
==== Front
Conserv Physiol
Conserv Physiol
conphys
Conservation Physiology
2051-1434
Oxford University Press

10.1093/conphys/coae063
coae063
Review
AcademicSubjects/SCI00840
An evolving roadmap: using mitochondrial physiology to help guide conservation efforts
Thoral Elisa Department of Biology, Section for Evolutionary Ecology, Lund University, Sölvegatan 37, Lund 223 62, Sweden

Dawson Neal J School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Garscube Campus, Bearsden Road, Glasgow, G61 1QH , UK

Bettinazzi Stefano Research Department of Genetics, Evolution and Environment, University College London, Darwin Building, 99-105 Gower Street, WC1E 6BT, London, UK

https://orcid.org/0000-0002-4605-2688
Rodríguez Enrique Research Department of Genetics, Evolution and Environment, University College London, Darwin Building, 99-105 Gower Street, WC1E 6BT, London, UK

Seebacher Frank Editor
Corresponding authors: Elisa Thoral, Department of Biology, Section for Evolutionary Ecology, Lund University, Sölvegatan 37, Lund 223 62, Sweden. Email: elisa.thoral@biol.lu.se; Neal J. Dawson, School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Garscube Campus, Bearsden Road, Glasgow, G61 1QH, UK. Email: neal.dawson@gmail.com; Stefano Bettinazzi, Research Department of Genetics, Evolution and Environment, University College London, Darwin Building, 99-105 Gower Street, WC1E 6BT, London, UK. Email: s.bettinazzi@ucl.ac.uk; Enrique Rodríguez, Research Department of Genetics, Evolution and Environment, University College London, Darwin Building, 99-105 Gower Street, WC1E 6BT, London, UK. Email: enrique.rodriguez@ucl.ac.uk
First co-authors.Last co-authors.

Last co-authors.

2024
07 9 2024
07 9 2024
12 1 coae06321 12 2023
19 8 2024
20 8 2024
© The Author(s) 2024. Published by Oxford University Press and the Society for Experimental Biology.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Mitochondrial processes play crucial roles in species’ responses to environmental variation. Here, we synthesize the methods and approaches involved in mitochondrial bioenergetics as tools to guide the field of conservation physiology.

Abstract

The crucial role of aerobic energy production in sustaining eukaryotic life positions mitochondrial processes as key determinants of an animal's ability to withstand unpredictable environments. The advent of new techniques facilitating the measurement of mitochondrial function offers an increasingly promising tool for conservation approaches. Herein, we synthesize the current knowledge on the links between mitochondrial bioenergetics, ecophysiology and local adaptation, expanding them to the wider conservation physiology field. We discuss recent findings linking cellular bioenergetics to whole-animal fitness, in the current context of climate change. We summarize topics, questions, methods, pitfalls and caveats to help provide a comprehensive roadmap for studying mitochondria from a conservation perspective. Our overall aim is to help guide conservation in natural populations, outlining the methods and techniques that could be most useful to assess mitochondrial function in the field.

Graphical Abstract

Graphical Abstract

Bioenergetics
climate change
conservation
ecophysiology
mitochondria
Biotechnology and Biological Sciences Research Council 10.13039/501100000268 BB/S003681/1 Advanced Grant from the European Research Council 834653 European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie #310331–01/Wellcome Trust
==== Body
pmcIntroduction

The study of metabolism integrates fundamental physicochemical principles and biology, to connect organismal physiology to the ecology of populations, environments and ecosystems. Metabolic rate, the rate at which organisms use energy, is considered a fundamental organismal trait: considered the ‘cost of living’ that unifies all levels of biological organization (Brown et al., 2024). For decades, investigations of the causes and consequences of variation in metabolic rate, both among and within species using whole-animal oxygen consumption rates have dominated the field of conservation biology and ecology (Biro and Stamps, 2010; Burton et al., 2011; Glazier, 2015; Pettersen et al., 2018). However, whole-organismal measures of metabolic rates have limitations for a number of reasons, including: (i) an unknown proportion of the consumed oxygen is actually associated with energy production in the form of ATP; (ii) the inability to determine where variation in oxygen consumption takes place and (iii) typically involve measuring maximum aerobic capacity, where oxygen supply may be a limiting factor. As a result, measures of total oxygen consumption frequently fail to find clear links to behaviours like wild fish activity (Baktoft et al., 2016), exploratory behaviour in wild mammals (Timonin et al., 2011), risk-taking in wild birds (Mathot and Dingemanse, 2015), temperature tolerance in ectotherms (Jutfelt et al., 2018), reproductive output in mice (Duarte et al., 2010) or rates of ageing in wild birds (Bouwhuis et al., 2011). Natural selection might act on energetic efficiency, rather than oxygen consumption; where individuals expending minimal energy to sustain these traits are likely to be favoured over individuals with high rates of energy or oxygen consumption. To address this issue, it is crucial to investigate the functioning of mitochondria within animals in their natural habitats.

For almost a century, the study of mitochondrial energy production has been an essential tool in physiology, as evidenced by more than a quarter million publications since 1925 including the keywords ‘mitochondria’ and ‘physiology’ (Web of Science). With the advent of new techniques allowing a more in-depth study of mitochondrial phenotype (Gnaiger and MitoEAGLE Task Group, 2020), biologists from different fields are increasingly interested in mitochondrial bioenergetics. Indeed, the measurement of mitochondrial function has extended across a wide range of disciplines, including ecophysiology, evolutionary ecology and conservation biology, as ways to gain insights into the mechanisms underpinning variation in life-history phenotypes (Koch et al., 2021). Conservation physiology uses physiological theory and tools to study how environmental perturbations link to ecological performance of vulnerable species and populations (Seebacher and Franklin, 2012). Among the physiological parameters of interest, mitochondrial metabolism appears to, at least partly, dictate the capacity of animals to face changes in environmental conditions (Iftikar et al., 2014; Jørgensen et al., 2021). Therefore, predicting the extent of mitochondrial and energy metabolism plasticity in response to the environment should contribute to the successful identification of vulnerable species and dictate the necessary interventions for conservation.

While it is out of the scope of this review to provide a detailed explanation of mitochondrial function, researchers investigating the role of mitochondrial bioenergetics in a conservation context can refer to past reviews on the precise mechanisms behind this organelle’s function (Wallace and Fan, 2010; Gnaiger and MitoEAGLE Task Group, 2020). However, as interest in harnessing mitochondrial physiology as a means to guide conservation efforts continues to develop, careful consideration should be given to adapting protocols designed in model organisms (Rodríguez et al., 2023) for use in the field (Nord et al., 2021; Parry et al., 2021). It is also critical to understand how and when to use the many different parameters underpinning mitochondrial phenotypes (e.g. oxygen flux, ATP production, reactive oxygen species, membrane potential, cristae morphology) to properly determine how cellular energy production links organismal performance to the ecology of populations, environments and ecosystems (Metcalfe et al., 2023). Consequently, it is essential to match measured mitochondrial parameters to the specific conservation outcomes and question(s) asked, as well as which species and tools are most appropriate for a given ecological niche.

Integrating mitochondrial and cellular bioenergetics to whole-animal fitness in a changing environment

A wide variety of approaches are used to study mitochondrial function, as can be appreciated by the different biological preparations involved, from isolated mitochondria (Rhodes et al., 2024), to intact cells (Nord et al., 2023), permeabilized tissue (Dawson et al., 2016) or shredded tissue samples (Thoral et al., 2021). The parameters measured can also vary, ranging from structure and morphology (Bock et al., 2019; Rodríguez et al., 2019; Christen et al., 2020), to oxygen consumption (Teulier et al., 2019), ATP production (Barbe et al., 2023b), membrane potential (Harford et al., 2023) as well as by-products of metabolism, such as reactive oxygen species (ROSs) production (Christen et al., 2018). In addition to energy production, mitochondrial function is essential in the maintenance of homeostasis, with fusion/fission dynamic, calcium regulation, heat production in endotherms (Nord et al., 2021), as well as regulation of several physiological mechanisms and signalling pathways (Mottis et al., 2019). This review combines new insights on mitochondrial function and addresses important and timely questions that remain unresolved. How do environmental stressors impact mitochondrial physiology? Which animal species, and which mitochondrial parameters, must be studied depending on the scientific questions asked? What is a good design to mimic environmental change and to test its effect on mitochondrial function? Does mitochondrial function correlate with whole-animal parameters, such as metabolic rate? Can we find a way to collect mitochondrial data properly in the field? And finally, how do we harmonise sample collection of threatened animals with conservation efforts? In other words, can we take advantage of mitochondrial physiology to understand/test the adaptive capacity of organisms, and can we develop a sustainable way to do so without threatening the organism we aim to preserve? Here, we address some of these questions and provide a toolbox for understanding how best to study mitochondrial function in organisms facing a changing world.

