Differential production of mitochondrial reactive oxygen species between mouse (Mus musculus) and crucian carp (Carassius carassius).

AIM: In most vertebrates, oxygen deprivation and subsequent re-oxygenation are associated with mitochondrial impairment and excess production of reactive oxygen species (ROS) like hydrogen peroxide (H2O2). This in turn triggers a cascade of cell-damaging events in a temperature-dependent manner. The crucian carp (Carassius carassius) is one of few vertebrates that survives months without oxygen at cold temperatures and overcomes oxidative damage during re-oxygenation periods. Mitochondria of this anoxia-tolerant species therefore serve as an excellent model in translational research to study adaptation and resilience to low oxygen conditions and thermal variability. METHODS: Here, we used high-resolution respirometry on isolated mitochondria from hearts of crucian carp and the anoxia-intolerant mouse (Mus musculus), at 37 and 8°C; two temperatures relevant for transplantation medicine (i.e., graft preservation and subsequent rewarming). RESULTS: We find: (1) a striking difference in H2O2 release between the two species at 37°C despite comparable mitochondrial efficiency and capacity, (2) a massive H2O2 release after inhibition of complex V in mouse at 37°C that is absent in crucian carp, and prevented in mouse by incubation at 8°C or uncoupling with a protonophore at 37°C, and (3) indications that differences in mitochondrial complex I and II capacity and thermal sensitivity influence the release of mitochondrial H2O2 relative to respiration. CONCLUSION: Our findings provide comparative insights into a spectrum of mitochondrial adaptations in vertebrates and the importance of thermal variability. Furthermore, the species- and temperature-related changes associated with mitochondria highlighted in this study may help identify mitochondria-based targets for translational medicine.

Expression of prolyl hydroxylase domains, the upstream regulators of HIF, in the brain of the anoxia-tolerant crucian carp during anoxia-reoxygenation.

The hypoxia-inducible factor (HIF) is considered key in the transcriptional response to low oxygen. Yet, the role of HIF in the absence of oxygen (anoxia) and in preparation for reoxygenation remains unclear. Recent studies suggest that mounting a HIF response may be counterproductive for anoxia survival. We here studied one of the champions of anoxia survival, the crucian carp (Carassius carassius), and hypothesized that expression of prolyl hydroxylase domains (PHDs; the upstream regulators of HIF) are upregulated to circumvent an energy-costly activation of HIF in anoxia and to prepare for reoxygenation. We measured whole brain mRNA and protein levels of the three isoforms PHD1, PHD2, and PHD3, coded for by multiple paralogs of the genes egln2, egln1, and egln3, using quantitative PCR and Western blotting in the brain of crucian carps exposed to 5 days normoxia or anoxia, and 5 days anoxia followed by 3 or 24 h of reoxygenation. The mRNA levels of most egln paralogs were increased in anoxia and upon reoxygenation, with egln3 showing the largest increase in mRNA level (up to 17-fold) and highest relative mRNA abundance (up to 75% of expressed egln). The protein level of all PHDs was maintained in anoxia and increased upon reoxygenation. We then explored PHD distribution in different brain regions and found PHD immunoreactivity to be associated with axonal branches and showing region-specific changes during anoxia-reoxygenation. Our results support an overall upregulation of egln under prolonged anoxia and PHDs upon reoxygenation in crucian carp, likely aimed at suppressing HIF responses, although regional differences are apparent in such a complex organ as the brain.NEW & NOTEWORTHY We report a profound upregulation of most egln paralog mRNA levels in anoxia and upon reoxygenation, with egln3ii showing the largest, a 17-fold increase, and highest relative mRNA abundance. The relative abundance of prolyl hydroxylase domain (PHD) proteins was maintained during anoxia and increased at reoxygenation. PHD immunoreactivity was localized to axonal branches with region-specific changes during anoxia-reoxygenation. These dynamic and regional changes in crucian carp, champion of anoxia tolerance, are most likely adaptive and call for further mechanistic studies.

Rapid physiological and transcriptomic changes associated with oxygen delivery in larval anemonefish suggest a role in adaptation to life on hypoxic coral reefs.

