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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. [ABSTRACT FROM AUTHOR]

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. Connectivity of coral reef fish populations relies on successful dispersal of a pelagic larval phase. This study shows that anemonefish larvae exhibit physiological and transcriptomic changes associated with swimming, oxygen uptake and hypoxia tolerance; these changes potentially support dispersal and connectivity among coral reefs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 reoxygenation 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, therewere 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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? [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (ṀO2), 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 (ṀO2,rest) and maximum (ṀO2,max) ṀO2, as well as ṀO2 during hypoxia and critical oxygen tension (PO2,crit), in snails kept at present-day ambient (28°C) or projectedfuture temperature (33°C). ṀO2,rest and ṀO2,max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in ṀO2, and ṀO2,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. [ABSTRACT FROM AUTHOR]

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 (ṀO2) in normoxia (19.8 kPa Po2) and hypoxia (2.5 kPa), with and without access to air; partitioning of standard Mo2 in normoxia and hypoxia; maximum Ṁo2 and partitioning after exercise; and critical oxygen tension (Pcrit). Additionally, the effects of temperature acclimation on haematocrit, haemoglobin oxygen affinity and gill morphology were assessed. Standard Ṁo2 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 Ṁo2 in hypoxia. Fish were able to maintain ṀO2 through aquatic respiration when air was denied in normoxia, but when air was denied in hypoxia, standard Ṁo2 was reduced by ~30-50%. Pcrit was relatively high (5 kPa) and there were no differences in Pcrit, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The air-breathing striped snakehead Channa striata is an important fish in Asian aquaculture with high market value. C. striata is typically cultured in simple mud ponds, where it is frequently exposed to severe hypoxia throughout the growth cycle. Here, the partitioning of oxygen uptake during digestion, and the size of the postprandial metabolism (specific dynamic action or SDA), were studied in hypoxic and normoxic snakehead (30 and 150mmHg, respectively). The ventilatory response to hypoxia was investigated separately. When fish were fed 5% of their body mass as fish fillet, total MO2 increased to 2–2.5 times above the standard metabolic rate. Peak MO2 was significantly lower in hypoxic fish (253mgO2 kg−1 h−1) compared to normoxic fish (322mgO2 kg−1 h−1), and the total SDA was also significantly lower in hypoxia. These results show that despite C. striata being an air-breather, the limited aquatic respiration in severe hypoxia impairs its general performance during digestion. These results have implications for the aquaculture practices of C. striata, and probably other species within this genus, because increased oxygen availability in the water would be expected to increase their digestion efficiency. Further studies are needed to understand the underlying mechanisms, and to determine optimal aeration in these aquaculture ponds. [Copyright &y& Elsevier]

Abstract: The tolerance and effects of nitrite on ion balance and haematology were investigated in the striped snakehead, Channa striata Bloch 1793, which is an air-breathing fish with reduced gills of importance for aquaculture in South East Asia. C. striata was nitrite tolerant with a 96h LC50 of 4.7mM. Effects of sub-lethal exposures to nitrite (0mM, 1.4mM, and 3.0mM) were determined during a 7-day exposure period. Plasma nitrite increased, but the internal concentration remained well below ambient levels. Extracellular nitrate rose by several mM, indicating that a large proportion of the nitrite taken up was converted to nitrate. Nitrite reacted with erythrocyte haemoglobin (Hb) causing methaemoglobin (metHb) to increase to 30% and nitrosylhaemoglobin (HbNO) to increase to 10% of total Hb. Both metHb and HbNO stabilised after 4 days, and functional Hb levels accordingly never fell below 60% of total Hb. Haematocrit and total Hb were unaffected by nitrite. Although the effects of nitrite exposure seemed minor in terms of plasma nitrite and metHb increases, ion balance was strongly affected. In the high exposure group, total osmolality decreased from 320mOsm to 260mOsm, and plasma sodium from 150mM to 120mM, while plasma chloride fell from 105mM to 60mM and plasma bicarbonate rose from 12mM in controls to 20mM in exposed fish. The extreme changes in ion balance in C. striata are different from the response reported in other fish, and further studies are needed to investigate the mechanism behind the observed changes in regulation. [Copyright &y& Elsevier]