Climate change is a major threat currently faced by living organisms on our planet, disrupting the balance between energy availability and demand. Together with a generalised global warming and increased extreme temperature fluctuations, climatic models also predict variation in global precipitation patterns, as well as ocean acidification, deoxygenation, and salinification (Rogelj et al., 2012; Masson-Delmotte, 2018; Bates and Johnson, 2020). All these events pose great challenges to organism physiology and performance, forcing changes in animal distribution, variation in trophic networks, but also population collapse and extinction (Pörtner and Farrell, 2008; Somero, 2010). The fundamental role played by aerobic metabolism in sustaining eukaryotic life, and the strong dependence of this process on both temperature and oxygen availability, make energy production and specifically mitochondrial physiology, a likely crucial determinant of animals’ ability to thrive in a changing environment. A better understanding of the extent by which the inability to sustain adequate mitochondrial energy production underpins failure of higher-level processes (e.g. cardiovascular failure) is crucial to both ecology and conservation studies.

Mitochondrial function is known to be affected by environmental changes, whether acute or long-term changes (Sokolova, 2018). Acute and extreme environmental variations, such as thermal variation (Thoral et al., 2021; Thoral et al., 2022b) or a fall in oxygen availability (Scott et al., 2018; Dawson and Scott, 2022; Cerra et al., 2023), can lead to mitochondrial changes linked to stress responses in order to cope with the shifting environment. On the contrary, environmental changes extending over long periods can lead to acclimation and adaptation of mitochondrial metabolism (Pichaud et al., 2017; Camus et al., 2017b; Bettinazzi et al., 2024). Therefore, changes in mitochondrial function differ depending on the duration and intensity of the environmental variation as well as on the thermal strategy of their host (endotherms versus ectotherms). This suggests that, in some species, long-term changes, such as global warming, could sometimes be adequately managed at the mitochondrial (and individual) level (Steffen et al., 2023). Indeed, as climate change also involves increased environmental fluctuations and extreme events such as heat waves, these rapid and unpredictable changes could be more deleterious for some animals (Masson-Delmotte, 2018). Thus, rapid and acute changes in temperature, oxygen availability, salinity, food availability and other parameters, as well as a combination of several variables at the same time can impact the physiology of the animals, particularly by affecting their mitochondrial function (Jørgensen et al., 2021; Menail et al., 2022; Menail et al., 2023; Steffen et al., 2023). Alongside other physiological parameters, mitochondrial function could therefore be one of the first to be modified in response to extreme variations in environmental parameters. These mitochondrial changes could then help determine if individuals are able to acclimatise to these new environmental conditions, which may or may not change in a predictable way, or lead to significant physiological stress, including respiratory dysregulation, oxidative stress and cellular senescence (Stier et al., 2021).

Among the abiotic factors, temperature plays a central role in determining worldwide species distribution (Somero, 2005; Schulte, 2015). The thermal limits of mitochondrial performance might dictate whole-animal thermal tolerance and species distribution; for instance, heat sensitivity appears especially linked to OXPHOS failure, notably in the heart (Iftikar and Hickey, 2013; Christen et al., 2020). Moreover, evidence of loss of mitochondrial performance and ATP production at temperatures close to the upper thermal limit is persistent in literature, especially for ectothermic species, whose metabolism is strictly linked with the external environment (Chung and Schulte, 2020). Evidence that mitochondrial function might determine whole-organism thermal susceptibility and biogeographical distribution ranges from intertidal copepods (Healy and Burton, 2023), insects (Jørgensen et al., 2021; Lubawy et al., 2022; Menail et al., 2022), fish (Iftikar and Hickey, 2013; Iftikar et al., 2014; Christen et al., 2018), bivalves (Hraoui et al., 2020; Hraoui et al., 2021) and crabs (Iftikar et al., 2010).

However, climate change is not solely constrained to the rise of temperatures and their fluctuation. Other phenomena such as the acidification and increased salinity of water (Melzner et al., 2013; Cunillera-Montcusí et al., 2022), or the impact of changes in precipitation frequency and intensity (McCluney et al., 2012) could also affect mitochondrial function; it is thus essential to consider the multiple factors that underlie the impact of climate change on mitochondrial phenotype. Additionally, environmental extremes could exacerbate the main phenotypic impact of intergenomic incompatibility in naturally hybridising populations (Rank et al., 2020; Bettinazzi et al., 2024). It is therefore crucial to quantify interpopulation divergence at the level of interacting mitochondrial and nuclear genes (and therefore account for potential intergenomic incompatibilities) when testing local adaptation and population dynamics in a context of mutating environment (Ellison and Burton, 2008; Healy and Burton, 2020).

To study the effects of environmental variations on mitochondrial function, it is, first of all, essential to choose the most appropriate mitochondrial parameters to match the study species and questions, keeping in mind that measuring several parameters is always better to optimally assess mitochondrial function. For example, in an environment where oxygen and substrates are not limiting, the maximum oxidative capacity of mitochondria, their abundance and volume could be examined (Dawson et al., 2022). The first can be measured through electron transfer system (ETS) complexes enzymatic activities (see table 1), while the second and third can be approximated through mitochondrial DNA copy number (Lubawy et al., 2022) or the activity of enzymes such as citrate synthase (CS) (Larsen et al., 2012; Milbergue et al., 2022). However, if these parameters are limiting, then assessing mitochondrial efficiency to produce energy (Sappal et al., 2015; Thoral et al., 2021) may be more appropriate. Because temperature variation can affect several mitochondrial parameters, each with their own specific changing trajectories (Chung and Schulte, 2020; Dawson and Scott, 2022), it appears that multiple mitochondrial traits in parallel would ideally be considered to understand environmental effects on individuals (Metcalfe et al., 2023). Combining measurements carried out at other biological scales to measure the relationship between mitochondrial metabolism and other functions such as immunity, thermal tolerance (Nord et al., 2021), swimming performance (Thoral et al., 2022a; Thoral et al., 2024a), metabolic rate (Thoral et al., 2024b), growth (Salin et al., 2019; Dawson et al., 2022), physical performance or activity (Bettinazzi et al., 2024) and longevity (Camus et al., 2023) of individuals and/or populations will provide a more complete picture of the relationship between environmental perturbations and organismal performance.

Table 1 Measures of mitochondrial function applied to a conservation framework and a (non-exhaustive) set of representative studies.

Mitochondrial parameter	Definition	Relevance	Techniques	Examples of studies	
Mitochondrial oxidative phosphorylation (OXPHOS)	
Pathway-specific OXPHOS	Respiration in a specific electron transfer pathway (complex I, II or alternative electron pathways), in the OXPHOS (phosphorylating) state	Assess loss of efficiency, breakdown or preferential utilisation of specific pathway(s)	Respirometry (eg. Oroboros O2k, Clark-type electrodes, Seahorse XF), Probes (MitoXpress)	Stier et al., 2019, Gnaiger and MitoEAGLE Task Group, 2020 (for theoretical underpinnings), Wagner et al., 2019, Cossin-Sevrin et al., 2022, Rhodes et al., 2024	
OXPHOS coupling efficiency	Coupling of electron transfer to phosphorylation of ADP to ATP. Calculated as: jP-L = (P − L)/P = 1 − L/P where L is LEAK, P is OXPHOS respiration	An index of mitochondrial efficiency to produce energy	Respirometry (eg. Oroboros O2k, Clark-type electrodes, Seahorse XF)	Gnaiger and MitoEAGLE Task Group, 2020, Dawson et al., 2022, Thoral et al., 2024a, McDiarmid et al., 2024	
ADP/O	ADP consumed per oxygen consumed	An index of oxidative phosphorylation efficiency	Respirometry (eg. Oroboros O2k, Clark-type electrodes, Seahorse XF)	Kraffe et al., 2007, Sussarellu et al., 2013	
ATP/O	ATP produced per oxygen consumed	Efficiency of ATP production per rate of respiration	High-resolution fluorespirometry (Oroboros O2k); kit-based assays (for eg. ATP hydrolysis and synthesis)	Salin et al., 2016b, Thoral et al., 2021, Roussel et al., 2018	
Flux control ratios (FCRs)	Ratio of oxygen flux in a given respiratory state, normalised for a common reference state	Shows coupling and substrate control independently of mitochondrial content	Respirometry (eg. Oroboros O2k, Clark-type electrodes, Seahorse XF)	Bettinazzi et al., 2019, Gnaiger and MitoEAGLE Task Group, 2020	
Substrates contribution	Relative contribution of different energy metabolism pathways to mitochondrial respiration	Assess changes in the preferential utilisation of specific electron pathway(s)	Respirometry (eg. Oroboros O2k, Clark-type electrodes, Seahorse XF)	Jørgensen et al., 2021, Rodríguez et al., 2021	
Inner membrane characteristics	
Membrane potential	Maintenance of mitochondrial membrane potential	Assessing the maintenance of the electron-proton circuit.	High-resolution fluorespirometry (e.g. Oroboros O2k); fluorescence microscopy and flow cytometry with cationic dyes (JC-1, Rhodamine 123, TMRM)	Lebenzon et al., 2022, Harford et al., 2023, Pouliot-Drouin et al., 2024	
Mitochondrial membrane composition	Lipid composition of the mitochondrial membrane (phospholipid classes, fatty acid abundance)	Assess the susceptibility of the mitochondrial membrane to oxidative damage, or temperature related changes (homeoviscous adaptations)	Lipidomics, GC-FID	Hazel, 1995, Munro and Blier, 2012	
(Continued)

