thermal tolerance faster and more efficiently after a brief exposure to sublethal thermal stress. This response is called 'heat hardening' as it leads to the generation of phenotypes with increased heat tolerance. The aim of this study was to investigate the impact of heat hardening on the metabolomic profile of Mytilus galloprovincialis in order to identify the associated adjustments of biochemical pathways that might benefit the mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat hardening and acclimation phases). To gain further insight into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of the tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non-heat-hardened and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels to undergo a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with intermittent and often extreme temperatures.

Heat hardening enhances mitochondrial potential for respiration and oxidative defence capacity in the mantle of thermally stressed Mytilus galloprovincialis.

Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub-lethal temperatures, a process known as "hardening". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the "hardening" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels' heat resistance is required.

Climate impacts on organisms, ecosystems and human societies: integrating OCLTT into a wider context.

Physiological studies contribute to a cause and effect understanding of ecological patterns under climate change and identify the scope and limits of adaptation. Across most habitats, this requires analyzing organism responses to warming, which can be modified by other drivers such as acidification and oxygen loss in aquatic environments or excess humidity or drought on land. Experimental findings support the hypothesis that the width and temperature range of thermal performance curves relate to biogeographical range. Current warming causes range shifts, hypothesized to include constraints in aerobic power budget which in turn are elicited by limitations in oxygen supply capacity in relation to demand. Different metabolic scopes involved may set the borders of both the fundamental niche (at standard metabolic rate) and the realized niche (at routine rate). Relative scopes for aerobic performance also set the capacity of species to interact with others at the ecosystem level. Niche limits and widths are shifting and probably interdependent across life stages, with young adults being least thermally vulnerable. The principles of thermal tolerance and performance may also apply to endotherms including humans, their habitat and human society. Overall, phylogenetically based comparisons would need to consider the life cycle of species as well as organism functional properties across climate zones and time scales. This Review concludes with a perspective on how mechanism-based understanding allows scrutinizing often simplified modeling approaches projecting future climate impacts and risks for aquatic and terrestrial ecosystems. It also emphasizes the usefulness of a consensus-building process among experimentalists for better recognition in the climate debate.

Advances in understanding the impacts of global warming on marine fishes farmed offshore: Sparus aurata as a case study.

Monitoring variations in proteins involved in metabolic processes, oxidative stress responses, cell signalling and protein homeostasis is a powerful tool for developing hypotheses of how environmental variations affect marine organisms' physiology and biology. According to the oxygen- and capacity-limited thermal tolerance hypothesis, thermal acclimation mechanisms such as adjusting the activities of enzymes of intermediary metabolism and of antioxidant defence mechanisms, inducing heat shock proteins (Hsps) or activating mitogen-activated protein kinases may all shift tolerance windows. Few studies have, however, investigated the molecular, biochemical and organismal responses by fishes to seasonal temperature variations in the field to link these to laboratory findings. Investigation of the impacts of global warming on fishes farmed offsore, in the open sea, can provide a stepping stone towards understanding effects on wild populations because they experience similar environmental fluctuations. Over the last 30 years, farming of the gilthead sea bream Sparus aurata (Linnaeus 1758) has become widespread along the Mediterranean coastline, rendering this species a useful case study. Based on available information, the prevailing seasonal temperature variations expose the species to the upper and lower limits of its thermal range. Evidence for this includes oxygen restriction, reduced feeding, reduced responsiveness to environmental stimuli, plus a range of molecular and biochemical indicators that change across the thermal range. Additionally, close relationships between biochemical pathways and seasonal patterns of metabolism indicate a connection between energy demand and metabolic processes on the one hand, and cellular stress responses such as oxidative stress, inflammation and autophagy on the other. Understanding physiological responses to temperature fluctuations in fishes farmed offshore can provide crucial background information for the conservation and successful management of aquaculture resources in the face of global change.

Energetic, antioxidant, inflammatory and cell death responses in the red muscle of thermally stressed Sparus aurata.