Connectivity of coral reef fish populations relies on successful dispersal of a pelagic larval phase. Pelagic larvae must exhibit high swimming abilities to overcome ocean and reef currents, but once settling onto the reef, larvae transition to endure habitats that become hypoxic at night. Therefore, coral reef fish larvae must rapidly and dramatically shift their physiology over a short period of time. Taking an integrative, physiological approach, using swimming respirometry, and examining hypoxia tolerance and transcriptomics, we show that larvae of cinnamon anemonefish (Amphiprion melanopus) rapidly transition between "physiological extremes" at the end of their larval phase. Daily measurements of swimming larval anemonefish over their entire early development show that they initially have very high mass-specific oxygen uptake rates. However, oxygen uptake rates decrease midway through the larval phase. This occurs in conjunction with a switch in haemoglobin gene expression and increased expression of myoglobin, cytoglobin, and neuroglobin, which may all contribute to the observed increase in hypoxia tolerance. Our findings indicate that critical ontogenetic changes in the gene expression of oxygen-binding proteins may underpin the physiological mechanisms needed for successful larval recruitment to reefs.

Two decades of research on anoxia tolerance - mitochondria, -omics and physiological diversity.

Just over two decades ago, Bob Boutilier published a much-cited Review in this journal on the mechanisms of cell survival in hypoxia and hypothermia. Here, we celebrate this important Review by describing how our knowledge of the mechanisms behind anoxia tolerance have progressed since 2001, including new key roles of mitochondria, something Boutilier had started exploring. Evidence now suggests that, in anoxia-tolerant brains, mitochondria initiate responses aimed at suppressing electrical activity and energy use. These responses are largely dependent on gamma-aminobutyric acid (GABA) release. Animals that survive anoxia must also tolerate reoxygenation - a major challenge that could cause a massive production of damaging reactive oxygen species (ROS). Here, the handling of succinate, which builds up during anoxia, is critical. Interestingly, there are clear species differences in succinate handling among anoxia-tolerant vertebrates (Trachemys and Chrysemys turtles and crucian carp, Carassius carassius). Trachemys turtles suppress succinate build-up during anoxia, presumably to limit ROS production during reoxygenation. By contrast, in crucian carp, reduction of fumarate to succinate during anoxia appears to be essential for keeping their mitochondria charged and viable. Consequently, during anoxia, crucian carp accumulate much more succinate than Trachemys turtles. Moreover, during anoxia, succinate is apparently transported from crucian carp brain and heart to the liver, which handles succinate upon reoxygenation. This is one example of the striking physiological diversity among vertebrates that survive long-term anoxia. More examples are given, and we argue that -omics approaches are, and will be, helpful in providing new insight and moving the field forward.

The Metabolomic Response of Crucian Carp (Carassius carassius) to Anoxia and Reoxygenation Differs between Tissues and Hints at Uncharacterized Survival Strategies.

The anoxia-tolerant crucian carp (Carassius carassius) has been studied in detail for numerous years, with particular focus on unravelling the underlying physiological mechanisms of anoxia tolerance. However, relatively little work has been focused on what occurs beyond anoxia, and often the focus is a single organ or tissue type. In this study, we quantified more than 100 metabolites by capillary electrophoresis-mass spectrometry (CE-MS) in brain, heart, liver, and blood plasma from four experimental groups, being normoxic (control) fish, anoxia-exposed fish, and two groups that had been exposed to anoxia followed by reoxygenation for either 3 h or 24 h. The heart, which maintains cardiac output during anoxia, unexpectedly, was slower to recover compared to the brain and liver, mainly due to a slower return to control concentrations of the energy-carrying compounds ATP, GTP, and phosphocreatine. Crucian carp accumulated amino acids in most tissues, and also surprisingly high levels of succinate in all tissues investigated during anoxia. Purine catabolism was enhanced, leading to accumulation of uric acid during anoxia and increasing urea formation that continued into 24 h of reoxygenation. These tissue-specific differences in accumulation and distribution of the metabolites may indicate an intricate system of transport between tissues, opening for new avenues of investigation of possible mechanisms aimed at reducing the generation of reactive oxygen species (ROS) and resultant tissue damage during reoxygenation.