Table 1 Continued

Mitochondrial parameter	Definition	Relevance	Techniques	Examples of studies	
Mitochondrial oxidative balance	
Mitochondrial reactive oxygen species (ROS) flux	ROS efflux (balance of production and consumption pathways) in a particular respiratory state	Dysregulation of these signalling molecules can lead to harmful defects	High-resolution fluorespirometry (eg. Oroboros O2k or other respirometers); spectrophotometric assays; flow cytometry; fluorescence microscopy	Loughland and Seebacher, 2020, Rodríguez et al., 2021, Kienzle et al., 2023, do Amaral et al., 2021	
Activity of enzymes linked with antioxidant capacity	Maximum activity of enzymes in isolation (e.g. SOD; CAT; GPx)	Shows maximal capacity of each enzyme	Spectrophotometric assays	Orr and Sohal, 1992, Doucet-Beaupré et al., 2010, Pichaud et al., 2010, Christen et al., 2020, Loughland and Seebacher, 2020	
Oxidative damage	Biological markers of oxidative damage (directly related or not to mitochondrial metabolism) to the membrane lipids, and the DNA	Assess the effects of oxidative damage	Kit-based assays: TBARS (for malondialdehyde species), 8-oxo-dG (for DNA)	Pelletier et al., 2023a	
Activity of energy metabolism enzymes	
OXPHOS complexes activity	Maximum enzymatic activity of specific ETS complexes in isolation (e.g. CI; CII; CI + CIII; CIV; ATPase))	Shows maximal capacity of each complex in isolation, outside of the ‘intact’ mitochondrial environment (potential pitfalls associated)	Spectrophotometric assays	Spinazzi et al., 2012, Hunter-Manseau et al., 2019, Rodríguez et al., 2019, Bettinazzi et al., 2021	
Activity of enzymes linked with energy metabolism upstream of the ETS (linked with glycolysis, fermentation, fatty acid metabolism, intermediate metabolism, Krebs cycle)	Maximum activity of enzymes in isolation (e.g. PK; PFK; LDH; CPT; 3-hydroxyacyl-CoA dehydrogenase; PDH; PK; CS; MDH etc)	Shows the maximal capacity of each enzyme	Spectrophotometric assays	Pelletier et al., 1994, Thibault et al., 1997, Hunter-Manseau et al., 2019, Bettinazzi et al., 2021	
Citrate synthase (CS) activity	Activity of a key enzyme of Krebs cycle, catalysing the conversion of acetyl-CoA and oxaloacetate to citrate	Proxies for the number of mitochondria, may show up- or down- regulation of mitochondrial content in the sample of interest	Kit or laboratory-made enzymatic assays	Bergmeyer, 2012, Larsen et al., 2012	
(Continued)

Table 1 Continued

Mitochondrial parameter	Definition	Relevance	Techniques	Examples of studies	
Mitochondrial protein content	
Uncoupling proteins (UCPs)	Transporter proteins of the inner mitochondrial membrane that dissipate the proton gradient and reduce ATP production	Dissipation of the proton gradient as heat, as a protective mechanism against high ROS levels. Related to energy expenditure and efficiency of exercise and/or metabolism and thermal adaptation	mRNA expression; Western blotting	Hilse et al., 2016, Bryant et al., 2018	
Mitochondrial protein content	Amount of mitochondrial proteins in purified mitochondrial isolates	Proxies for the number of mitochondria, may show up- or downregulation of mitochondrial content in the sample of interest	Kit or laboratory-made enzymatic assays	Pichaud et al., 2010	
Structural or quantitative aspects of mitochondria	
Supercomplex assemblies	Supramolecular organization of the electron transfer system	Structural assessment of the ETS organization, potential role in ROS, electron flux	Blue-native PAGE, proteomics	Bundgaard et al., 2020	
Cristae morphology	Mitochondrial cristae surface area and shape	Assess structural abnormalities and functional decline	Cryo-ET and EM	Perkins et al., 2012, Brandt et al., 2017	
Mitochondrial DNA (mtDNA) copy number	Amount of mtDNA (relative to nuclear gene copies) can indicate increased mitochondrial abundance (or balance between mitogenesis and mitophagy)	Proxies for the number of mitochondria, may show up- or downregulation of mitochondrial content in the sample of interest	DNA extraction and qPCR	Ballard et al., 2007, Meyer et al., 2013, Noguera and Velando, 2019, Cossin-Sevrin et al., 2022, McDiarmid et al., 2024	
See the main text for more examples of relevant articles.

Mitochondrial function as a guide to population-level changes and conservation

Conservation science is continuously looking for ways to determine if a particular species or population is at risk due to changes in various biotic and abiotic factors. Assessing mitochondrial function could be a good approach to predict the effects of changes in habitat quality on individual health and potentially extend these predictions up to population and species fitness. Indeed, individuals with high quality mitochondria that can maintain efficient cellular energy production should be selected for (Fangue et al., 2009; Iftikar et al., 2014; Salin et al., 2016b; Dawson et al., 2022). However, how we measure mitochondrial quality remains context and species dependent. For example, having a greater number of mitochondria or a greater overall capacity to produce ATP would be beneficial to process large amounts of food when resources are abundant; however, when resources are low, sustaining a high level of food consumption may prove impossible (Salin et al., 2016a; Salin et al., 2019; Závorka et al., 2021). The trend towards augmented mitochondrial efficiency rather than abundance appears to hold true for endotherms experiencing higher environmental temperatures (Fangue et al., 2009; Dawson et al., 2022), which increases energy requirements to sustain basic metabolic needs (Boyles et al., 2011). Yet, endothermic animals inhabiting cold and challenging environments often increase capacity and reduce efficiency, possibly to produce heat (Nord et al., 2021). These counterintuitive changes in mitochondrial phenotype in endothermic animals facing harsh environmental conditions are often accompanied by unique physiological specialisations in oxygen uptake, transport and usage (Scott et al., 2010; Mahalingam et al., 2017). Therefore, it is important to understand and identify what constitutes a ‘well suited’ mitochondrial phenotype, depending mainly on the environmental conditions experienced, but also on the species and tissue studied, and that could be defined by several parameters.

Increasing evidence points to mitochondrial function as a tool to guide conservation efforts, with studies in threatened species highlighting important roles for these organelles. For example, thermal stress in imperilled fish affects gene expression and alternative splicing related to mitochondrial processes (Thorstensen et al., 2022). Unfavourable breeding conditions affect oxidative stress management and overall physiological condition, triggering compensatory behavioural adjustments in species of northern gannets (Pelletier et al., 2023a; Pelletier et al., 2023b). In contrast, other species such as king penguins can adjust mitochondrial function to cope with stress (Stier et al., 2019). Mitochondrial ‘health’ can thus provide useful information on the feasibility of various conservation interventions targeting species and populations. Among these interventions, translocating populations can be a useful tool in conservation (George et al., 2009), as well as genetic rescue through adaptive introgression (Hamilton and Miller, 2016). Introgressing foreign mitochondria with a lower mutational load or better adapted to a specific environment can potentially improve population performance and lead to adaptation (Hill, 2019). Nonetheless, evidence suggests that translocation can have catastrophic consequences on populations, as introducing incompatible mitochondrial variants might affect fitness (Ellison and Burton, 2006; Smith et al., 2010; Innocenti et al., 2011; Bettinazzi et al., 2024) and potentially lead to extinction due to genetic incompatibility (Gemmell and Allendorf, 2001; Hughes et al., 2003). Proper mitochondrial function necessarily relies on intergenomic coadaptation (Burton, 2022). For instance, interactions between mitochondrial and nuclear genes affect metabolic rate and organismal performance in response to temperature in both seed and leaf beetle species (Arnqvist et al., 2010; Rank et al., 2020), affecting their potential distribution and thermal adaptation. Successful conservation plans involving mitochondrial introgression should account for mitonuclear compatibility, and for that, a priori genetic screening and mitochondrial profiling of both parental and hybrid populations could be useful to test the potential of ‘mitonuclear outbreeding depression’ when planning genetic rescue of small, inbred populations. In line with this idea, the concepts of mitonuclear ecology and conservation mitonuclear replacement (CmNR) have recently emerged (Hill, 2015; Hill et al., 2019; Iverson, 2024).

As illustrated by the famous Krogh’s principle (‘for a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied’) (Krogh, 1929), a natural system that is ideal to answer the question(s) of interest should be adopted when studying conservation physiology through the lens of mitochondrial physiology. Model species represent a powerful starting tool. For example, a versatile species like Drosophila which are easy to breed and maintain, and for which gold-standard genetic tools are widely employed (such as balancer chromosomes which prevent recombination, and various mutant lines readily available from stock centres) can enable researchers to select and study specific genetic variation of interest more precisely than in non-model species. Furthermore, model species are usually fast reproducing, making them a good tool to explore how environmental changes (including diet, which is easily modifiable) can impact the selection of mitochondrial phenotypes at various generational timescales (Camus et al., 2017a; Camus and Inwongwan, 2023; Bettinazzi et al., 2024). Fundamental principles about mitochondrial function can and have been discovered using laboratory animal models and can then be applied to other species in more natural settings, to help understand which selection pressures may act on populations in the wild (Rauhamäki et al., 2014; Mesquita et al., 2021; McDiarmid et al., 2024).