For a comprehensive understanding of fish responses to increasing thermal stress in marine environments, we investigated tissue energetics, antioxidant levels, inflammatory and cell death responses in Sparus aurata (gilthead seabream) red muscle during exposure to elevated temperatures (24 °C, 26 °C, 30 °C) compared to the control temperature of 18 °C. Energetic aspects were assessed by determining lactate, glucose and lipids levels in blood plasma, ATP, ADP and AMP levels, and AMPK phosphorylation as an indicator of regulatory changes in energy metabolism, in tissue extracts. Oxidative defence was assessed by determining superoxide dismutase, catalase and glutathione reductase maximum activities. Moreover, xanthine levels were determined as an indicator of purine conversion to xanthine and associated ROS production. In the context of inflammatory response and cell death due to oxidative stress, pro-inflammatory cytokines (IkBα phosphorylation, IL-6 and TNFα) levels, and LC3 II/I ratio and SQSTM1/p62 as indicators of autophagic-lysosomal pathway were also determined. A recovery in the efficacy of ATP production after a marked decrease during the 1st day of exposure to 24 °C is observed. This biphasic pattern is paralleled by antioxidant enzymes' activities and inflammatory and autophagy responses, indicating a close correlation between ATP turnover and stress responses, which may benefit tissue function and survival. However, exposure beyond 24 °C caused tissue's antioxidant capacity loss, triggering the inflammatory and cell death response, leading to increased fish mortality. The results of the present study set the thermal limits of the gilthead seabream at 22-24 °C and establish the used cellular and metabolic indicators as tools for the definition of the extreme thermal limits in marine organisms.

Single and combined effects of the "Deadly trio" hypoxia, hypercapnia and warming on the cellular metabolism of the great scallop Pecten maximus.

In the ocean the main climate drivers affecting marine organisms are warming, hypercapnia, and hypoxia. We investigated the acute effects of warming (W), warming plus hypercapnia (WHc, ~1800 µatm CO2), warming plus hypoxia (WHo, ~12.1 kPa O2), and a combined exposure of all three drivers (Deadly Trio, DT) on king scallops (Pecten maximus). All exposures started at 14 °C and temperature was increased by 2 °C once every 48 h until the lethal temperature was reached (28 °C). Gill samples were taken at 14 °C, 18 °C, 22 °C, and 26 °C and analyzed for their metabolic response by 1H-nuclear magnetic resonance (NMR) spectroscopy. Scallops were most tolerant to WHc and most susceptible to oxygen reduction (WHo and DT). In particular under DT, scallops' mitochondrial energy metabolism was affected. Changes became apparent at 22 °C and 26 °C involving significant accumulation of glycogenic amino acids (e.g. glycine and valine) and anaerobic end-products (e.g. acetic acid and succinate). In line with these observations the LT50 was lower under the exposure to DT (22.5 °C) than to W alone (~ 25 °C) indicating a narrowing of the thermal niche due to an imbalance between oxygen demand and supply.

In vivo31P-MRS of muscle bioenergetics in marine invertebrates: Future ocean limits scallops' performance.

OBJECT: Dynamic in vivo31P-NMR spectroscopy in combination with Magnetic Resonance Imaging (MRI) was used to study muscle bioenergetics of boreal and Arctic scallops (Pecten maximus and Chlamys islandica) to test the hypothesis that future Ocean Warming and Acidification (OWA) will impair the performance of marine invertebrates. MATERIALS & METHODS: Experiments were conducted following the recommendations for studies of muscle bioenergetics in vertebrates. Animals were long-term incubated under different environmental conditions: controls at 0 °C for C. islandica and 15 °C for P. maximus under ambient PCO2 of 0.039 kPa, a warm exposure with +5 °C (5 °C and 20 °C, respectively) under ambient PCO2 (OW group), and a combined exposure to warmed acidified conditions (5 °C and 20 °C, 0.112 kPa PCO2, OWA group). Scallops were placed in a 4.7 T MR animal scanner and the energetic status of the adductor muscle was determined under resting conditions using in vivo31P-NMR spectroscopy. The surplus oxidative flux (Qmax) was quantified by recording the recovery of arginine phosphate (PLA) directly after moderate swimming exercise of the scallops. RESULTS: Measurements led to reproducible results within each experimental group. Under projected future conditions resting PLA levels (PLArest) were reduced, indicating reduced energy reserves in warming exposed scallops per se. In comparison to vertebrate muscle tissue surplus Qmax of scallop muscle was about one order of magnitude lower. This can be explained by lower mitochondrial contents and capacities in invertebrate than vertebrate muscle tissue. Warm exposed scallops showed a slower recovery rate of PLA levels (kPLA) and a reduced surplus Qmax. Elevated PCO2 did not affected PLA recovery further. CONCLUSION: Dynamic in vivo31P-NMR spectroscopy revealed constrained residual aerobic power budgets in boreal and Arctic scallops under projected ocean warming and acidification indicating that scallops are susceptible to future climate change. The observed reduction in muscular PLArest of scallops coping with a warmer and acidified ocean may be linked to an enhanced energy demand and reduced oxygen partial pressures (PO2) in their body fluids. Delayed recovery from moderate swimming at elevated temperature is a result of reduced PLArest concentrations associated with a warm-induced reduction of a residual aerobic power budget.

