Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)—a toxicant that impairs mitochondrial function—across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and—in some instances—codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

SUMMARYThe freshwater turtle Trachemys scripta can survive in the complete absence of O 2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F 1 F O -ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O 2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival.

Speers-Roesch B, Sandblom E, Lau GY, Farrell AP, Richards JG. Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia. Am J Physiol Regul Integr Comp Physiol 298: R104 -R119, 2010. First published October 28, 2009 doi:10.1152/ajpregu.00418.2009.-The ability of an animal to depress ATP turnover while maintaining metabolic energy balance is important for survival during hypoxia. In the present study, we investigated the responses of cardiac energy metabolism and performance in the hypoxia-tolerant tilapia (Oreochromis hybrid sp.) during exposure to environmental hypoxia. Exposure to graded hypoxia (Ն92% to 2.5% air saturation over 3.6 Ϯ 0.2 h) followed by exposure to 5% air saturation for 8 h caused a depression of whole animal oxygen consumption rate that was accompanied by parallel decreases in heart rate, cardiac output, and cardiac power output (CPO, analogous to ATP demand of the heart). These cardiac parameters remained depressed by 50 -60% compared with normoxic values throughout the 8-h exposure. During a 24-h exposure to 5% air saturation, cardiac ATP concentration was unchanged compared with normoxia and anaerobic glycolysis contributed to ATP supply as evidenced by considerable accumulation of lactate in the heart and plasma. Reductions in the provision of aerobic substrates were apparent from a large and rapid (in Ͻ1 h) decrease in plasma nonesterified fatty acids concentration and a modest decrease in activity of pyruvate dehydrogenase. Depression of cardiac ATP demand via bradycardia and an associated decrease in CPO appears to be an integral component of hypoxia-induced metabolic rate depression in tilapia and likely contributes to hypoxic survival. fish; cardiovascular function; adenosine 5Ј-triphosphate; lipid; pyruvate dehydrogenase DURING PERIODS OF LOW OXYGEN, hypoxia-tolerant animals undergo a profound, rapid, and reversible metabolic rate depression as shown by large decreases in oxygen consumption rate (Ṁ O 2 ) and heat production (44,54). This metabolic rate depression reflects a downregulation of cellular ATP turnover to a level that can be sustained by oxygen-independent ATP production. The ability to balance ATP demand with supply and thus maintain stable cellular ATP concentration ([ATP]) is a key response ensuring hypoxic survival in tolerant animals, including many species of fishes that regularly encounter environmental hypoxia (7).A major component of the hypoxia-induced depression of ATP turnover is a reduction of cellular ATP demand, including the regulated arrest of ion pumping and anabolic pathways such as protein synthesis (30,45,49). Metabolic control analyses demonstrate, however, that ATP turnover in both active and metabolically depressed organisms can be controlled both by regulating ATP demand as well as by modulation of metabolic pathways involved in ATP supply such as mitochondrial substrate oxidation (6, 49). Our knowledge is incomplete as to how processes of ATP demand and ATP supply respond during hypoxia exposure in order to achiev...

Dr Wood's Commentary (Wood, 2018) provides six reasons to question the usefulness of P crit and proposes alternative ṀO 2 versus P O2 analyses as its replacement. While we agree with some of Dr Wood's arguments, we feel that none of them warrant abandoning P crit , especially in favour of his proposed alternatives, which provide different information than P crit . A more useful way forward would involve (1) clearly defining P crit , to avoid misinterpretation, and (2) standardizing (or at least clearly describing) the methods used to determine and report P crit , to optimize its comparative value. This topic demands further discussion because Dr Wood's conclusion could have unwarranted influence on how future hypoxia research is conducted and past hypoxia research is interpreted.Dr Wood's arguments are either theoretical (reasons 3-6) or methodological (reasons 1, 2). The theoretical arguments, if true, may warrant the abandonment of P crit . However, contrary to Dr Wood's claims, across species, P crit is strongly correlated with the environmental O 2 level to which species are exposed and is therefore ecologically relevant (Childress and Seibel, 1998;Mandic et al., 2009;Wishner et al., 2018). P crit is also correlated with multiple steps of the O 2 transport cascade, from gill surface area through haemoglobin P 50 to mitochondrial P 50 (Childress and Seibel, 1998;Lau et al., 2017;Mandic et al., 2009), and is a sensitive measure of an animal's overall ability to extract O 2 because altering physiological traits along the cascade can change P crit . For example, anatomical restructuring of the gill to favour O 2 diffusion [e.g. reducing gill epithelial thickness through seawater acclimation in sculpins (Henriksson et al., 2008); increasing lamellar surface area through hypoxia acclimation in crucian carp (Sollid et al., 2003)] can lower P crit . These relationships between P crit and plastic traits along the O 2 transport cascade clearly reflect the physiological relevance of P crit and indicate that P critespecially when it shifts with acclimationdoes indeed represent the P O2 at which O 2 uptake becomes constrained. Therefore, contrary to reason 6, P crit per se does carry biologically relevant information. Hence, P crit remains a useful tool for understanding hypoxic performance because it allows for predictive statements.Much of Dr Wood's reasoning centres on the over-interpretation of P crit . P crit does not necessarily quantify an animal's overall hypoxia tolerance (the product of some combination of aerobic metabolism, anaerobic metabolism and metabolic depression; reason 5), reveal what biological processes the O 2 consumed at P crit is supporting (reason 4), or indicate the onset P O2 of enhanced glycolytic reliance (reason 4) or metabolic depression (reason 5).These ideas have long been excluded from the definition of P crit . Simply, P crit defines the lowest water P O2 at which the animal can maintain some benchmark ṀO 2 state (e.g. ṀO 2,std , the ṀO 2 of an inactive and post-absorptive ectotherm...