Popular laboratory models that differ from the more traditional rodents and insect species include goldfish (Thoral et al., 2022a; Thoral et al., 2024a), zebrafish (Cadiz et al., 2019; Thoral et al., 2022b), zebra finches (Salmón et al., 2023) or Japanese quails (Stier et al., 2022). Species that are relatively easy to keep in laboratory conditions allows for unmatched sensitivity and control over the characterisation of the mechanisms underpinning mitochondrial function (like the Drosophila outlined above) which can then be harnessed to explore possible genetic/phenotypic selection pressures in wild systems. Indeed, recent years have seen a marked increase in the number of studies carried out on nontraditional species, such as wild animals captured in their natural environment and sometimes kept in captivity, including: brown trout (Dawson et al., 2022); triplefin fish (Harford et al., 2023); several species of birds (Barbe et al., 2023a; Barbe et al., 2023b); honey bees (Menail et al., 2023); lampreys (Belyaeva et al., 2014) and bivalves (Steffen et al., 2023). The expanding diversity of study organisms and successes in determining their mitochondrial function would suggest that it should be possible to extend these measurements to most desired species to assess their energy metabolism. This would also allow us to discover common issues faced and strategies employed by species inhabiting similar habitats, or organisms facing similar pressures due to environmental change. In addition, the use of these wild species provides a wider genetic background than that obtained with species that have been kept in captivity for dozens or even hundreds of generations, which can affect certain physiological parameters such as their ability to acclimate (Morgan et al., 2019).

Resources and tools to study mitochondrial function in the laboratory and in the field

Researchers in the field of conservation physiology interested in characterising mitochondrial function in their species of interest might first be wondering which parameter(s) to target. To this end, we list some of the most informative parameters about mitochondrial function in Table 1. These are common and popular measurements, but researchers should keep in mind that finding clear distinction between ‘functional’ or ‘dysfunctional’ mitochondria might not be the definite objective. Indeed, there is a recent push to move mitochondrial science beyond function and dysfunction and better adapt the terminology to reflect that mitochondria are multifaceted, multifunctional, species and tissue-specific, dynamic organelles (see Monzel et al., 2023 for a review and perspective).

The recognition of the pivotal role mitochondrial metabolism plays across an array of physiological processes (Garlid, 2001) along with the recent advancements in user-friendly technologies to study respiration (Gnaiger, 2011) and other mitochondrial parameters such as membrane potential (Pendergrass et al., 2004) has resulted in substantial methodological improvements to measure mitochondrial function in the past decades. Multiple methods to assess mitochondrial parameters in different settings are available to researchers depending on their equipment, resources, time, and whether working in a fully equipped laboratory or in the field (see Palmeira and Moreno, 2018, for more details). Measurement of mitochondrial oxygen consumption rates provide a robust and detailed analysis of mitochondrial function, as the activity of specific enzymes, isolated mitochondria or permeabilized cells require different metabolic pathways for oxidation but all terminate in oxygen consumption (Makrecka-Kuka et al., 2015; Vandenberg et al., 2021). The two most commonly used instruments to measure mitochondrial respirometry use either chamber-based platinum electrodes (Seebacher and James, 2008; Rissoli et al., 2017) or microplate-based fluorescence readings (Brand and Nicholls, 2011; Divakaruni et al., 2014). These techniques are accessible for researchers with appropriate training obtainable either via one of the instruments’ manufacturers, or through collaboration with researchers in the ever-growing mitochondrial physiology field. The major advantage of both of these methods is the ability to produce real-time data that is not possible when using more traditional endpoint metabolic assays.

A popular instrument to measure rates of oxygen consumption using platinum electrodes in 0.5 ml or 2 ml chambers is the high-resolution respirometer Oxygraph-2 k (O2k, Oroboros Instruments, Innsbruck, Austria) that is available with an optional fluorescence module (O2k-Fluo). Over the last few years, dozens of different protocols have been set up and adjusted to measure various mitochondrial parameters on these O2k oxygraphs (Blier and Lemieux, 2001; Stier et al., 2017b; Teulier et al., 2019; Bettinazzi et al., 2019b; Dawson et al., 2020b; Thoral et al., 2021; Harford et al., 2023; Nord et al., 2023; Rodríguez et al., 2023; Steffen et al., 2023). This instrument’s advantages are the assessment of several parameters of mitochondrial function simultaneously from a single sample, i.e. oxygen consumption and either ATP production (Magnesium Green; Thoral et al., 2021), ROS production (Amplex UltraRed/Ampliflu Red; Steffen et al., 2023), calcium uptake (Calcium Green; Cheng et al., 2023) or membrane potential (TMRM, Harford et al., 2023). These devices also allow acute monitoring of several parameters, including temperature and oxygen concentration. The different chamber sizes available also allow different biological samples to be studied, from isolated mitochondria (Christen et al., 2018; Barbe et al., 2023b) to whole individuals or cells (Patil et al., 2013), as well as permeabilized, shredded or homogenised tissue samples (Dawson et al., 2020b; Thoral et al., 2022b). An equally popular alternative to the O2k is the Seahorse XF, which offers greater use for high-throughput analysis and is often used in the biomedical field (Divakaruni et al., 2014). This instrument can measure far more samples simultaneously when compared to the O2k using 8- to 96-well plates and offers measures of mitochondrial respiration, glycolysis and ATP production in cells and isolated mitochondria. However, it does not measure the fluorescence-based process outlined above simultaneously and conditions inside the sample well such as oxygen saturation cannot be as readily modified as in the O2k chambers. Thus, these different techniques offer a wide range of possibilities for studying mitochondrial function, depending on the species studied, the biological samples used and the researchers’ budget.

As mentioned previously, numerous methods for preparing mitochondria for analysis exist, ranging from intact cells (Nord et al., 2021) with mitochondrial respiratory uncouplers and inhibitors that can permeate through the plasma membrane, to detergent- or mechanically permeabilized tissues and cells (Dawson et al., 2020b), and even isolated organelles to assess fine kinetic function or different subpopulations of mitochondria (Scott et al., 2018; Rodríguez et al., 2020; Dawson and Scott, 2022). Each method offers advantages and disadvantages that must be carefully evaluated. Indeed, permeabilized tissues can be useful to circumvent the collection of high quantities of tissue and minimise the use of other instruments (such as centrifuges) that are needed for mitochondrial isolation (Kuznetsov et al., 2008). Isolated mitochondria, however, are taken out of their cellular context and are thus free of many of the confounding biochemical pathways and processes that may interfere or compete with mitochondrial function, such as ATPases or NADH-consuming enzymes. Working with tissue samples and blood cells can be done with minimal manipulation, and can in some cases provide a non-terminal method of sampling individuals, ideal for longitudinal studies (Stier et al., 2017a; Stier et al., 2019; Stier et al., 2022; Nord et al., 2023; Thoral et al., 2024a; Thoral et al., 2024b).

Although the typical mitochondrial respirometry-based approaches outlined above rely on fresh samples, access to these is not always possible and the cryopreservation of tissues for later analysis of mitochondrial function can sometimes be a more desirable solution. A key problem is the effect of freeze thawing of mitochondria on their membranes and subsequent functionality of intact mitochondria. Recently developed approaches in wild animals (Bettinazzi et al., 2019a) provide a solution to this. Assessing mitochondrial function, with the measure of mitochondrial respiration for example, using minimal amounts of mitochondria is now possible in frozen mitochondria, tissue and cells (2–30 μg of isolates or homogenates, or 30 000 cells per well; Acin-Perez et al., 2020). This analysis on previously frozen samples relies on carefully optimised conditions including the addition of NADH as a substrate (rather than pyruvate, glutamate and malate) to assess complex I activity, along with a need for pre-incubation with succinate for complex II respiration. It also comes with a major limitation, as the freeze thawing of samples causes an uncoupling from oxidative phosphorylation since ATP synthase can no longer exert control over respiration (Acin-Perez et al., 2020).

Another means of measuring mitochondrial function on previously frozen samples is the measurement of enzymatic activities (Spinazzi et al., 2012) which are commercially available in assay kits and straightforward to replicate. The assessment of enzyme capacities from frozen tissues is particularly useful when working in remote locations or time- and resource-constrained situations (Dawson et al., 2020a; Schell et al., 2023). Enzyme assays have a potential drawback in that they are assessed under in vitro conditions that deviate from physiological settings (pH, osmolarity, substrate concentrations, etc.) and they do not allow the evaluation of respiratory coupling. Nonetheless, they offer vital quantitative data regarding catalytic and flux capacities of the mitochondrial respiratory complexes where more complex respirometry experiments are not possible. The levels of the different ETS complexes can also be compared through western blotting techniques and can reveal key changes in mitochondrial organisation (Jové et al., 2014). Finally, accurate measurement of mitochondrial content remains a topic of debate. Indeed, CS activity or mitochondrial copy number are a commonly used proxies of mitochondrial content across tissues and species (Larsen et al., 2012); however, a recent method using mitochondrial-targeted nLC-MS/MS (nano-liquid chromatography/mass spectrometry) might provide more accurate measurements of this crucial parameter (McLaughlin et al., 2020).