De novo transcriptome assembly and gene expression profile of thermally challenged green abalone (Haliotis fulgens: Gastropoda) under acute hypoxia and hypercapnia.

Transcriptional regulation constitutes a rapid response of marine organisms facing stressful environmental conditions, such as the concomitant exposure to warming, ocean acidification and hypoxia under climate change. In previous studies, we investigated whole-organism physiological patterns and cellular metabolism in gill and muscle of the marine gastropod Haliotis fulgens in response to increasing temperature (18 °C to 32 °C at +3 °C per day) under hypoxia (50% air saturation), hypercapnia (1000 µatm pCO2) and both factors combined. Here, we report investigations of the molecular responses of H. fulgens to temperature and identify mechanisms concomitantly affected by hypoxia and hypercapnia. A de novo transcriptome assembly with subsequent quantitative PCR and correlation network analysis of genes involved in the molecular response were used to unravel the correlations between gene expression patterns under the different experimental conditions. The correlation networks identified a shift from the expression of genes involved in energy metabolism (down-regulated) to the up-regulation of Hsp70 during warming under all experimental conditions in gill and muscle, indicating a strong up-regulation of damage prevention and repair systems at sustained cellular energy production. However, a higher capacity for anaerobic succinate production was evicted in gill, matching with observations from our previous studies indicating succinate accumulation in gill but not in muscle. Additionally, warming under hypoxia and hypercapnia kept mRNA levels of citrate synthase in both tissues unchanged following a similar pattern as muscle enzyme capacity from a previous study, suggesting an emphasis on maintaining rather than down-regulating mitochondrial activity.

Assessment of muscular energy metabolism and heat shock response of the green abalone Haliotis fulgens (Gastropoda: Philipi) at extreme temperatures combined with acute hypoxia and hypercapnia.

The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18 °C to 32 °C at +3 °C day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 µatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32 °C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32 °C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30 °C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.

Impact of Ocean Acidification and Warming on the bioenergetics of developing eggs of Atlantic herring Clupea harengus.

Atlantic herring (Clupea harengus) is a benthic spawner, therefore its eggs are prone to encounter different water conditions during embryonic development, with bottom waters often depleted of oxygen and enriched in CO2. Some Atlantic herring spawning grounds are predicted to be highly affected by ongoing Ocean Acidification and Warming with water temperature increasing by up to +3°C and CO2 levels reaching ca. 1000 µatm (RCP 8.5). Although many studies investigated the effects of high levels of CO2 on the embryonic development of Atlantic herring, little is known about the combination of temperature and ecologically relevant levels of CO2. In this study, we investigated the effects of Ocean Acidification and Warming on embryonic metabolic and developmental performance such as mitochondrial function, respiration, hatching success (HS) and growth in Atlantic herring from the Oslo Fjord, one of the spawning grounds predicted to be greatly affected by climate change. Fertilized eggs were incubated under combinations of two PCO2 conditions (400 µatm and 1100 µatm) and three temperatures (6, 10 and 14°C), which correspond to current and end-of-the-century conditions. We analysed HS, oxygen consumption (MO2) and mitochondrial function of embryos as well as larval length at hatch. The capacity of the electron transport system (ETS) increased with temperature, reaching a plateau at 14°C, where the contribution of Complex I to the ETS declined in favour of Complex II. This relative shift was coupled with a dramatic increase in MO2 at 14°C. HS was high under ambient spawning conditions (6-10°C), but decreased at 14°C and hatched larvae at this temperature were smaller. Elevated PCO2 increased larval malformations, indicating sub-lethal effects. These results indicate that energetic limitations due to thermally affected mitochondria and higher energy demand for maintenance occur at the expense of embryonic development and growth.

CO2 induced pHi changes in the brain of polar fish: a TauCEST application.

