The silent loss of cell physiology hampers marine biosciences.

An ongoing loss of experts in marine cellular biochemistry and physiology (CBP) is stagnating the generation of knowledge upon which rapidly growing "omics" approaches rely, ultimately hampering our ability to predict organismal responses to climate change.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Windows of opportunity: Ocean warming shapes temperature-sensitive epigenetic reprogramming and gene expression across gametogenesis and embryogenesis in marine stickleback.

Rapid climate change is placing many marine species at risk of local extinction. Recent studies show that epigenetic mechanisms (e.g. DNA methylation, histone modifications) can facilitate both within and transgenerational plasticity to cope with changing environments. However, epigenetic reprogramming (erasure and re-establishment of epigenetic marks) during gamete and early embryo development may hinder transgenerational epigenetic inheritance. Most of our knowledge about reprogramming stems from mammals and model organisms, whereas the prevalence and extent of reprogramming among non-model species from wild populations is rarely investigated. Moreover, whether reprogramming dynamics are sensitive to changing environmental conditions is not well known, representing a key knowledge gap in the pursuit to identify mechanisms underlying links between parental exposure to changing climate patterns and environmentally adapted offspring phenotypes. Here, we investigated epigenetic reprogramming (DNA methylation/hydroxymethylation) and gene expression across gametogenesis and embryogenesis of marine stickleback (Gasterosteus aculeatus) under three ocean warming scenarios (ambient, +1.5 and +4°C). We found that parental acclimation to ocean warming led to dynamic and temperature-sensitive reprogramming throughout offspring development. Both global methylation/hydroxymethylation and expression of genes involved in epigenetic modifications were strongly and differentially affected by the increased warming scenarios. Comparing transcriptomic profiles from gonads, mature gametes and early embryonic stages showed sex-specific accumulation and temperature sensitivity of several epigenetic actors. DNA methyltransferase induction was primarily maternally inherited (suggesting maternal control of remethylation), whereas induction of several histone-modifying enzymes was shaped by both parents. Importantly, massive, temperature-specific changes to the epigenetic landscape occurred in blastula, a critical stage for successful embryo development, which could, thus, translate to substantial consequences for offspring phenotype resilience in warming environments. In summary, our study identified key stages during gamete and embryo development with temperature-sensitive reprogramming and epigenetic gene regulation, reflecting potential 'windows of opportunity' for adaptive epigenetic responses under future climate change.

Effects of ocean acidification over successive generations decrease resilience of larval European sea bass to ocean acidification and warming but juveniles could benefit from higher temperatures in the NE Atlantic.

European sea bass (Dicentrarchus labrax) is a large, economically important fish species with a long generation time whose long-term resilience to ocean acidification (OA) and warming (OW) is not clear. We incubated sea bass from Brittany (France) for two generations (>5 years in total) under ambient and predicted OA conditions (PCO2: 650 and 1700 µatm) crossed with ambient and predicted OW conditions in F1 (temperature: 15-18°C and 20-23°C) to investigate the effects of climate change on larval and juvenile growth and metabolic rate. We found that in F1, OA as a single stressor at ambient temperature did not affect larval or juvenile growth and OW increased developmental time and growth rate, but OAW decreased larval size at metamorphosis. Larval routine and juvenile standard metabolic rate were significantly lower in cold compared with warm conditioned fish and also lower in F0 compared with F1 fish. We did not find any effect of OA as a single stressor on metabolic rate. Juvenile PO2,crit was not affected by OA or OAW in both generations. We discuss the potential underlying mechanisms resulting in the resilience of F0 and F1 larvae and juveniles to OA and in the beneficial effects of OW on F1 larval growth and metabolic rate, but contrastingly in the vulnerability of F1, but not F0 larvae to OAW. With regard to the ecological perspective, we conclude that recruitment of larvae and early juveniles to nursery areas might decrease under OAW conditions but individuals reaching juvenile phase might benefit from increased performance at higher temperatures.

High gene flow in polar cod (Boreogadus saida) from West-Svalbard and the Eurasian Basin.