Mitochondrial respiration and ATP production are compromised by hypoxia. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals and reduce metabolic rate in hypoxic environments; however, little is known regarding mitochondrial function during hypoxia exposure in this species. To address this knowledge gap, we asked whether the function of NMR brain mitochondria exhibits metabolic plasticity during acute hypoxia. Respirometry was utilized to assess whole-animal oxygen consumption rates and high-resolution respirometry was utilized to assess electron transport system (ETS) function in saponin-permeabilized NMR brain. We found that NMR whole-animal oxygen consumption rate reversibly decreased by ∼85% in acute hypoxia (4 h at 3% O). Similarly, relative to untreated controls, permeabilized brain respiratory flux through the ETS was decreased by ∼90% in acutely hypoxic animals. Relative to carbonyl cyanide -trifluoro-methoxyphenylhydrazone-uncoupled total ETS flux, this functional decrease was observed equally across all components of the ETS except for complex IV (cytochrome oxidase), at which flux was further reduced, supporting a regulatory role for this enzyme during acute hypoxia. The maximum enzymatic capacities of ETS complexes I-V were not altered by acute hypoxia; however, the mitochondrial H gradient decreased in step with the decrease in ETS respiration. Taken together, our results indicate that NMR brain ETS flux and H leak are reduced in a balanced and regulated fashion during acute hypoxia. Changes in NMR mitochondrial metabolic plasticity mirror whole-animal metabolic responses to hypoxia.

22Extreme environments test the limits of life. Still, some organisms thrive in harsh conditions, 23 begging the question whether the repeated colonization of extreme environments is facilitated by 24 predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying 25 convergent evolution of tolerance to hydrogen sulfide (H2S)-a potent toxicant that impairs 26 mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, 27 Poeciliidae) from H2S-rich freshwater springs. We found that mitochondrial function is maintained 28 in the presence of H2S in sulfide spring P. mexicana, but not ancestral lineages in adjacent nonsulfidic 29 habitats, due to convergent adaptations in both the primary toxicity target and a major detoxification 30 enzyme. Additionally, we show that H2S tolerance in 10 independent lineages of sulfide spring fishes 31 across multiple genera of Poeciliidae is mediated by convergent modification and expression changes 32 of genes associated with H2S toxicity and detoxification. Our results demonstrate that the repeated 33 modification of highly conserved physiological pathways associated with essential mitochondrial 34 processes enabled the colonization of novel environments. 35 36 3 Stephen J. Gould was a fierce proponent of the importance of contingency in evolution, famously 37 quipping that replaying the "tape of life" would lead to different outcomes every time (1). 38Mitochondrial genomes were historically thought to be a prime example of such contingency 39 evolution, because alternative genetic variants were assumed to be selectively neutral (2). This 40 paradigm has been shifting, with mounting evidence that mitochondria-and genes encoded in the 41 mitochondrial genome-play an important role in adaptation, especially in the context of 42 physiochemical stress (3). However, it often remains unclear how genetic variation in mitochondrial 43 genomes and nuclear genes that contribute to mitochondrial function translates to variation in 44 physiological and organismal function. Furthermore, it is not known whether exposure to similar 45 selective regimes may cause convergent modifications of mitochondrial genomes and emergent 46 biochemical and physiological functions in evolutionarily independent lineages. Extreme 47 environments that represent novel ecological niches are natural experiments to address questions 48 about mechanisms underlying mitochondrial adaptations and illuminate the predictability of adaptive 49 evolution of mitochondria. Among the most extreme freshwater ecosystems are springs with high 50 levels of hydrogen sulfide (H2S), a potent respiratory toxicant lethal to metazoans due to its 51 inhibition of mitochondrial ATP production (4). Multiple lineages of livebearing fishes (Poeciliidae) 52 have colonized H2S-rich springs throughout the Americas and independently evolved tolerance to 53 sustained H2S concentrations orders of magnitudes higher than those encountered by ancestral 54 lineages in nonsulfid...

Nitrate (NO3−) and nitrite (NO2−) are known to be cardioprotective and to alter energy metabolism in vivo. NO3− action results from its conversion to NO2− by salivary bacteria, but the mechanism(s) by which NO2− affects metabolism remains obscure. NO2− may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2−-dependent S-nitrosation of proteins thiols in vivo. Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2− under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2− in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2− on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2−, combined with the lack of S-nitrosation during anoxia alone or by NO2− during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2− exposure.

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2.