Recalibrating the significance of the decline effect in fish ocean acidification research.

The recently described decline effect in ocean acidification impacts on fish behaviour should not be equated with negligible effects. Here, existing mechanistic data are used to argue for continued research and cautions against "throwing the baby out with the bathwater."

Acute toxicity testing of 6PPD-quinone on the estuarine-dependent sport fish, Sciaenops ocellatus.

Recently, large-scale fish kills in the Pacific Northwest were linked to tire wear particles (TWPs) left on roadways, with the lethality attributed to 6PPD-quinone. which has a median lethal concentration of <1 µg/L for selected salmonids. However, there remains a paucity of 6PPD-quinone toxicity values developed for estuarine fish species, which is particularly significant because estuaries receiving inflows from highly urbanized watersheds are especially vulnerable to TWP contamination. Therefore, the present study aimed to determine the toxicity of 6PPD-quinone to an economically and ecologically important estuarine-dependent fish-red drum (Sciaenops ocellatus). Here, we examined the relative sensitivities of three early life stages within red drum: embryonic, larval, and post-settlement for 24-72 hours, depending on the life stage. Exposure concentrations ranged from 10 µg/L to 500 µg/L. We also assessed the sub-lethal impacts of 6PPD-quinone exposure on development during embryonic and larval stages, including body and organ sizes. Our results indicate that red drum are not acutely sensitive to 6PPD-quinone at each early life stage tested. We also found that yolk-sac larvae did not exhibit sub-lethal morphological impacts in a dose-dependent manner, regardless of exposure during embryonic and larval stages. These data are the first to assess the impacts of 6PPD-quinone on estuarine-dependent non-model fishes.

Hypoxia acclimation improves mitochondrial efficiency in the aerobic swimming muscle of red drum (Sciaenops ocellatus).

Environmental hypoxia (low dissolved oxygen) is a significant threat facing fishes. As fishes require oxygen to efficiently produce ATP, hypoxia can significantly limit aerobic capacity. However, some fishes show respiratory flexibility that rescues aerobic performance, including plasticity in mitochondrial performance. This plasticity may result in increased mitochondrial efficiency (e.g., less proton leak), increased oxygen storage capacity (increased myoglobin), and oxidative capacity (e.g., higher citrate synthase activity) under hypoxia. We acclimated a hypoxia-tolerant fish, red drum (Sciaenops ocellatus), to 8-days of constant hypoxia to induce a hypoxic phenotype. Fish were terminally sampled for cardiac and red muscle tissue to quantify oxidative phosphorylation, proton leak, and maximum respiration in tissue from both hypoxia-acclimated and control fish. Tissue was also collected to assess the plasticity of citrate synthase enzyme activity and mRNA expression for select oxygen storage and antioxidant pathway transcripts. We found that mitochondrial respiration rates were not affected by hypoxia exposure in cardiac tissue, though citrate synthase activity and myoglobin expression were higher following hypoxia acclimation. Interestingly, measures of mitochondrial efficiency in red muscle significantly improved in hypoxia-acclimated individuals. Hypoxia-acclimated fish had significantly higher OXPHOS Control Efficiency, OXPHOS Capacity and Coupling Control Ratios (i.e., LEAK/OXPHOS). There was no significant change to citrate synthase activity or myoglobin expression in red muscle. Overall, these results suggest that red muscle mitochondria of hypoxia-acclimated fish more efficiently utilize oxygen, which may explain previous reports in red drum of improved aerobic swimming performance in the absence of improved maximum metabolic rate following hypoxia acclimation.

Functional divergence of teleost carbonic anhydrase 4.

The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.

The role of carbonic anhydrase-mediated tissue oxygen extraction in a marine teleost acclimated to hypoxia.