Perspectives, pitfalls and perorates

The diverse range of stressors explored herein (temperature, oxygen availability, salinity, food availability) create an imbalance between energy requirements and energy production, highlighting the potential importance of studying differences in mitochondrial energy production as a potential tool to help guide conservation efforts. However, what is unclear from a conservation standpoint is how different organisms can cope with changes in the environment both temporally and in intensity. The main aim of conservation practices should therefore be to determine how quickly a population or species can react to acute environmental shifts, if they can acclimate in case these changes are permanent, and what the consequences are of possible phenotypic selection acting on these populations.

As such, it is critical to identify what constitutes a ‘high quality’ mitochondrial phenotype for each species, population and environment in order to properly guide conservation efforts. The works presented herein highlight a potential issue with lab-based studies in that mitochondrial function is almost always analysed under highly controlled conditions that can be quite distinct from the physiological conditions in which mitochondria naturally exist.

In the case of studying mitochondrial thermal sensitivity (specifically when trying to link the upper thermal limit of organisms with their mitochondrial thermal performance), one concern lies in the choice of substrates given to the mitochondrial preparation. Indeed, at a temperature close to their thermal limit, Jørgensen and colleagues showed that some Drosophila species exhibit a breakdown in Complex I-linked respiration (Jørgensen et al., 2021). However, maximal coupled respiration was maintained through the oxidation of alternative substrates such as glycerophosphate, even when exposed to temperatures above their thermal limit. Thus, the careful dissection of the different steps of the ETS is paramount, as this potential ‘rescue’ of respiration by alternative complexes at higher temperatures might not be accompanied by sufficiently efficient ATP production (as not all mitochondrial complexes pump protons and hence differ in their contribution to ATP production). Respiration protocols used to guide conservation efforts must therefore be as informative as possible on the relative contributions of each ETS complex, and measure ATP production rates and/or ROS efflux to properly assess mitochondrial quality.

Moreover, at the individual level, laboratory conditions are also generally far from the natural conditions in which individuals live, due to their stability and/or the precise control of different environmental parameters. Future projects should therefore focus on new approaches to get as close as possible to the physiological and environmental conditions experienced naturally by individuals (Drake et al., 2017), while bearing in mind that the conditions for in vitro measurements of mitochondrial function are likely distinct from natural conditions due to the technical limitations of the instruments used and should be confirmed with wild studies (Dawson et al., 2016; Nord et al., 2021). Therefore, it is important to initially consider what the stable conditions representative of the organism under study are, and to compare the biotic or abiotic factors under question to these ‘standard’ conditions (e.g. stickleback and temperature studies in Cominassi et al., 2022 and Dawson et al., 2022).

Recent progress has been made by several groups looking at linking mitochondrial function to traditionally studied parameters. One of these important traits is metabolic rate, a key parameter measured in studies concerned with conservation. It seems that the relationship between mitochondrial function and whole-animal metabolic rate depends on many factors such as the organ, tissue or cellular compartment of interest; the type of preparation used for respirometry; the status of the individual (for e.g. stress) and the parameter (respiration state) or mitochondrial pathway under scrutiny (Malkoc et al., 2021; Cominassi et al., 2022; Casagrande et al., 2023). Even behavioural traits such as territorial ‘performance’, linked to standard metabolic rate (Metcalfe et al., 1995) have been correlated to maximal OXPHOS capacity and mitochondrial density (Larsen et al., 2012). Other fitness traits such as reproductive output can also be linked to mitochondrial function. For example, male reproductive success can be impaired by temperature in Drosophila (Van Heerwaarden and Sgrò, 2021); and studies on temperature and other stressors have shown that reproductive tissue quality (number of eggs, sperm motility, etc.) can vary with mitochondrial function, often measured in the same tissue (Bettinazzi et al., 2019b; Bettinazzi et al., 2020; Rank et al., 2020; Bettinazzi et al., 2023; Camus et al., 2023). Investigating mitochondria should therefore go beyond the traditional dichotomy between ‘function’ and ‘dysfunction’ when being used in a conservation setting: mitochondrial phenotypes, behaviour, features and activities (Monzel et al., 2023) can and should be investigated from the perspective of overall animal performance (see Heine and Hood, 2020 for a review and a hypothesis). Moreover, and closely linked to mitochondrial physiology (due to mitochondria’s central role as a metabolic hub), there is a need for other approaches and markers such as oxidative status (Beaulieu and Costantini, 2014) and metabolomics analysis (Lawson et al., 2022) to be used in conservation strategies, with some successful examples recently published in birds and in mussels (Putnam et al., 2023; Waller et al., 2023; Pelletier et al., 2023a; Pelletier et al., 2023b).

Finally, the example of wild species captured and then brought back to the laboratory shows the current limits that scientists face in studying energy metabolism. The different measurement methods presented above require stable conditions which are mainly found in the laboratory. However, it seems essential to find solutions so that these measurements take place directly in the field, in order to allow the non-terminal measurements of individuals in the wild (Dawson et al., 2016), by taking small samples of tissues such as muscle biopsies (Quéméneur et al., 2022; Thoral et al., 2024a) or blood samples (Stier et al., 2017b; Nord et al., 2021), while also releasing individuals back into their natural environment immediately after collection. Although some scientists have already started experimenting with portable field laboratories such as the ‘MitoMobile’ (Parry et al., 2021), it now seems essential to continue to devise new methods and techniques that can be easily brought into the field to study mitochondrial function with the aim of guiding and improving conservation efforts, without terminally sampling from the very populations we aim to protect.

Acknowledgements

This synthesis stems from a symposium held at the Society of Experimental Biology’s 2023 Centenary Conference in Edinburgh, UK entitled ‘Keeping the pace: integrating mitochondrial and cellular bioenergetics to whole-animal fitness in a changing environment’. We thank the conference organisers, as well as the delegates who attended our session, and the speakers: Pierre Blier, Christian Bock, Agata Burzawa, Florencia Camus, Amélie Crespel, Mariacristine Filice, Alice Harford, Darryl McLennnan, Andreas Nord, Inna Sokolova, Loïc Teulier and Amanda Wiesenthal.

Author contributions

All authors contributed equally to this manuscript, from the organization of the conference symposium to the manuscript’s theoretical conception and writing.

Conflicts of interest

The authors have no conflicts of interest to declare.

Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101030803 to S.B. N.J.D. was also supported by an ISSF ECR Catalyst Grant (#310331–01/Wellcome Trust) and an Advanced Grant from the European Research Council (ERC; no. 834653). E.R. was supported by funding from the Biotechnology and Biological Sciences Research Council (BB/S003681/1).