The current and projected environmental change of the Arctic Ocean contrasts sharply with the limited knowledge of its genetic biodiversity. Polar cod Boreogadus saida (Lepechin, 1774) is an abundant circumpolar marine fish and ecological key species. The central role of polar cod in the Arctic marine food web warrants a better understanding of its population structure and connectivity. In this study, the genetic population structure of 171 juveniles, collected from several fjords off West-Svalbard (Billefjorden, Hornsund and Kongsfjorden), the northern Sophia Basin and the Eurasian Basin of the Arctic Ocean, was analysed using nine DNA microsatellite loci. Genetic analyses indicated moderate to high genetic diversity, but absence of spatial population structure and isolation-by-distance, suggesting ongoing gene flow between the studied sampling regions. High levels of connectivity may be key for polar cod to maintain populations across wide spatial scales. The adaptive capacity of the species will be increasingly important to face challenges such as habitat fragmentation, ocean warming and changes in prey composition. In view of a limited understanding of the population dynamics and evolution of polar cod, a valuable next step to predict future developments should be an integrated biological evaluation, including population genomics, a life-history approach, and habitat and biophysical dispersal modelling.

Future ocean warming may prove beneficial for the northern population of European seabass, but ocean acidification will not.

The world's oceans are acidifying and warming as a result of increasing atmospheric CO2 concentrations. The thermal tolerance of fish greatly depends on the cardiovascular ability to supply the tissues with oxygen. The highly oxygen-dependent heart mitochondria thus might play a key role in shaping an organism's tolerance to temperature. The present study aimed to investigate the effects of acute and chronic warming on the respiratory capacity of European sea bass (Dicentrarchus labrax L.) heart mitochondria. We hypothesized that acute warming would impair mitochondrial respiratory capacity, but be compensated for by life-time conditioning. Increasing PCO2  may additionally cause shifts in metabolic pathways by inhibiting several enzymes of the cellular energy metabolism. Among other shifts in metabolic pathways, acute warming of heart mitochondria of cold life-conditioned fish increased leak respiration rate, suggesting a lower aerobic capacity to synthesize ATP with acute warming. However, thermal conditioning increased mitochondrial functionality, e.g. higher respiratory control ratios in heart mitochondria of warm life-conditioned compared with cold life-conditioned fish. Exposure to high PCO2  synergistically amplified the effects of acute and long-term warming, but did not result in changes by itself. This high ability to maintain mitochondrial function under ocean acidification can be explained by the fact that seabass are generally able to acclimate to a variety of environmental conditions. Improved mitochondrial energy metabolism after warm conditioning could be due to the origin of this species in the warm waters of the Mediterranean. Our results also indicate that seabass are not yet fully adapted to the colder temperatures in their northern distribution range and might benefit from warmer temperatures in these latitudes.

Effects of ocean acidification increase embryonic sensitivity to thermal extremes in Atlantic cod, Gadus morhua.

Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO2 -driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO2 conditions (400 µatm vs. 1100 µatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO2 ) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid-base-relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3-6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO2 caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO2 and mitochondrial capacities. Elevated PCO2 stimulated MO2 at cold and intermediate temperatures, but exacerbated warming-induced constraints on MO2 , indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO2 . Increased MO2 in response to elevated PCO2 was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO2 treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO2 conditions and suggest that acclimation to elevated PCO2 requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains the thermal performance window of embryos, which has important implication for the susceptibility of cod to projected climate change.

Mitochondrial acclimation potential to ocean acidification and warming of Polar cod (Boreogadus saida) and Atlantic cod (Gadus morhua).

BACKGROUND: Ocean acidification and warming are happening fast in the Arctic but little is known about the effects of ocean acidification and warming on the physiological performance and survival of Arctic fish. RESULTS: In this study we investigated the metabolic background of performance through analyses of cardiac mitochondrial function in response to control and elevated water temperatures and PCO2 of two gadoid fish species, Polar cod (Boreogadus saida), an endemic Arctic species, and Atlantic cod (Gadus morhua), which is a temperate to cold eurytherm and currently expanding into Arctic waters in the wake of ocean warming. We studied their responses to the above-mentioned drivers and their acclimation potential through analysing the cardiac mitochondrial function in permeabilised cardiac muscle fibres after 4 months of incubation at different temperatures (Polar cod: 0, 3, 6, 8 °C and Atlantic cod: 3, 8, 12, 16 °C), combined with exposure to present (400µatm) and year 2100 (1170µatm) levels of CO2. OXPHOS, proton leak and ATP production efficiency in Polar cod were similar in the groups acclimated at 400µatm and 1170µatm of CO2, while incubation at 8 °C evoked increased proton leak resulting in decreased ATP production efficiency and decreased Complex IV capacity. In contrast, OXPHOS of Atlantic cod increased with temperature without compromising the ATP production efficiency, whereas the combination of high temperature and high PCO2 depressed OXPHOS and ATP production efficiency. CONCLUSIONS: Polar cod mitochondrial efficiency decreased at 8 °C while Atlantic cod mitochondria were more resilient to elevated temperature; however, this resilience was constrained by high PCO2. In line with its lower habitat temperature and higher degree of stenothermy, Polar cod has a lower acclimation potential to warming than Atlantic cod.