The role of mechanistic physiology in investigating impacts of global warming on fishes.

Warming of aquatic environments as a result of climate change is already having measurable impacts on fishes, manifested as changes in phenology, range shifts and reductions in body size. Understanding the physiological mechanisms underlying these seemingly universal patterns is crucial if we are to reliably predict the fate of fish populations with future warming. This includes an understanding of mechanisms for acute thermal tolerance, as extreme heatwaves may be a major driver of observed effects. The hypothesis of gill oxygen limitation (GOL) is claimed to explain asymptotic fish growth, and why some fish species are decreasing in size with warming; but its underlying assumptions conflict with established knowledge and direct mechanistic evidence is lacking. The hypothesis of oxygen- and capacity-limited thermal tolerance (OCLTT) has stimulated a wave of research into the role of oxygen supply capacity and thermal performance curves for aerobic scope, but results vary greatly between species, indicating that it is unlikely to be a universal mechanism. As thermal performance curves remain important for incorporating physiological tolerance into models, we discuss potentially fruitful alternatives to aerobic scope, notably specific dynamic action and growth rate. We consider the limitations of estimating acute thermal tolerance by a single rapid measure whose mechanism of action is not known. We emphasise the continued importance of experimental physiology, particularly in advancing our understanding of underlying mechanisms, but also the challenge of making this knowledge relevant to the more complex reality.

Effects of lactate ions on the cardiorespiratory system in rainbow trout (Oncorhynchus mykiss).

Lactate ions are involved in several physiological processes, including a direct stimulation of the carotid body, causing increased ventilation in mammals. A similar mechanism eliciting ventilatory stimulation in other vertebrate classes has been demonstrated, but it remains to be thoroughly investigated. Here, we investigated the effects of lactate ions on the cardiorespiratory system in swimming rainbow trout by manipulating the blood lactate concentration. Lactate elicited a vigorous, dose-dependent elevation of ventilation and bradycardia at physiologically relevant concentrations at constant pH. After this initial confirmation, we examined the chiral specificity of the response and found that only l-lactate induced these effects. By removal of the afferent inputs from the first gill arch, the response was greatly attenuated, and a comparison of the responses to injections up- and downstream of the gills collectively demonstrated that the lactate response was initiated by branchial cells. Injection of specific receptor antagonists revealed that a blockade of serotonergic receptors, which are involved in the hypoxic ventilatory response, significantly reduced the lactate response. Finally, we identified two putative lactate receptors based on sequence homology and found that both were expressed at substantially higher levels in the gills. We propose that lactate ions modulate ventilation by stimulating branchial oxygen-sensing cells, thus eliciting a cardiorespiratory response through receptors likely to have originated early in vertebrate evolution.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

In modelling effects of global warming, invalid assumptions lead to unrealistic projections.

In their recent Opinion, Pauly and Cheung () provide new projections of future maximum fish weight (W∞ ). Based on criticism by Lefevre et al. (2017) they changed the scaling exponent for anabolism, dG . Here we find that changing both dG and the scaling exponent for catabolism, b, leads to the projection that fish may even become 98% smaller with a 1°C increase in temperature. This unrealistic outcome indicates that the current W∞ is unlikely to be explained by the Gill-Oxygen Limitation Theory (GOLT) and, therefore, GOLT cannot be used as a mechanistic basis for model projections about fish size in a warmer world.

Re-oxygenation after anoxia induces brain cell death and memory loss in the anoxia-tolerant crucian carp.