Data availability

No data were generated or analysed during this work.
==== Refs
REFERENCES

Acin-Perez R , BenadorIY, PetcherskiA, VeliovaM, BenavidesGA, LagarrigueS, CaudalA, VergnesL, MurphyAN, KaramanlidisGet al. (2020) A novel approach to measure mitochondrial respiration in frozen biological samples. EMBO J 39 : e104073. 10.15252/embj.2019104073.32432379
do Amaral MA , ParedesLC, PadovaniBN, Mendonça-GomesJM, MontesLF, NosC, Morales FéneroC (2021) Mitochondrial connections with immune system in zebrafish. Fish Shellfish Immunol Rep 2 : 100019. 10.1016/j.fsirep.2021.100019.36420514
Arnqvist G , DowlingDK, EadyP, GayL, TregenzaT, TudaM, HoskenDJ (2010) Genetic architecture of metabolic rate: environment specific epistasis between mitochondrial and nuclear genes in an insect. Evolution 64 : 3354–3363. 10.1111/j.1558-5646.2010.01135.x.20874734
Baktoft H , JacobsenL, SkovC, KoedA, JepsenN, BergS, BoelM, AarestrupK, SvendsenJC (2016) Phenotypic variation in metabolism and morphology correlating with animal swimming activity in the wild: relevance for the ocltt (oxygen- and capacity-limitation of thermal tolerance), allocation and performance models. Conserv Physiol 4 : cov055. 10.1093/conphys/cov055.27382465
Ballard JWO , MelvinRG, KatewaSD, MaasK (2007) Mitochondrial DNA variation is associated with measurable differences in life-history traits and mitochondrial metabolism in Drosophila simulans. Evolution 61 : 1735–1747. 10.1111/j.1558-5646.2007.00133.x.17598752
Barbe J , RousselD, VoituronY (2023a) Effect of physiological hyperthermia on mitochondrial fuel selection in skeletal muscle of birds and mammals. J Therm Biol 117 : 103719. 10.1016/j.jtherbio.2023.103719.37776632
Barbe J , WatsonJ, RousselD, VoituronY (2023b) The allometry of mitochondrial efficiency is tissue dependent: a comparison between skeletal and cardiac muscles of birds. J Exp Biol 226 : jeb246299. 10.1242/jeb.246299.37921223
Bates NR , JohnsonRJ (2020) Acceleration of ocean warming, salinification, deoxygenation and acidification in the surface subtropical North Atlantic Ocean. Commun Earth Environ 1 : 33. 10.1038/s43247-020-00030-5.
Beaulieu M , CostantiniD (2014) Biomarkers of oxidative status: missing tools in conservation physiology. Conserv Physiol 2 : cou014. 10.1093/conphys/cou014.27293635
Belyaeva EA , EmelyanovaLV, KorotkovSM, BrailovskayaIV, SavinaMV (2014) On the mechanism(s) of membrane permeability transition in liver mitochondria of lamprey, Lampetra fluviatilis L.: insights from cadmium. Biomed Res Int 2014 : 691724.24995321
Bergmeyer HUI (2012) Methods of Enzymatic Analysis. Elsevier Science, Amsterdam, The Netherlands.
Bettinazzi S , GendronAD, BretonS (2019a) The effect of cryopreservation on mitochondrial function in freshwater mussel tissue samples (Bivalvia: Unionida). Cryobiology 88 : 106–109. 10.1016/j.cryobiol.2019.04.006.31028719
Bettinazzi S , LiangJ, RodriguezE, BonneauM, HoltR, WhiteheadB, DowlingD, LaneN, CamusMF (2024) Assessing the role of mitonuclear interactions on mitochondrial function and organismal fitness in natural drosophila populations. Evol Lett qrae043. 10.1093/evlett/qrae043.
Bettinazzi S , LiangJ, RodriguezE, BonneauM, HoltR, WhiteheadB, DowlingDK, LaneN, CamusM (2023) Assessing the role of mitonuclear interactions on mitochondrial function and organismal fitness in natural drosophila populations. bioRxiv 2023.2009.2025.559268.
Bettinazzi S , MilaniL, BlierPU, BretonS (2021) Bioenergetic consequences of sex-specific mitochondrial DNA evolution. Proc R Soc B Biol Sci 288 : 20211585. 10.1098/rspb.2021.1585.
Bettinazzi S , NadarajahS, DalpéA, MilaniL, BlierPU, BretonS (2020) Linking paternally inherited mtdna variants and sperm performance. Philos Trans R Soc B Biol Sci 375 : 20190177. 10.1098/rstb.2019.0177.
Bettinazzi S , RodríguezE, MilaniL, BlierPU, BretonS (2019b) Metabolic remodelling associated with mtdna: insights into the adaptive value of doubly uniparental inheritance of mitochondria. Proc R Society B Biol Sci 286 : 20182708. 10.1098/rspb.2018.2708.
Biro PA , StampsJA (2010) Do consistent individual differences in metabolic rate promote consistent individual differences in behavior? Trends Ecol Evol 25 : 653–659. 10.1016/j.tree.2010.08.003.20832898
Blier PU , LemieuxH (2001) The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of arctic charr red muscle mitochondria. J Comp Physiol B Biochem Syst Environ Physiol 171 : 247–253. 10.1007/s003600000169.
Bock C , WermterFC, SchalkhausserB, BlicherME, PörtnerH-O, LannigG, SejrMK (2019) In vivo 31p-mrs of muscle bioenergetics in marine invertebrates: Future Ocean limits scallops' performance. Magn Reson Imaging 61 : 239–246. 10.1016/j.mri.2019.06.003.31173850
Bouwhuis S , SheldonBC, VerhulstS (2011) Basal metabolic rate and the rate of senescence in the great tit. Funct Ecol 25 : 829–838. 10.1111/j.1365-2435.2011.01850.x.
Boyles JG , SeebacherF, SmitB, McKechnieAE (2011) Adaptive thermoregulation in endotherms may alter responses to climate change. Integr Comp Biol 51 : 676–690. 10.1093/icb/icr053.21690108
Brand M , NichollsD (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435 : 297–312. 10.1042/BJ20110162.21726199
Brandt T , MourierA, TainLS, PartridgeL, LarssonN-G, KühlbrandtW (2017) Changes of mitochondrial ultrastructure and function during ageing in mice and drosophila. Elife 6 : e24662. 10.7554/eLife.24662.28699890
Bryant HJ , ChungDJ, SchultePM (2018) Subspecies differences in thermal acclimation of mitochondrial function and the role of uncoupling proteins in killifish. J Exp Biol 221 : jeb186320. 10.1242/jeb.186320.30352820
Brown JH , Gillooly, JF, AllenAP, SavageVM, West, GB (2004). Toward a metabolic theory of ecology. Ecology 85 (7 ), 1771–1789. 10.1890/03-9000.
Bundgaard A , JamesAM, HarbourME, MurphyMP, FagoA (2020) Stable mitochondrial ciciii2 supercomplex interactions in reptiles versus homeothermic vertebrates. J Exp Biol 223 : jeb223776.32393546
Burton RS (2022) The role of mitonuclear incompatibilities in allopatric speciation. Cell Mol Life Sci 79 : 103. 10.1007/s00018-021-04059-3.35091831
Burton T , KillenSS, ArmstrongJD, MetcalfeNB (2011) What causes intraspecific variation in resting metabolic rate and what are its ecological consequences? Proc R Soc B Biol Sci 278 : 3465–3473. 10.1098/rspb.2011.1778.
Cadiz L , BundgaardA, MalteH, FagoA (2019) Hypoxia enhances blood o2 affinity and depresses skeletal muscle o2 consumption in zebrafish (Danio rerio). Comp Biochem Physiol B Biochem Mol Biol 234 : 18–25. 10.1016/j.cbpb.2019.05.003.31075501
Camus MF , FowlerK, PiperMWD, ReuterM (2017a) Sex and genotype effects on nutrient-dependent fitness landscapes in drosophila melanogaster. Proc R Soc B Biol Sci 284 : 20172237. 10.1098/rspb.2017.2237.
Camus MF , InwongwanS (2023) Mitonuclear interactions modulate nutritional preference. Biol Lett 19 : 20230375. 10.1098/rsbl.2023.0375.38053364
Camus MF , RodriguezE, KotiadisV, CarterH, LaneN (2023) Redox stress shortens lifespan through suppression of respiratory complex i in flies with mitonuclear incompatibilities. Exp Gerontol 175 : 112158. 10.1016/j.exger.2023.112158.36965604
Camus MF , WolffJN, SgròCM, DowlingDK (2017b) Experimental support that natural selection has shaped the latitudinal distribution of mitochondrial haplotypes in Australian drosophila melanogaster. Mol Biol Evol 34 : 2600–2612. 10.1093/molbev/msx184.28637217
Casagrande S , DzialoM, TrostL, MalkocK, SadowskaET, HauM, PierceB, McWilliamsS, BauchingerU (2023) Mitochondrial metabolism in blood more reliably predicts whole-animal energy needs compared to other tissues. iScience 26 : 108321. 10.1016/j.isci.2023.108321.38025793
Cerra MC , FiliceM, CaferroA, MazzaR, GattusoA, ImbrognoS (2023) Cardiac hypoxia tolerance in fish: from functional responses to cell signals. Int J Mol Sci 24 : 1460. 10.3390/ijms24021460.36674975
Cheng H , PerkinsGA, JuS, KimK, EllismanMH, PamenterME (2023) Enhanced mitochondrial buffering prevents ca2+ overload in naked mole-rat brain. J Physiol . 10.1113/JP285002.