With the growing prevalence of hypoxia (O2 levels ≤2 mg l-1) in aquatic and marine ecosystems, there is increasing interest in the adaptive mechanisms fish may employ to better their performance in stressful environments. Here, we investigated the contribution of a proposed strategy for enhancing tissue O2 extraction - plasma-accessible carbonic anhydrase (CA-IV) - under hypoxia in a species of estuarine fish (red drum, Sciaenops ocellatus) that thrives in fluctuating habitats. We predicted that hypoxia-acclimated fish would increase the prevalence of CA-IV in aerobically demanding tissues to confer more efficient tissue O2 extraction. Furthermore, we predicted the phenotypic changes to tissue O2 extraction that occur with hypoxia acclimation may improve respiratory and swim performance under 100% O2 conditions (i.e. normoxia) when compared with performance in fish that have not been acclimated to hypoxia. Interestingly, there were no significant differences in relative CA-IV mRNA expression, protein abundance or enzyme activity between the two treatments, suggesting CA-IV function is maintained under hypoxia. Likewise, respiratory performance of hypoxia-acclimated fish was similar to that of control fish when tested in normoxia. Critical swim speed (Ucrit) was significantly higher in hypoxia-acclimated fish but translated to marginal ecological benefits with an increase of ∼0.3 body lengths per second. Instead, hypoxia-acclimated fish may have relied more heavily on anaerobic metabolism during their swim trials, utilizing burst swimming 1.5 times longer than control fish. While the maintenance of CA-IV may still be an important contributor for hypoxia tolerance, our evidence suggests hypoxia-acclimated red drum are using other mechanisms to cope in an O2-depleted environment.

Is hypoxia vulnerability in fishes a by-product of maximum metabolic rate?

The metabolic index concept combines metabolic data and known thermal sensitivities to estimate the factorial aerobic scope of animals in different habitats, which is valuable for understanding the metabolic demands that constrain species' geographical distributions. An important assumption of this concept is that the O2 supply capacity (which is equivalent to the rate of oxygen consumption divided by the environmental partial pressure of oxygen: ) is constant at O2 tensions above the critical O2 threshold (i.e. the where O2 uptake can no longer meet metabolic demand). This has led to the notion that hypoxia vulnerability is not a selected trait, but a by-product of selection on maximum metabolic rate. In this Commentary, we explore whether this fundamental assumption is supported among fishes. We provide evidence that O2 supply capacity is not constant in all fishes, with some species exhibiting an elevated O2 supply capacity in hypoxic environments. We further discuss the divergent selective pressures on hypoxia- and exercise-based cardiorespiratory adaptations in fishes, while also considering the implications of a hypoxia-optimized O2 supply capacity for the metabolic index concept.

A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

Behavioral Changes in a Coastal Marine Fish Lead to Increased Predation Risk Following Oil Exposure.

Fishes exposed to crude oil have shown reduced sociability and poor habitat selection, which corresponded with increased predation risk. However, the contribution of oil-induced cardiorespiratory impairments to these findings is uncertain. This study explores the effect of oil exposure on predation risk in a model fish species, Sciaenops ocellatus, across a suite of physiological and behavioral end points to elucidate the mechanisms through which any observed effects are manifested. Using mesocosms to assess group predator avoidance, oil exposure to 36.3 µg l-1 ΣPAH reduced the time to 50% mortality from a mean time of 80.0 (74.1-86.0 95% confidence interval [CI]) min to 39.2 (35.6-42.8 95% CI) min. The influence of oil impaired cardiorespiratory and behavioral pathways on predation risk was assessed based on respiratory performance, swim performance, sociability, and routine activity. Swim trials demonstrated that cardiorespiratory and swim performance were unaffected by exposures to 26.6 or 100.8 µg l-1 ΣPAH. Interestingly, behavioral tests revealed that exposure to 26.6 µg l-1 ΣPAH increased distance moved, speed, acceleration, and burst activity. These data indicate that behavioral impairment is more sensitive than cardiorespiratory injury and may be a more important driver of downstream ecological risk following oil exposure in marine species.

The effects of warming on red blood cell carbonic anhydrase activity and respiratory performance in a marine fish.

Measures of fitness are valuable tools to predict species' responses to environmental changes, like increased water temperature. Aerobic scope (AS) is a measure of an individual's capacity for aerobic processes, and frequently used as a proxy for fitness. However, AS is complicated by individual variation found not only within a species, but within similar body sizes as well. Maximum metabolic rate (MMR), one of the factors determining AS, is constrained by an individual's ability to deliver and extract oxygen (O2) at the tissues. Recently, data has shown that red blood cell carbonic anhydrase (RBC CA) is rate-limiting for O2 delivery in red drum (Sciaenops ocellatus). We hypothesized increased temperature impacts MMR and RBC CA activity in a similar manner, and that an individual's RBC CA activity drives individual variation in AS. Red drum were acutely exposed to increased temperature (+6 °C; 22 °C to 28 °C) for 24 h prior to exhaustive exercise and intermittent-flow respirometry at 28 °C. RBC CA activity was measured before temperature exposure and after aerobic performance. Due to enzymatic thermal sensitivity, acute warming increased individual RBC CA activity by 36%, while there was no significant change in the control (22 °C) treatment. Interestingly, average MMR of the acute warming treatment was 36% greater than that of control drum. However, we found no relationships between individual RBC CA activity and their respective MMR and AS at either temperature. While warming similarly affects RBC CA activity and MMR, RBC CA activity is not a predictor of individual MMR.