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.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

BACKGROUND: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. RESULTS: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. CONCLUSIONS: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Blue blood on ice: modulated blood oxygen transport facilitates cold compensation and eurythermy in an Antarctic octopod.

INTRODUCTION: The Antarctic Ocean hosts a rich and diverse fauna despite inhospitable temperatures close to freezing, which require specialist adaptations to sustain animal activity and various underlying body functions. While oxygen transport has been suggested to be key in setting thermal tolerance in warmer climates, this constraint is relaxed in Antarctic fishes and crustaceans, due to high levels of dissolved oxygen. Less is known about how other Antarctic ectotherms cope with temperatures near zero, particularly the more active invertebrates like the abundant octopods. A continued reliance on the highly specialised blood oxygen transport system of cephalopods may concur with functional constraints at cold temperatures. We therefore analysed the octopod's central oxygen transport component, the blue blood pigment haemocyanin, to unravel strategies that sustain oxygen supply at cold temperatures. RESULTS: To identify adaptive compensation of blood oxygen transport in octopods from different climatic regions, we compared haemocyanin oxygen binding properties, oxygen carrying capacities as well as haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata. In the Antarctic Pareledone charcoti at 0°C, oxygen unloading by haemocyanin was poor but supported by high levels of dissolved oxygen. However, lower oxygen affinity and higher oxygen carrying capacity compared to warm water octopods, still enabled significant contribution of haemocyanin to oxygen transport at 0°C. At warmer temperatures, haemocyanin of Pareledone charcoti releases most of the bound oxygen, supporting oxygen supply at 10°C. In warm water octopods, increasing oxygen affinities reduce the ability to release oxygen from haemocyanin at colder temperatures. Though, unlike Eledone moschata, Octopus pallidus attenuated this increase below 15°C. CONCLUSIONS: Adjustments of haemocyanin physiological function and haemocyanin concentrations but also high dissolved oxygen concentrations support oxygen supply in the Antarctic octopus Pareledone charcoti at near freezing temperatures. Increased oxygen supply by haemocyanin at warmer temperatures supports extended warm tolerance and thus eurythermy of Pareledone charcoti. Limited haemocyanin function towards colder temperatures in Antarctic and warm water octopods highlights the general role of haemocyanin oxygen transport in constraining cold tolerance in octopods.

Simultaneous high-resolution pH and spectrophotometric recordings of oxygen binding in blood microvolumes.

Oxygen equilibrium curves have been widely used to understand oxygen transport in numerous organisms. A major challenge has been to monitor oxygen binding characteristics and concomitant pH changes as they occur in vivo, in limited sample volumes. Here we report a technique allowing highly resolved and simultaneous monitoring of pH and blood pigment saturation in minute blood volumes. We equipped a gas diffusion chamber with a broad-range fibre-optic spectrophotometer and a micro-pH optode and recorded changes of pigment oxygenation along oxygen partial pressure (PO2) and pH gradients to test the setup. Oxygen binding parameters derived from measurements in only 15 µl of haemolymph from the cephalopod Octopus vulgaris showed low instrumental error (0.93%) and good agreement with published data. Broad-range spectra, each resolving 2048 data points, provided detailed insight into the complex absorbance characteristics of diverse blood types. After consideration of photobleaching and intrinsic fluorescence, pH optodes yielded accurate recordings and resolved a sigmoidal shift of 0.03 pH units in response to changing PO2 from 0 to 21 kPa. Highly resolved continuous recordings along pH gradients conformed to stepwise measurements at low rates of pH changes. In this study we showed that a diffusion chamber upgraded with a broad-range spectrophotometer and an optical pH sensor accurately characterizes oxygen binding with minimal sample consumption and manipulation. We conclude that the modified diffusion chamber is highly suitable for experimental biologists who demand high flexibility, detailed insight into oxygen binding as well as experimental and biological accuracy combined in a single setup.

Hidden impacts of ocean warming and acidification on biological responses of marine animals revealed through meta-analysis.