Crucian carp (Carassius carassius) survive without oxygen for several months, but it is unknown whether they are able to protect themselves from cell death normally caused by the absence, and particularly return, of oxygen. Here, we quantified cell death in brain tissue from crucian carp exposed to anoxia and re-oxygenation using the terminal deoxy-nucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and cell proliferation by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) as well as PCNA mRNA expression. We also measured mRNA and protein expression of the apoptosis executer protease caspase 3, in laboratory fish exposed to anoxia and re-oxygenation and fish exposed to seasonal anoxia and re-oxygenation in their natural habitat over the year. Finally, a behavioural experiment was used to assess the ability to learn and remember how to navigate in a maze to find food, before and after exposure to anoxia and re-oxygenation. The number of TUNEL-positive cells in the telencephalon increased after 1 day of re-oxygenation following 7 days of anoxia, indicating increased cell death. However, there were no consistent changes in whole-brain expression of caspase 3 in either laboratory-exposed or naturally exposed fish, indicating that cell death might occur via caspase-independent pathways or necrosis. Re-oxygenated crucian carp appeared to have lost the memory of how to navigate in a maze (learnt prior to anoxia exposure), while the ability to learn remained intact. PCNA mRNA was elevated after re-oxygenation, indicating increased neurogenesis. We conclude that anoxia tolerance involves not only protection from damage but also repair after re-oxygenation.

Models projecting the fate of fish populations under climate change need to be based on valid physiological mechanisms.

Some recent modelling papers projecting smaller fish sizes and catches in a warmer future are based on erroneous assumptions regarding (i) the scaling of gills with body mass and (ii) the energetic cost of 'maintenance'. Assumption (i) posits that insurmountable geometric constraints prevent respiratory surface areas from growing as fast as body volume. It is argued that these constraints explain allometric scaling of energy metabolism, whereby larger fishes have relatively lower mass-specific metabolic rates. Assumption (ii) concludes that when fishes reach a certain size, basal oxygen demands will not be met, because of assumption (i). We here demonstrate unequivocally, by applying accepted physiological principles with reference to the existing literature, that these assumptions are not valid. Gills are folded surfaces, where the scaling of surface area to volume is not constrained by spherical geometry. The gill surface area can, in fact, increase linearly in proportion to gill volume and body mass. We cite the large body of evidence demonstrating that respiratory surface areas in fishes reflect metabolic needs, not vice versa, which explains the large interspecific variation in scaling of gill surface areas. Finally, we point out that future studies basing their predictions on models should incorporate factors for scaling of metabolic rate and for temperature effects on metabolism, which agree with measured values, and should account for interspecific variation in scaling and temperature effects. It is possible that some fishes will become smaller in the future, but to make reliable predictions the underlying mechanisms need to be identified and sought elsewhere than in geometric constraints on gill surface area. Furthermore, to ensure that useful information is conveyed to the public and policymakers about the possible effects of climate change, it is necessary to improve communication and congruity between fish physiologists and fisheries scientists.

Physiological Challenges to Fishes in a Warmer and Acidified Future.

With the projected levels of global warming and ocean acidification, fishes have to face warmer waters with CO2 levels that are the highest in over 30 million years. The resultant rise in body temperature means that metabolic rates of fish will increase, and some may become energetically compromised. No less worrying, and maybe more surprising, is that rising CO2 concentrations appear to trigger pH regulatory mechanisms that disrupts neural ion gradients, leading to altered neurotransmitter function and maladaptive behavioral changes. We point out the many outstanding questions, including the ultimate one: Will fish be able to adapt to these challenges?

Conservation physiology of marine fishes: state of the art and prospects for policy.

The state of the art of research on the environmental physiology of marine fishes is reviewed from the perspective of how it can contribute to conservation of biodiversity and fishery resources. A major constraint to application of physiological knowledge for conservation of marine fishes is the limited knowledge base; international collaboration is needed to study the environmental physiology of a wider range of species. Multifactorial field and laboratory studies on biomarkers hold promise to relate ecophysiology directly to habitat quality and population status. The 'Fry paradigm' could have broad applications for conservation physiology research if it provides a universal mechanism to link physiological function with ecological performance and population dynamics of fishes, through effects of abiotic conditions on aerobic metabolic scope. The available data indicate, however, that the paradigm is not universal, so further research is required on a wide diversity of species. Fish physiologists should interact closely with researchers developing ecological models, in order to investigate how integrating physiological information improves confidence in projecting effects of global change; for example, with mechanistic models that define habitat suitability based upon potential for aerobic scope or outputs of a dynamic energy budget. One major challenge to upscaling from physiology of individuals to the level of species and communities is incorporating intraspecific variation, which could be a crucial component of species' resilience to global change. Understanding what fishes do in the wild is also a challenge, but techniques of biotelemetry and biologging are providing novel information towards effective conservation. Overall, fish physiologists must strive to render research outputs more applicable to management and decision-making. There are various potential avenues for information flow, in the shorter term directly through biomarker studies and in the longer term by collaborating with modellers and fishery biologists.