Assessment of hypoxia avoidance behaviours in a eurythermal fish at two temperatures using a modified shuttlebox system.

Behavioural avoidance responses of red drum (Sciaenops ocellatus) to aquatic hypoxia were investigated at 22 and 30°C using a modified shuttlebox system. Fish movement between a control side maintained at normoxia and a hypoxic side with stepwise decreasing water oxygen tension was analysed for entries into the hypoxic side, residence time per entry into the hypoxic side and total time in the hypoxic side. Acclimation to 30°C increased the oxygen threshold for the onset of hypoxia avoidance behaviours for entries and total time, while residence time per entry was unchanged.

Red blood cell carbonic anhydrase mediates oxygen delivery via the Root effect in red drum.

Oxygen (O2) and carbon dioxide (CO2) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O2 binding even at high O2 tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O2 delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O2 offloading following a physiologically relevant CO2-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O2 offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O2 offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O2 offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O2 delivery in red drum.

Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes.

We investigated whether fatigue from sustained aerobic swimming provides a sub-lethal endpoint to define tolerance of acute warming in fishes, as an alternative to loss of equilibrium (LOE) during a critical thermal maximum (CTmax) protocol. Two species were studied, Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus). Each fish underwent an incremental swim test to determine gait transition speed (UGT), where it first engaged the unsteady anaerobic swimming mode that preceded fatigue. After suitable recovery, each fish was exercised at 85% of their own UGT and warmed 1°C every 30 min, to identify the temperature at which they fatigued, denoted as CTswim Fish were also submitted to a standard CTmax, warming at the same rate as CTswim, under static conditions until LOE. All individuals fatigued in CTswim, at a mean temperature approximately 2°C lower than their CTmax Therefore, if exposed to acute warming in the wild, the ability to perform aerobic metabolic work would be constrained at temperatures significantly below those that directly threatened survival. The collapse in performance at CTswim was preceded by a gait transition qualitatively indistinguishable from that during the incremental swim test. This suggests that fatigue in CTswim was linked to an inability to meet the tissue oxygen demands of exercise plus warming. This is consistent with the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, regarding the mechanism underlying tolerance of warming in fishes. Overall, fatigue at CTswim provides an ecologically relevant sub-lethal threshold that is more sensitive to extreme events than LOE at CTmax.

Mechanisms of acid-base regulation following respiratory alkalosis in red drum (Sciaenops ocellatus).

Respiratory acidosis and subsequent metabolic compensation are well-studied processes in fish exposed to elevated CO2 (hypercapnia). Yet, such exposures in the marine environment are invariably accompanied by a return of environmental CO2 to atmospheric baselines. This understudied phenomenon has the potential to cause a respiratory alkalosis that would necessitate base excretion. Here we sought to explore this question and the associated physiological mechanisms that may accompany base excretions using the red drum (Sciaenops ocellatus). As expected, when high pCO2 (15,000 µatm CO2) acclimated red drum were transferred to normal pCO2, their net H+ excretion shifted from positive (0.157 ± 0.044 µmol g-1 h-1) to negative (-0.606 ± 0.116 µmol g-1 h-1) in the 2 h post-transfer period. Net H+ excretion returned to control rates during the 3 to 24 h flux period. Gene expression and enzyme activity assays demonstrated that while the acidosis resulted in significant changes in several relevant transporters, no significant changes accompanied the alkalosis phase. Confocal microscopy was used to assess alkalosis-stimulated translocation of V-type H+ ATPase to the basolateral membrane previously seen in other marine species; however, no apparent translocation was observed. Overall, these data demonstrate that fluctuations in environmental CO2 result in both acidic and alkalotic respiratory disturbances; however, red drum maintain sufficient regulatory capacity to accommodate base excretion. Furthermore, this work does not support a role for basolateral VHA translocation in metabolic compensation from a systemic alkalosis in teleosts.

Na+ K+ ATPase isoform switching in zebrafish during transition to dilute freshwater habitats.