Chemical exchange saturation transfer (CEST) from taurine to water (TauCEST) can be used for in vivo mapping of taurine concentrations as well as for measurements of relative changes in intracellular pH (pHi ) at temperatures below 37°C. Therefore, TauCEST offers the opportunity to investigate acid-base regulation and neurological disturbances of ectothermic animals living at low temperatures, and in particular to study the impact of ocean acidification (OA) on neurophysiological changes of fish. Here, we report the first in vivo application of TauCEST imaging. Thus, the study aimed to investigate the TauCEST effect in a broad range of temperatures (1-37°C) and pH (5.5-8.0), motivated by the high taurine concentration measured in the brains of polar fish. The in vitro data show that the TauCEST effect is especially detectable in the low temperature range and strictly monotonic for the relevant pH range (6.8-7.5). To investigate the specificity of TauCEST imaging for the brain of polar cod (Boreogadus saida) at 1.5°C simulations were carried out, indicating a taurine contribution of about 65% to the in vivo expected CEST effect, if experimental parameters are optimized. B. saida was acutely exposed to three different CO2 concentrations in the sea water (control normocapnia; comparatively moderate hypercapnia OAm  = 3300 µatm; high hypercapnia OAh  = 4900 µatm). TauCEST imaging of the brain showed a significant increase in the TauCEST effect under the different CO2 concentrations of about 1.5-3% in comparison with control measurements, indicative of changes in pHi or metabolite concentration. Consecutive recordings of 1 H MR spectra gave no support for a concentration induced change of the in vivo observed TauCEST effect. Thus, the in vivo application of TauCEST offers the possibility of mapping relative changes in pHi in the brain of polar cod during exposure to CO2 .

Water bicarbonate modulates the response of the shore crab Carcinus maenas to ocean acidification.

Ocean acidification causes an accumulation of CO2 in marine organisms and leads to shifts in acid-base parameters. Acid-base regulation in gill breathers involves a net increase of internal bicarbonate levels through transmembrane ion exchange with the surrounding water. Successful maintenance of body fluid pH depends on the functional capacity of ion-exchange mechanisms and associated energy budget. For a detailed understanding of the dependence of acid-base regulation on water parameters, we investigated the physiological responses of the shore crab Carcinus maenas to 4 weeks of ocean acidification [OA, P(CO2)w = 1800 µatm], at variable water bicarbonate levels, paralleled by changes in water pH. Cardiovascular performance was determined together with extra-(pHe) and intracellular pH (pHi), oxygen consumption, haemolymph CO2 parameters, and ion composition. High water P(CO2) caused haemolymph P(CO2) to rise, but pHe and pHi remained constant due to increased haemolymph and cellular [HCO3-]. This process was effective even under reduced seawater pH and bicarbonate concentrations. While extracellular cation concentrations increased throughout, anion levels remained constant or decreased. Despite similar levels of haemolymph pH and ion concentrations under OA, metabolic rates, and haemolymph flow were significantly depressed by 40 and 30%, respectively, when OA was combined with reduced seawater [HCO3-] and pH. Our findings suggest an influence of water bicarbonate levels on metabolic rates as well as on correlations between blood flow and pHe. This previously unknown phenomenon should direct attention to pathways of acid-base regulation and their potential feedback on whole-animal energy demand, in relation with changing seawater carbonate parameters.

Seasonal Changes in Metabolism and Cellular Stress Phenomena in the Gilthead Sea Bream (Sparus aurata).

Seasonal temperature changes may take organisms to the upper and lower limit of their thermal range, with respective variations in their biochemical and metabolic profile. To elucidate these traits, we investigated metabolic and antioxidant patterns in tissues of sea bream Sparus aurata during seasonal acclimatization for 1 yr in the field. Metabolic patterns were assessed by determining lactate dehydrogenase, citrate synthase, and ß-hydroxyacyl CoA dehydrogenase activities, their kinetic properties and plasma levels of glucose, lactate, and triglycerides and tissue succinate levels. Oxidative stress was assessed by determining antioxidant enzymes superoxide dismutase, catalase, and glutathione reductase activities and levels of thiobarbituric acid reactive substances. Xanthine oxidase (XO) activity was determined as another source of reactive oxygen species (ROS) production. Furthermore, we studied the antiapoptotic protein indicator Bcl-2 and the apoptotic protein indicators Bax, Bad, ubiquitin, and caspase as well as indexes of autophagy (LC3B II/LC3B I and SQSTM1/p62) in the liver and the heart to identify possible relationships between oxidative stress and cell death. The results indicate clear seasonal metabolic patterns involving oxidative stress during summer as well as winter. During cold acclimatization, lipid oxidation is induced, while during increased temperatures, warm-induced metabolic activation and carbohydrate oxidation are observed. Thus, oxidative stress seems to be more prominent during warming because of the increased aerobic metabolism. The seasonal profile of apoptosis and XO as another source of ROS matches the results obtained in the laboratory and are interpreted within the framework of oxygen- and capacity-limited thermal tolerance.

Sensitivity to ocean acidification differs between populations of the Sydney rock oyster: Role of filtration and ion-regulatory capacities.