Conflicting results remain on the impacts of climate change on marine organisms, hindering our capacity to predict the future state of marine ecosystems. To account for species-specific responses and for the ambiguous relation of most metrics to fitness, we develop a meta-analytical approach based on the deviation of responses from reference values (absolute change) to complement meta-analyses of directional (relative) changes in responses. Using this approach, we evaluate responses of fish and invertebrates to warming and acidification. We find that climate drivers induce directional changes in calcification, survival, and metabolism, and significant deviations in twice as many biological responses, including physiology, reproduction, behavior, and development. Widespread deviations of responses are detected even under moderate intensity levels of warming and acidification, while directional changes are mostly limited to more severe intensity levels. Because such deviations may result in ecological shifts impacting ecosystem structures and processes, our results suggest that climate change will likely have stronger impacts than those previously predicted based on directional changes alone.

Protein Synthesis Determined from Non-Radioactive Phenylalanine Incorporated by Antarctic Fish.

Direct measurements of temperature-dependent weight gains are experimentally challenging and time-consuming in long-lived/slow-growing organisms such as Antarctic fish. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We tested whether it is possible to avoid hazardous radioactive materials and whether the analytical pathway chosen is robust against analytical errors. In the eelpout, Pachycara brachycephalum, 13C9H1115N1O2 phenylalanine was injected intraperitoneally and muscle tissue was sampled before injection and at 1.5 h time intervals up to 6 h thereafter. The incorporation of 13C15N-labeled-phenylalanine into muscle was monitored by quantification of bound and free phenylalanine through liquid chromatography-mass spectrometry. We found an increase in the pool of labeled, free phenylalanine in the cytosolic fraction that leveled off after 4.5 h. The labeled phenylalanine bound in the proteins increased linearly over time. The resulting protein synthesis rate (Ks) for P. brachycephalum was as low as 0.049 ± 0.021% day-1. This value and its variability were in good agreement with literature data obtained from studies using radioactive labels, indicating that this methodology is well suited for characterizing growth in polar fish under in situ conditions in remote areas or on research vessels.

Evolutionary Adaptation of Protein Turnover in White Muscle of Stenothermal Antarctic Fish: Elevated Cold Compensation at Reduced Thermal Responsiveness.

Protein turnover is highly energy consuming and overall relates to an organism's growth performance varying largely between species, e.g., due to pre-adaptation to environmental characteristics such as temperature. Here, we determined protein synthesis rates and capacity of protein degradation in white muscle of the cold stenothermal Antarctic eelpout (Pachycara brachycephalum) and its closely related temperate counterpart, the eurythermal common eelpout (Zoarces viviparus). Both species were exposed to acute warming (P. brachycephalum, 0 °C + 2 °C day-1; Z. viviparus, 4 °C + 3 °C day-1). The in vivo protein synthesis rate (Ks) was monitored after injection of 13C-phenylalanine, and protein degradation capacity was quantified by measuring the activity of cathepsin D in vitro. Untargeted metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy was used to identify the metabolic processes involved. Independent of temperature, the protein synthesis rate was higher in P. brachycephalum (Ks = 0.38-0.614 % day-1) than in Z. viviparus (Ks= 0.148-0.379% day-1). Whereas protein synthesis remained unaffected by temperature in the Antarctic species, protein synthesis in Z. viviparus increased to near the thermal optimum (16 °C) and tended to fall at higher temperatures. Most strikingly, capacities for protein degradation were about ten times higher in the Antarctic compared to the temperate species. These differences are mirrored in the metabolic profiles, with significantly higher levels of complex and essential amino acids in the free cytosolic pool of the Antarctic congener. Together, the results clearly indicate a highly cold-compensated protein turnover in the Antarctic eelpout compared to its temperate confamilial. Constant versus variable environments are mirrored in rigid versus plastic functional responses of the protein synthesis machinery.

Genomic evidence for West Antarctic Ice Sheet collapse during the Last Interglacial.

The marine-based West Antarctic Ice Sheet (WAIS) is considered vulnerable to irreversible collapse under future climate trajectories, and its tipping point may lie within the mitigated warming scenarios of 1.5° to 2°C of the United Nations Paris Agreement. Knowledge of ice loss during similarly warm past climates could resolve this uncertainty, including the Last Interglacial when global sea levels were 5 to 10 meters higher than today and global average temperatures were 0.5° to 1.5°C warmer than preindustrial levels. Using a panel of genome-wide, single-nucleotide polymorphisms of a circum-Antarctic octopus, we show persistent, historic signals of gene flow only possible with complete WAIS collapse. Our results provide the first empirical evidence that the tipping point of WAIS loss could be reached even under stringent climate mitigation scenarios.