Are global warming and ocean acidification conspiring against marine ectotherms? A meta-analysis of the respiratory effects of elevated temperature, high CO2 and their interaction.

With the occurrence of global change, research aimed at estimating the performance of marine ectotherms in a warmer and acidified future has intensified. The concept of oxygen- and capacity-limited thermal tolerance, which is inspired by the Fry paradigm of a bell-shaped increase-optimum-decrease-type response of aerobic scope to increasing temperature, but also includes proposed negative and synergistic effects of elevated CO2 levels, has been suggested as a unifying framework. The objectives of this meta-analysis were to assess the following: (i) the generality of a bell-shaped relationship between absolute aerobic scope (AAS) and temperature; (ii) to what extent elevated CO2 affects resting oxygen uptake MO2rest and AAS; and (iii) whether there is an interaction between elevated temperature and CO2. The behavioural effects of CO2 are also briefly discussed. In 31 out of 73 data sets (both acutely exposed and acclimated), AAS increased and remained above 90% of the maximum, whereas a clear thermal optimum was observed in the remaining 42 data sets. Carbon dioxide caused a significant rise in MO2rest in only 18 out of 125 data sets, and a decrease in 25, whereas it caused a decrease in AAS in four out of 18 data sets and an increase in two. The analysis did not reveal clear evidence for an overall correlation with temperature, CO2 regime or duration of CO2 treatment. When CO2 had an effect, additive rather than synergistic interactions with temperature were most common and, interestingly, they even interacted antagonistically on MO2rest and AAS. The behavioural effects of CO2 could complicate experimental determination of respiratory performance. Overall, this meta-analysis reveals heterogeneity in the responses to elevated temperature and CO2 that is not in accordance with the idea of a single unifying principle and which cannot be ignored in attempts to model and predict the impacts of global warming and ocean acidification on marine ectotherms.

Ambient CO2, fish behaviour and altered GABAergic neurotransmission: exploring the mechanism of CO2-altered behaviour by taking a hypercapnia dweller down to low CO2 levels.

Recent studies suggest that projected rises of aquatic CO2 levels cause acid-base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl(-) and HCO3 (-) gradients across neural membranes interfere with the function of GABA-gated anion channels (GABAA receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO2 environments, like many oceanic habitats, to high levels of CO2 (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO2 levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO2 conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO2 (3.1 kPa) or low-CO2 (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO2 fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABAA receptor blocker. This indicates an involvement of the GABAA receptor and altered Cl(-) and HCO3 (-) ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO2 may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO2 conditions and is prone to disruption if these conditions change.

Will jumping snails prevail? Influence of near-future CO2, temperature and hypoxia on respiratory performance in the tropical conch Gibberulus gibberulus gibbosus.

Tropical coral reef organisms are predicted to be especially sensitive to ocean warming because many already live close to their upper thermal limit, and the expected rise in ocean CO2 is proposed to further reduce thermal tolerance. Little, however, is known about the thermal sensitivity of a diverse and abundant group of reef animals, the gastropods. The humpbacked conch (Gibberulus gibberulus gibbosus), inhabiting subtidal zones of the Great Barrier Reef, was chosen as a model because vigorous jumping, causing increased oxygen uptake (MO2 ), can be induced by exposure to odour from a predatory cone snail (Conus marmoreus). We investigated the effect of present-day ambient (417-454 µatm) and projected-future (955-987 µatm) PCO2 on resting (MO2 , rest) and maximum (MO2 , max) MO2 , as well as MO2 during hypoxia and critical oxygen tension (PO2 , crit), in snails kept at present-day ambient (28°C) or projected-future temperature (33°C). MO2 , rest and MO2 , max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in MO2 , and MO2 , max increased with temperature so that absolute aerobic scope was maintained even at 38°C, although factorial scope was reduced. The humpbacked conch has a high hypoxia tolerance with a PO2 , crit of 2.5 kPa at 28°C and 3.5 kPa at 33°C. There was no effect of elevated CO2 on respiratory performance at any temperature. Long-term temperature records and our field measurements suggest that habitat temperature rarely exceeds 32.6°C during the summer, indicating that these snails have aerobic capacity in excess of current and future needs.