Understanding mechanisms of intraspecific variation in resilience to environmental drivers is key to predict species' adaptive potential. Recent studies show a higher CO2 resilience of Sydney rock oysters selectively bred for increased growth and disease resistance ('selected oysters') compared to the wild population. We tested whether the higher resilience of selected oysters correlates with an increased ability to compensate for CO2-induced acid-base disturbances. After 7 weeks of exposure to elevated seawater PCO2 (1100 µatm), wild oysters had a lower extracellular pH (pHe = 7.54 ±â€¯0.02 (control) vs. 7.40 ±â€¯0.03 (elevated PCO2)) and increased hemolymph PCO2 whereas extracellular acid-base status of selected oysters remained unaffected. However, differing pHe values between oyster types were not linked to altered metabolic costs of major ion regulators (Na+/K+-ATPase, H+-ATPase and Na+/H+-exchanger) in gill and mantle tissues. Our findings suggest that selected oysters possess an increased systemic capacity to eliminate metabolic CO2, possibly through higher and energetically more efficient filtration rates and associated gas exchange. Thus, effective filtration and CO2 resilience might be positively correlated traits in oysters.

Ocean acidification but not warming alters sex determination in the Sydney rock oyster, Saccostrea glomerata.

Whether sex determination of marine organisms can be altered by ocean acidification and warming during this century remains a significant, unanswered question. Here, we show that exposure of the protandric hermaphrodite oyster, Saccostrea glomerata to ocean acidification, but not warming, alters sex determination resulting in changes in sex ratios. After just one reproductive cycle there were 16% more females than males. The rate of gametogenesis, gonad area, fecundity, shell length, extracellular pH and survival decreased in response to ocean acidification. Warming as a sole stressor slightly increased the rate of gametogenesis, gonad area and fecundity, but this increase was masked by the impact of ocean acidification at a level predicted for this century. Alterations to sex determination, sex ratios and reproductive capacity will have flow on effects to reduce larval supply and population size of oysters and potentially other marine organisms.

Oxygen- and capacity-limited thermal tolerance: bridging ecology and physiology.

Observations of climate impacts on ecosystems highlight the need for an understanding of organismal thermal ranges and their implications at the ecosystem level. Where changes in aquatic animal populations have been observed, the integrative concept of oxygen- and capacity-limited thermal tolerance (OCLTT) has successfully characterised the onset of thermal limits to performance and field abundance. The OCLTT concept addresses the molecular to whole-animal mechanisms that define thermal constraints on the capacity for oxygen supply to the organism in relation to oxygen demand. The resulting 'total excess aerobic power budget' supports an animal's performance (e.g. comprising motor activity, reproduction and growth) within an individual's thermal range. The aerobic power budget is often approximated through measurements of aerobic scope for activity (i.e. the maximum difference between resting and the highest exercise-induced rate of oxygen consumption), whereas most animals in the field rely on lower (i.e. routine) modes of activity. At thermal limits, OCLTT also integrates protective mechanisms that extend time-limited tolerance to temperature extremes - mechanisms such as chaperones, anaerobic metabolism and antioxidative defence. Here, we briefly summarise the OCLTT concept and update it by addressing the role of routine metabolism. We highlight potential pitfalls in applying the concept and discuss the variables measured that led to the development of OCLTT. We propose that OCLTT explains why thermal vulnerability is highest at the whole-animal level and lowest at the molecular level. We also discuss how OCLTT captures the thermal constraints on the evolution of aquatic animal life and supports an understanding of the benefits of transitioning from water to land.

Ocean acidification narrows the acute thermal and salinity tolerance of the Sydney rock oyster Saccostrea glomerata.

Coastal and estuarine environments are characterised by acute changes in temperature and salinity. Organisms living within these environments are adapted to withstand such changes, yet near-future ocean acidification (OA) may challenge their physiological capacity to respond. We tested the impact of CO2-induced OA on the acute thermal and salinity tolerance, energy metabolism and acid-base regulation capacity of the oyster Saccostrea glomerata. Adult S. glomerata were acclimated to three CO2 levels (ambient 380µatm, moderate 856µatm, high 1500µatm) for 5weeks (24°C, salinity 34.6) before being exposed to a series of acute temperature (15-33°C) and salinity (34.2-20) treatments. Oysters acclimated to elevated CO2 showed a significant metabolic depression and extracellular acidosis with acute exposure to elevated temperature and reduced salinity, especially at the highest CO2 of 1500µatm. Our results suggest that the acute thermal and salinity tolerance of S. glomerata and thus its distribution will reduce as OA continues to worsen.