Na+ K+ ATPase (NKA) is crucial to branchial ion transport as it uses the energy from ATP to move Na+ against its electrochemical gradient. When fish encounter extremely dilute environments the energy available from ATP hydrolysis may not be sufficient to overcome thermodynamic constraints on ion transport. Yet many fish species-including zebrafish-are capable of surviving in dilute environments. Despite much study, the physiological mechanisms by which this occurs remain poorly understood. Here, we demonstrate that zebrafish acclimated to less than 10 µM Na+ water exhibit upregulation of a specific NKA α subunit ( zatp1a1a.5) that, unlike most NKA heterotrimers, would result in transfer of only a single Na+ and K+ per ATP hydrolysis reaction. Thermodynamic models demonstrate that this change is sufficient to reduce the activation energy of NKA, allowing it to overcome the adverse electrochemical gradient imposed by dilute freshwater. Importantly, upregulation of zatp1a1a.5 also coincides with the recovery of whole body Na+ post-transfer, which occurs within 24 h. While these structural modifications are crucial for allowing zebrafish to survive in ion-poor environments, phylogenetic and structural analysis of available α subunits from a range of teleosts suggests this adaptation is not widely distributed.

mRNA-miRNA-Seq Reveals Neuro-Cardio Mechanisms of Crude Oil Toxicity in Red Drum ( Sciaenops ocellatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil can cause global gene dysregulation and developmental impairment in fish. However, the mechanisms that alter gene regulation are not well understood. In this study, larval red drum ( Sciaenops ocellatus) were exposed to water accommodated fractions of source oil (6.8, 13.7, and 35.9 µg/L total PAHs) and weathered slick oil (4.7, 8.1, and 18.0 µg/L total PAHs) from the Deepwater Horizon (DWH) oil spill. The global mRNA-microRNA functional networks associated with the toxicity of DWH oil were explored by next-generation sequencing and in-depth bioinformatics analyses. Both source and slick oil significantly altered the expression of miR-18a, miR-27b, and miR-203a across all exposure concentrations. Consistent with the observed concentration-dependent morphological changes, the target mRNAs of these microRNAs were predominantly involved in neuro-cardio system development processes and associated key signaling pathways such as axonal guidance signaling, cAMP-response-element-binding protein signaling in neurons, calcium signaling, and nuclear-factor-of-activated T cells signaling in cardiac hypertrophy. The results indicated that the developmental toxicity of crude oil may result from the abnormal expression of microRNAs and associated target genes, especially for the nervous system. Moreover, we provide a case study for systematic toxicity evaluation leveraging mRNA-microRNA-seq data using nonmodel species.

A solution to Nature's haemoglobin knockout: a plasma-accessible carbonic anhydrase catalyses CO2 excretion in Antarctic icefish gills.

In all vertebrates studied to date, CO2 excretion depends on the enzyme carbonic anhydrase (CA) that catalyses the rapid conversion of HCO3- to CO2 at the gas-exchange organs. The largest pool of CA is present within red blood cells (RBCs) and, in some vertebrates, plasma-accessible CA (paCA) isoforms participate in CO2 excretion. However, teleost fishes typically do not have paCA at the gills and CO2 excretion is reliant entirely on RBC CA - a strategy that is not possible in icefishes. As the result of a natural knockout, Antarctic icefishes (Channichthyidae) are the only known vertebrates that do not express haemoglobin (Hb) as adults, and largely lack RBCs in the circulation (haematocrit <1%). Previous work has indicated the presence of high levels of membrane-bound CA activity in the gills of icefishes, but without determining its cellular orientation. Thus, we hypothesised that icefishes express a membrane-bound CA isoform at the gill that is accessible to the blood plasma. The CA distribution was compared in the gills of two closely related notothenioid species, one with Hb and RBCs (Notothenia rossii) and one without (Champsocephalus gunnari). Molecular, biochemical and immunohistochemical markers indicate high levels of a Ca4 isoform in the gills of the icefish (but not the red-blooded N. rossii), in a plasma-accessible location that is consistent with a role in CO2 excretion. Thus, in the absence of RBC CA, the icefish gill could exclusively provide the catalytic activity necessary for CO2 excretion - a pathway that is unlike that of any other vertebrate.

Aggression supersedes individual oxygen demand to drive group air-breathing in a social catfish.