Air breathing in the Arctic: influence of temperature, hypoxia, activity and restricted air access on respiratory physiology of the Alaska blackfish Dallia pectoralis.

The Alaska blackfish (Dallia pectoralis) is an air-breathing fish native to Alaska and the Bering Sea islands, where it inhabits lakes that are ice-covered in the winter, but enters warm and hypoxic waters in the summer to forage and reproduce. To understand the respiratory physiology of this species under these conditions and the selective pressures that maintain the ability to breathe air, we acclimated fish to 5°C and 15°C and used respirometry to measure: standard oxygen uptake (M(O2)) in normoxia (19.8 kPa P(O2)) and hypoxia (2.5 kPa), with and without access to air; partitioning of standard M(O2) in normoxia and hypoxia; maximum M(O2) and partitioning after exercise; and critical oxygen tension (P(crit)). Additionally, the effects of temperature acclimation on haematocrit, haemoglobin oxygen affinity and gill morphology were assessed. Standard M(O2) was higher, but air breathing was not increased, at 15°C or after exercise at both temperatures. Fish acclimated to 5°C or 15°C increased air breathing to compensate and fully maintain standard M(O2) in hypoxia. Fish were able to maintain M(O2) through aquatic respiration when air was denied in normoxia, but when air was denied in hypoxia, standard M(O2) was reduced by ∼30-50%. P(crit) was relatively high (5 kPa) and there were no differences in P(crit), gill morphology, haematocrit or haemoglobin oxygen affinity at the two temperatures. Therefore, Alaska blackfish depends on air breathing in hypoxia and additional mechanisms must thus be utilised to survive hypoxic submergence during the winter, such as hypoxia-induced enhancement in the capacities for carrying and binding blood oxygen, behavioural avoidance of hypoxia and suppression of metabolic rate.

Anaemia only causes a small reduction in the upper critical temperature of sea bass: is oxygen delivery the limiting factor for tolerance of acute warming in fishes?

To address how the capacity for oxygen transport influences tolerance of acute warming in fishes, we investigated whether a reduction in haematocrit, by means of intra-peritoneal injection of the haemolytic agent phenylhydrazine, lowered the upper critical temperature of sea bass. A reduction in haematocrit from 42±2% to 20±3% (mean ± s.e.m.) caused a significant but minor reduction in upper critical temperature, from 35.8±0.1 to 35.1±0.2°C, with no correlation between individual values for haematocrit and upper thermal limit. Anaemia did not influence the rise in oxygen uptake between 25 and 33°C, because the anaemic fish were able to compensate for reduced blood oxygen carrying capacity with a significant increase in cardiac output. Therefore, in sea bass the upper critical temperature, at which they lost equilibrium, was not determined by an inability of the cardio-respiratory system to meet the thermal acceleration of metabolic demands.

Marine mollusc predator-escape behaviour altered by near-future carbon dioxide levels.

Ocean acidification poses a range of threats to marine invertebrates; however, the potential effects of rising carbon dioxide (CO2) on marine invertebrate behaviour are largely unknown. Marine gastropod conch snails have a modified foot and operculum allowing them to leap backwards rapidly when faced with a predator, such as a venomous cone shell. Here, we show that projected near-future seawater CO2 levels (961 µatm) impair this escape behaviour during a predator-prey interaction. Elevated-CO2 halved the number of snails that jumped from the predator, increased their latency to jump and altered their escape trajectory. Physical ability to jump was not affected by elevated-CO2 indicating instead that decision-making was impaired. Antipredator behaviour was fully restored by treatment with gabazine, a GABA antagonist of some invertebrate nervous systems, indicating potential interference of neurotransmitter receptor function by elevated-CO2, as previously observed in marine fishes. Altered behaviour of marine invertebrates at projected future CO2 levels could have potentially far-reaching implications for marine ecosystems.