Christen F , DesrosiersV, Dupont-CyrBA, VandenbergGW, Le FrançoisNR, TardifJ-C, DufresneF, LamarreSG, BlierPU (2018) Thermal tolerance and thermal sensitivity of heart mitochondria: mitochondrial integrity and ros production. Free Radic Biol Med 116 : 11–18. 10.1016/j.freeradbiomed.2017.12.037.29294390
Christen F , DufresneF, LeducG, Dupont-CyrBA, VandenbergGW, Le FrançoisNR, TardifJ-C, LamarreSG, BlierPU (2020) Thermal tolerance and fish heart integrity: fatty acids profiles as predictors of species resilience. Conserv Physiol 8 : coaa108. 10.1093/conphys/coaa108.33408863
Chung DJ , SchultePM (2020) Mitochondria and the thermal limits of ectotherms. J Exp Biol 223 : jeb227801. 10.1242/jeb.227801.33109621
Cominassi L , ResselKN, BrookingAA, MarbacherP, Ransdell-GreenEC, O'BrienKM (2022) Metabolic rate increases with acclimation temperature and is associated with mitochondrial function in some tissues of threespine stickleback. J Exp Biol 225 : jeb244659. 10.1242/jeb.244659.36268761
Cossin-Sevrin N , HsuB-Y, MarciauC, ViblancVA, RuuskanenS, StierA (2022) Effect of prenatal glucocorticoids and thyroid hormones on developmental plasticity of mitochondrial aerobic metabolism, growth and survival: an experimental test in wild great tits. J Exp Biol 225 : jeb243414. 10.1242/jeb.243414.35420125
Cunillera-Montcusí D , BeklioğluM, Cañedo-ArgüellesM, JeppesenE, PtacnikR, AmorimCA, ArnottSE, BergerSA, BrucetS, DuganHAet al. (2022) Freshwater salinisation: a research agenda for a saltier world. Trends Ecol Evol 37 : 440–453. 10.1016/j.tree.2021.12.005.35058082
Dawson NJ , AlzaL, NandalG, ScottGR, McCrackenKG (2020a) Convergent changes in muscle metabolism depend on duration of high-altitude ancestry across andean waterfowl. Elife 9 : e56259. 10.7554/eLife.56259.32729830
Dawson NJ , IvyCM, AlzaL, CheekR, YorkJM, ChuaB, MilsomWK, McCrackenKG, ScottGR (2016) Mitochondrial physiology in the skeletal and cardiac muscles is altered in torrent ducks, Merganetta armata, from high altitudes in the Andes. J Exp Biol 219 : 3719–3728. 10.1242/jeb.142711.27618861
Dawson NJ , MilletC, SelmanC, MetcalfeNB (2020b) Measurement of mitochondrial respiration in permeabilized fish gills. J Exp Biol 223 : jeb216762. 10.1242/jeb.216762.31974221
Dawson NJ , MilletC, SelmanC, MetcalfeNB (2022) Inter-individual variation in mitochondrial phosphorylation efficiency predicts growth rates in ectotherms at high temperatures. FASEB J 36 : e22333. 10.1096/fj.202101806RR.35486025
Dawson NJ , ScottGR (2022) Adaptive increases in respiratory capacity and o2 affinity of subsarcolemmal mitochondria from skeletal muscle of high-altitude deer mice. FASEB J 36 : e22391. 10.1096/fj.202200219R.35661419
Divakaruni AS , RogersGW, MurphyAN (2014) Measuring mitochondrial function in permeabilized cells using the seahorse xf analyzer or a Clark-type oxygen electrode. Curr Protoc Toxicol 60 : 25.2.1–25.2.16. 10.1002/0471140856.tx2502s60.
Doucet-Beaupré H , DubéC, BretonS, PörtnerHO, BlierPU (2010) Thermal sensitivity of metabolic enzymes in subarctic and temperate freshwater mussels (bivalvia: Unionoida). J Therm Biol 35 : 11–20. 10.1016/j.jtherbio.2009.10.002.
Drake MJ , MillerNA, TodghamAE (2017) The role of stochastic thermal environments in modulating the thermal physiology of an intertidal limpet, Lottia digitalis. J Exp Biol 220 : 3072–3083. 10.1242/jeb.159020.28855321
Duarte LC , VaanholtLM, SinclairRE, GamoY, SpeakmanJR (2010) Limits to sustained energy intake xii: is the poor relation between resting metabolic rate and reproductive performance because resting metabolism is not a repeatable trait? J Exp Biol 213 : 278–287. 10.1242/jeb.037069.20038662
Ellison CK , BurtonRS (2006) Disruption of mitochondrial function in interpopulation hybrids of Tigriopus califronicus. Evolution 60 : 1382–1391.16929655
Ellison CK , BurtonRS (2008) Interpopulation hybrid breakdown maps to the mitochondrial genome. Evolution 62 : 631–638. 10.1111/j.1558-5646.2007.00305.x.18081717
Fangue NA , RichardsJG, SchultePM (2009) Do mitochondrial properties explain intraspecific variation in thermal tolerance? J Exp Biol 212 : 514–522. 10.1242/jeb.024034.19181899
Garlid KD (2001) Physiology of mitochondria. In SperelakisN, eds, Cell Physiology Source Book. Academic Press, New York, USA, pp. 139–151
Gemmell NJ , AllendorfFW (2001) Mitochondrial mutations may decrease population viability. Trends Ecol Evol 16 : 115–117. 10.1016/S0169-5347(00)02087-5.11179569
George AL , KuhajdaBR, WilliamsJD, CantrellMA, RakesPL, ShuteJR (2009) Guidelines for propagation and translocation for freshwater fish conservation. Fisheries 34 : 529–545. 10.1577/1548-8446-34.11.529.
Glazier DS (2015) Is metabolic rate a universal ‘pacemaker’ for biological processes? Biol Rev 90 : 377–407. 10.1111/brv.12115.24863680
Gnaiger E (2011) The oxygraph for high-resolution respirometry. Mitochondr Physiol Netw 6 : 1–18.
Gnaiger E , MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020 : 1. 10.26124/bec:2020-0001.v1.
Hamilton JA , MillerJM (2016) Adaptive introgression as a resource for management and genetic conservation in a changing climate. Conserv Biol 30 : 33–41. 10.1111/cobi.12574.26096581
Harford AR , DevauxJBL, HickeyAJR (2023) Dynamic defence? Intertidal triplefin species show better maintenance of mitochondrial membrane potential than subtidal species at low oxygen pressures. J Exp Biol 226 : jeb245926. 10.1242/jeb.245926.37498237
Hazel JR (1995) Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu Rev Physiol 57 : 19–42. 10.1146/annurev.ph.57.030195.000315.7778864
Healy TM , BurtonRS (2020) Strong selective effects of mitochondrial DNA on the nuclear genome. Proc Natl Acad Sci 117 : 6616–6621. 10.1073/pnas.1910141117.32156736
Healy TM , BurtonRS (2023) Loss of mitochondrial performance at high temperatures is correlated with upper thermal tolerance among populations of an intertidal copepod. Comp Biochem Physiol B Biochem Mol Biol 266 : 110836. 10.1016/j.cbpb.2023.110836.36801253
Heine KB , HoodWR (2020) Mitochondrial behaviour, morphology, and animal performance. Biol Rev 95 : 730–737. 10.1111/brv.12584.32022456
Hill GE (2015) Mitonuclear ecology. Mol Biol Evol 32 : 1917–1927. 10.1093/molbev/msv104.25931514
Hill GE (2019) Reconciling the mitonuclear compatibility species concept with rampant mitochondrial introgression. Integr Comp Biol 59 : 912–924.30937430
Hill GE , HavirdJC, SloanDB, BurtonRS, GreeningC, DowlingDK (2019) Assessing the fitness consequences of mitonuclear interactions in natural populations. Biol Rev 94 : 1089–1104. 10.1111/brv.12493.30588726
Hilse KE , KalinovichAV, RupprechtA, SmorodchenkoA, ZeitzU, StaniekK, ErbenRG, PohlEE (2016) The expression of ucp3 directly correlates to ucp1 abundance in brown adipose tissue. Biochim Biophys Acta 1857 : 72–78. 10.1016/j.bbabio.2015.10.011.26518386
Hraoui G , BettinazziS, GendronAD, BoisclairD, BretonS (2020) Mitochondrial thermo-sensitivity in invasive and native freshwater mussels. J Exp Biol 223 : jeb215921. 10.1242/jeb.215921.31915201
Hraoui G , BretonS, MironG, BoudreauLH, Hunter-ManseauF, PichaudN (2021) Mitochondrial responses towards intermittent heat shocks in the eastern oyster, Crassostrea virginica. J Exp Biol 224 : jeb242745. 10.1242/jeb.242745.34401903
Hughes J , GoudkampK, HurwoodD, HancockM, BunnS (2003) Translocation causes extinction of a local population of the freshwater shrimp Paratya australiensis. Conserv Biol 17 : 1007–1012. 10.1046/j.1523-1739.2003.01636.x.
Hunter-Manseau F , DesrosiersV, Le FrançoisNR, DufresneF, DetrichHW, NozaisC, BlierPU (2019) From Africa to Antarctica: exploring the metabolism of fish heart mitochondria across a wide thermal range. Front Physiol 10 : 1220. 10.3389/fphys.2019.01220.31636568
Iftikar FI , HickeyAJR (2013) Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus. PloS One 8 : e64120. 10.1371/journal.pone.0064120.23724026
Iftikar FI , MacDonaldJ, HickeyAJR (2010) Thermal limits of portunid crab heart mitochondria: could more thermo-stable mitochondria advantage invasive species? J Exp Mar Biol Ecol 395 : 232–239. 10.1016/j.jembe.2010.09.005.