Abstract: In this study we investigated nitrite (NO2 −) effects in striped catfish, a facultative air-breather. Fish were exposed to 0, 0.4, and 0.9mM nitrite for 0, 1, 2, 4, and 7 days, and levels of functional haemoglobin, methaemoglobin (metHb) and nitrosyl haemoglobin (HbNO) were assessed using spectral deconvolution. Plasma concentrations of nitrite, nitrate, chloride, potassium, and sodium were also measured. Partitioning of oxygen consumption was determined to reveal whether elevated metHb (causing functional hypoxia) induced air-breathing. The effects of nitrite on maximum oxygen uptake (MO2max) and critical swimming speed (U crit) were also assessed. Striped catfish was highly tolerant to nitrite exposure, as reflected by a 96h LC50 of 1.65mM and a moderate nitrite uptake into the blood. Plasma levels of nitrite reached a maximum after 1 day of exposure, and then decreased, never exceeding ambient levels. MetHb, HbNO and nitrate (a nitrite detoxification product) also peaked after 1 day and then decreased. Only high levels of nitrite and metHb caused reductions in MO2max and U crit. The response of striped catfish contrasts with that seen in most other fish species and discloses efficient mechanisms of combating nitrite threats. Furthermore, even though striped catfish is an efficient air-breather, this species has the ability to sustain aerobic scope and swimming performance without air-breathing, even when faced with nitrite-induced reductions in blood oxygen carrying capacity. Our study is the first to confirm that high levels of nitrite and metHb reduce MO2max and thereby aerobic scope, while more moderate elevations fail to do so. Further studies are needed to elucidate the mechanisms underlying the low nitrite accumulation in striped catfish. [Copyright &y& Elsevier]

Abstract: Air-breathing fish are common in the tropics, and their importance in Asian aquaculture is increasing, but the respiratory physiology of some of the key species such as the striped catfish, Pangasianodon hypophthalmus Sauvage 1878 is unstudied. P. hypophthalmus is an interesting species as it appears to possess both well-developed gills and a modified swim bladder that functions as an air-breathing organ indicating a high capacity for both aquatic and aerial respiration. Using newly developed bimodal intermittent-closed respirometry, the partitioning of oxygen consumption in normoxia and hypoxia was investigated in P. hypophthalmus. In addition the capacity for aquatic breathing was studied through measurements of oxygen consumption when access to air was denied, both in normoxia and hypoxia, and the critical oxygen tension, P crit, was also determined during these experiments. Finally, gill ventilation and air-breathing frequency were measured in a separate experiment with pressure measurements from the buccal cavity. The data showed that P. hypophthalmus is able to maintain standard metabolic rate (SMR) through aquatic breathing alone in normoxia, but that air-breathing is important during hypoxia. Gill ventilation was reduced during air-breathing, which occurred at oxygen levels below 8 kPa, coinciding with the measured P crit of 7.7 kPa. The findings in this study indicate that the introduction of aeration into the aquaculture of P. hypophthalmus could potentially reduce the need to air-breathe. The possibility of reducing air-breathing frequency may be energetically beneficial for the fish, leaving more of the aerobic scope for growth and other activities, due to the proposed energetic costs of surfacing behavior. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogen sulfide (H2S), nitric oxide (NO) and nitrite (NO2 −) are formed in vivo and are of crucial importance in the tissue response to hypoxia, particularly in the cardiovascular system, where these signaling molecules are involved in a multitude of processes including the regulation of vascular tone, cellular metabolic function and cytoprotection. This report summarizes current advances on the mechanisms by which these signaling pathways act and may have evolved in animals with different tolerance to hypoxia, as presented and discussed during the scientific sessions of the annual meeting of the Society for Experimental Biology in 2011 in Glasgow. It also highlights the need and potential for a comparative approach of study and collaborative effort to identify potential link(s) between the signaling pathways involving NO, nitrite and H2S in the whole-body responses to hypoxia. [Copyright &y& Elsevier]

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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.

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., (© 2016. Published by The Company of Biologists Ltd.)

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 (ṀO2 ), 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 (ṀO2 , rest) and maximum (ṀO2 , max) ṀO2 , as well as ṀO2 during hypoxia and critical oxygen tension (PO2 , crit), in snails kept at present-day ambient (28°C) or projected-future temperature (33°C). ṀO2 , rest and ṀO2 , max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in ṀO2 , and ṀO2 , 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., (© 2015. Published by The Company of Biologists Ltd.)

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.