Metabolic shifts in the Antarctic fish Notothenia rossii in response to rising temperature and PCO2.

INTRODUCTION: Ongoing ocean warming and acidification increasingly affect marine ecosystems, in particular around the Antarctic Peninsula. Yet little is known about the capability of Antarctic notothenioid fish to cope with rising temperature in acidifying seawater. While the whole animal level is expected to be more sensitive towards hypercapnia and temperature, the basis of thermal tolerance is set at the cellular level, with a putative key role for mitochondria. This study therefore investigates the physiological responses of the Antarctic Notothenia rossii after long-term acclimation to increased temperatures (7°C) and elevated PCO2 (0.2 kPa CO2) at different levels of physiological organisation. RESULTS: For an integrated picture, we analysed the acclimation capacities of N. rossii by measuring routine metabolic rate (RMR), mitochondrial capacities (state III respiration) as well as intra- and extracellular acid-base status during acute thermal challenges and after long-term acclimation to changing temperature and hypercapnia. RMR was partially compensated during warm- acclimation (decreased below the rate observed after acute warming), while elevated PCO2 had no effect on cold or warm acclimated RMR. Mitochondrial state III respiration was unaffected by temperature acclimation but depressed in cold and warm hypercapnia-acclimated fish. In both cold- and warm-exposed N. rossii, hypercapnia acclimation resulted in a shift of extracellular pH (pHe) towards more alkaline values. A similar overcompensation was visible in muscle intracellular pH (pHi). pHi in liver displayed a slight acidosis after warm normo- or hypercapnia acclimation, nevertheless, long-term exposure to higher PCO2 was compensated for by intracellular bicarbonate accumulation. CONCLUSION: The partial warm compensation in whole animal metabolic rate indicates beginning limitations in tissue oxygen supply after warm-acclimation of N. rossii. Compensatory mechanisms of the reduced mitochondrial capacities under chronic hypercapnia may include a new metabolic equilibrium to meet the elevated energy demand for acid-base regulation. New set points of acid-base regulation under hypercapnia, visible at the systemic and intracellular level, indicate that N. rossii can at least in part acclimate to ocean warming and acidification. It remains open whether the reduced capacities of mitochondrial energy metabolism are adaptive or would impair population fitness over longer timescales under chronically elevated temperature and PCO2.

Mitochondrial dynamics underlying thermal plasticity of cuttlefish (Sepia officinalis) hearts.

In the eurythermal cuttlefish Sepia officinalis, performance depends on hearts that ensure systemic oxygen supply over a broad range of temperatures. We therefore aimed to identify adjustments in energetic cardiac capacity and underlying mitochondrial function supporting thermal acclimation and adaptation that could be crucial for the cuttlefish's competitive success in variable environments. Two genetically distinct cuttlefish populations were acclimated to 11, 16 and 21°C. Subsequently, skinned and permeabilised heart fibres were used to assess mitochondrial functioning by means of high-resolution respirometry and a substrate-inhibitor protocol, followed by measurements of cardiac citrate synthase and cytosolic enzyme activities. Temperate English Channel cuttlefish had lower mitochondrial capacities but larger hearts than subtropical Adriatic cuttlefish. Warm acclimation to 21°C decreased mitochondrial complex I activity in Adriatic cuttlefish and increased complex IV activity in English Channel cuttlefish. However, compensation of mitochondrial capacities did not occur during cold acclimation to 11°C. In systemic hearts, the thermal sensitivity of mitochondrial substrate oxidation was high for proline and pyruvate but low for succinate. Oxygen efficiency of catabolism rose as temperature changed from 11 to 21°C via shifts to oxygen-conserving oxidation of proline and pyruvate and via reduced relative proton leak. The changes observed for substrate oxidation, mitochondrial complexes, relative proton leak and heart mass improve energetic efficiency and essentially seem to extend tolerance to high temperatures and reduce associated tissue hypoxia. We conclude that cuttlefish sustain cardiac performance and, thus, systemic oxygen delivery over short- and long-term changes of temperature and environmental conditions by multiple adjustments in cellular and mitochondrial energetics.