Group-living is widespread among animals and comes with numerous costs and benefits. To date, research examining group-living has focused on trade-offs surrounding foraging, while other forms of resource acquisition have been largely overlooked. Air-breathing has evolved in many fish lineages, allowing animals to obtain oxygen in hypoxic aquatic environments. Breathing air increases the threat of predation, so some species perform group air-breathing, to reduce individual risk. Within species, individual air-breathing can be influenced by metabolic rate as well as personality, but the mechanisms of group air-breathing remain unexplored. It is conceivable that keystone individuals with high metabolic demand or intrinsic tendency to breathe air may drive social breathing, especially in hypoxia. We examined social air-breathing in African sharptooth catfish Clarias gariepinus, to determine whether individual physiological traits and spontaneous tendency to breathe air influence the behaviour of entire groups, and whether such influences vary in relation to aquatic oxygen availability. We studied 11 groups of four catfish in a laboratory arena and recorded air-breathing behaviour, activity and agonistic interactions at varying levels of hypoxia. Bimodal respirometry was used to estimate individual standard metabolic rate (SMR) and the tendency to utilize aerial oxygen when alone. Fish took more air breaths in groups as compared to when they were alone, regardless of water oxygen content, and displayed temporally clustered air-breathing behaviour, consistent with existing definitions of synchronous air-breathing. However, groups displayed tremendous variability in surfacing behaviour. Aggression by dominant individuals within groups was the main factor influencing air-breathing of the entire group. There was no association between individual SMR, or the tendency to obtain oxygen from air when in isolation, and group air-breathing. For C. gariepinus, synchronous air-breathing is strongly influenced by agonistic interactions, which may expose subordinate individuals to risk of predation. Influential individuals exerted an overriding effect on risk-taking by the entire group, for reasons independent of their physiological oxygen requirements. Overall, this illustrates that social context can obscure interactions between an individual's physiological and behavioural traits and their tendency to take risks to obtain resources.

Effects of salinity and hypoxia-induced hyperventilation on oxygen consumption and cost of osmoregulation in the estuarine red drum (Sciaenops ocellatus).

Understanding the physiological responses of fishes to salinity changes and aquatic hypoxia is essential for the conservation of marine species. Salinity changes affect the osmotic gradient across the gill epithelium, while hypoxia increases gill ventilation and the flow of water over the gills. Both processes affect the diffusive movement of ions and water across the gill epithelium, and the rate of active ion transport required for maintaining osmotic homeostasis. Consequently, salinity and hypoxia may affect the energetic cost of osmoregulation, and consequently the energy available for other physiological functions such as migration, growth, and reproduction. Historically, studies have assessed the costs of osmoregulation and ventilation in fishes via standard metabolic rate (SMR); however, few studies have used a multi-stressor approach that fully accounts for the osmorespiratory compromise. Here, we determined the combined effects of salinity and hypoxia on SMR, routine metabolic rate (RMR), and plasma ion concentrations in red drum (Sciaenops ocellatus) acclimated to salinities ranging from freshwater to hypersalinity. Surprisingly, there was no significant change in any parameter as a consequence of salinity or hypoxia, including the relatively extreme scenario of combined hypersalinity and hypoxia exposure. We conclude that changes in the osmotic gradient across the gill epithelium and the flow of water over the gills have a negligible effect on the whole animal energy budget of S. ocellatus, suggesting that the cost of osmoregulation is a minor component of basal metabolism regardless of oxygenation status.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.

Effects of hypoxia and ocean acidification on the upper thermal niche boundaries of coral reef fishes.

Rising ocean temperatures are predicted to cause a poleward shift in the distribution of marine fishes occupying the extent of latitudes tolerable within their thermal range boundaries. A prevailing theory suggests that the upper thermal limits of fishes are constrained by hypoxia and ocean acidification. However, some eurythermal fish species do not conform to this theory, and maintain their upper thermal limits in hypoxia. Here we determine if the same is true for stenothermal species. In three coral reef fish species we tested the effect of hypoxia on upper thermal limits, measured as critical thermal maximum (CTmax). In one of these species we also quantified the effect of hypoxia on oxygen supply capacity, measured as aerobic scope (AS). In this species we also tested the effect of elevated CO2 (simulated ocean acidification) on the hypoxia sensitivity of CTmax We found that CTmax was unaffected by progressive hypoxia down to approximately 35 mmHg, despite a substantial hypoxia-induced reduction in AS. Below approximately 35 mmHg, CTmax declined sharply with water oxygen tension (PwO2). Furthermore, the hypoxia sensitivity of CTmax was unaffected by elevated CO2 Our findings show that moderate hypoxia and ocean acidification do not constrain the upper thermal limits of these tropical, stenothermal fishes.