Iftikar FI , MacDonaldJR, BakerDW, RenshawGMC, HickeyAJR (2014) Could thermal sensitivity of mitochondria determine species distribution in a changing climate? J Exp Biol 217 : 2348–2357. 10.1242/jeb.098798.25141346
Innocenti P , MorrowEH, DowlingDK (2011) Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution. Science 332 : 845–848. 10.1126/science.1201157.21566193
Iverson ENK (2024) Conservation mitonuclear replacement: facilitated mitochondrial adaptation for a changing world. Evol Appl 17 : e13642. 10.1111/eva.13642.38468713
Jørgensen LB , OvergaardJ, Hunter-ManseauF, PichaudN (2021) Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using drosophila. J Exp Biol 224 : jeb240960. 10.1242/jeb.240960.33563650
Jové M , NaudíA, Ramírez-NúñezO, Portero-OtínM, SelmanC, WithersDJ, PamplonaR (2014) Caloric restriction reveals a metabolomic and lipidomic signature in liver of male mice. Aging Cell 13 : 828–837. 10.1111/acel.12241.25052291
Jutfelt F , NorinT, ErnR, OvergaardJ, WangT, McKenzieDJ, LefevreS, NilssonGE, MetcalfeNB, HickeyAJet al. (2018) Oxygen-and capacity-limited thermal tolerance: blurring ecology and physiology. J Exp Biol 221 : jeb169615. 10.1242/jeb.169615.29321291
Kienzle L , BettinazziS, ChoquetteT, BrunetM, KhoramiHH, JacquesJ-F, MoreauM, RoucouX, LandryCR, AngersAet al. (2023) A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics. BMC Biol 21 : 111. 10.1186/s12915-023-01609-y.37198654
Koch RE , BuchananKL, CasagrandeS, CrinoO, DowlingDK, HillGE, HoodWR, McKenzieM, MarietteMM, NobleDWAet al. (2021) Integrating mitochondrial aerobic metabolism into ecology and evolution. Trends Ecol Evol 36 : 321–332. 10.1016/j.tree.2020.12.006.33436278
Kraffe E , MartyY, GuderleyH (2007) Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout oncorhynchus mykiss: roles of membrane proteins, phospholipids and their fatty acid compositions. J Exp Biol 210 : 149–165. 10.1242/jeb.02628.17170158
Krogh A (1929) The progress of physiology. Science 70 : 200–204. 10.1126/science.70.1809.200.17732865
Kuznetsov AV , VekslerV, GellerichFN, SaksV, MargreiterR, KunzWS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3 : 965–976. 10.1038/nprot.2008.61.18536644
Larsen S , NielsenJ, HansenCN, NielsenLB, WibrandF, StrideN, SchroderHD, BoushelR, HelgeJW, DelaFet al. (2012) Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 590 : 3349–3360. 10.1113/jphysiol.2012.230185.22586215
Lawson CA , CampE, DavySK, Ferrier-PagèsC, MatthewsJ, SuggettDJ (2022) Informing coral reef conservation through metabolomic approaches. In RieglBM, DodgeRE, eds, Coral Reef Conservation and Restoration in the Omics Age. Springer International Publishing, London UK, pp. 179–202
Lebenzon JE , DenezisPW, MohammadL, MathersKE, TurnbullKF, StaplesJF, SinclairBJ (2022) Reversible mitophagy drives metabolic suppression in diapausing beetles. Proc Natl Acad Sci 119 : e2201089119. 10.1073/pnas.2201089119.35858446
Loughland I , SeebacherF (2020) Differences in oxidative status explain variation in thermal acclimation capacity between individual mosquitofish (Gambusia holbrooki). Funct Ecol 34 : 1380–1390. 10.1111/1365-2435.13563.
Lubawy J , ChowańskiS, AdamskiZ, SłocińskaM (2022) Mitochondria as a target and central hub of energy division during cold stress in insects. Front Zool 19 : 1. 10.1186/s12983-021-00448-3.34991650
Mahalingam S , McClellandGB, ScottGR (2017) Evolved changes in the intracellular distribution and physiology of muscle mitochondria in high-altitude native deer mice. J Physiol 595 : 4785–4801. 10.1113/JP274130.28418073
Makrecka-Kuka M , KrumschnabelG, GnaigerE (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5 : 1319–1338. 10.3390/biom5031319.26131977
Malkoc K , CasagrandeS, HauM (2021) Inferring whole-organism metabolic rate from red blood cells in birds. Front Physiol 12 : 691633. 10.3389/fphys.2021.691633.34335298
Masson-Delmotte V (2018) Global Warming of 1.5° c: An IPCC Special Report on Impacts of Global Warming of 1.5° c Above Pre-Industrial Levels and Related Global Greenhouse Gas Emission Pathways, in the Contex of Strengthening the Global Response to the Thereat of Blimate Change, Sustainable Development, and Efforts to Eradicate Poverty. Cambridge University Press.
Mathot KJ , DingemanseNJ (2015) Energetics and behavior: unrequited needs and new directions. Trends Ecol Evol 30 : 199–206. 10.1016/j.tree.2015.01.010.25687159
McCluney KE , BelnapJ, CollinsSL, GonzálezAL, HagenEM, Nathaniel HollandJ, KotlerBP, MaestreFT, SmithSD, WolfBO (2012) Shifting species interactions in terrestrial dryland ecosystems under altered water availability and climate change. Biol Rev 87 : 563–582. 10.1111/j.1469-185X.2011.00209.x.22098619
McDiarmid CS , HooperDM, StierA, GriffithSC (2024) Mitonuclear interactions impact aerobic metabolism in hybrids and may explain mitonuclear discordance in young, naturally hybridizing bird lineages. Mol Ecol 33 (12 ): e17374. 10.1111/mec.17374.38727686
McLaughlin KL , HagenJT, CoalsonHS, NelsonMAM, KewKA, WootenAR, Fisher-WellmanKH (2020) Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs. Sci Rep 10 : 17599. 10.1038/s41598-020-74718-1.33077793
Melzner F , ThomsenJ, KoeveW, OschliesA, GutowskaMA, BangeHW, HansenHP, KörtzingerA (2013) Future ocean acidification will be amplified by hypoxia in coastal habitats. Mar Biol 160 : 1875–1888. 10.1007/s00227-012-1954-1.
Menail HA , CormierSB, Ben YoussefM, JørgensenLB, VickruckJL, MorinP, BoudreauLH, PichaudN (2022) Flexible thermal sensitivity of mitochondrial oxygen consumption and substrate oxidation in flying insect species. Front Physiol 13 : 897174. 10.3389/fphys.2022.897174.35547573
Menail HA , CormierSB, LégerA, RobichaudS, Hebert-ChatelainE, LamarreSG, PichaudN (2023) Age-related flexibility of energetic metabolism in the honey bee Apis mellifera. FASEB J 37 : e23222. 10.1096/fj.202300654R.37781970
Mesquita RD , GaviraghiA, GonçalvesRLS, Vannier-SantosMA, MignacoJA, FontesCFL, MachadoL, OliveiraMF (2021) Cytochrome c oxidase at full thrust: regulation and biological consequences to flying insects. Cells 10 : 470. 10.3390/cells10020470.33671793
Metcalfe NB , BellmanJ, BizeP, BlierPU, CrespelA, DawsonNJ, DunnRE, HalseyLG, HoodWR, HopkinsMet al. (2023) Solving the conundrum of intra-specific variation in metabolic rate: a multidisciplinary conceptual and methodological toolkit. Bioessays 45 : 2300026. 10.1002/bies.202300026.
Metcalfe NB , TaylorAC, ThorpeJE (1995) Metabolic rate, social status and life-history strategies in Atlantic salmon. Anim Behav 49 : 431–436. 10.1006/anbe.1995.0056.
Meyer JN , LeungMC, RooneyJP, SendoelA, HengartnerMO, KisbyGE, BessAS (2013) Mitochondria as a target of environmental toxicants. Toxicol Sci 134 : 1–17. 10.1093/toxsci/kft102.23629515
Milbergue MS , VézinaF, DesrosiersV, BlierPU (2022) How does mitochondrial function relate to thermogenic capacity and basal metabolic rate in small birds? J Exp Biol 225 : jeb242612. 10.1242/jeb.242612.35762381
Monzel AS , EnríquezJA, PicardM (2023) Multifaceted mitochondria: moving mitochondrial science beyond function and dysfunction. Nat Metab 5 : 546–562. 10.1038/s42255-023-00783-1.37100996
Morgan R , SundinJ, FinnøenMH, DreslerG, VendrellMM, DeyA, SarkarK, JutfeltF (2019) Are model organisms representative for climate change research? Testing thermal tolerance in wild and laboratory zebrafish populations. Conserv Physiol 7 : coz036. 10.1093/conphys/coz036.31249690
Mottis A , HerzigS, AuwerxJ (2019) Mitocellular communication: shaping health and disease. Science 366 : 827–832. 10.1126/science.aax3768.31727828
Munro D , BlierPU (2012) The extreme longevity of Arctica islandica is associated with increased peroxidation resistance in mitochondrial membranes. Aging Cell 11 : 845–855. 10.1111/j.1474-9726.2012.00847.x.22708840
Noguera JC , VelandoA (2019) Bird embryos perceive vibratory cues of predation risk from clutch mates. Nat Ecol Evol 3 : 1225–1232. 10.1038/s41559-019-0929-8.31332329
Nord A , ChamkhaI, ElmérE (2023) A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian blood cells. FASEB J 37 : e22766. 10.1096/fj.202201749R.36734850
Nord A , MetcalfeNB, PageJL, HuxtableA, McCaffertyDJ, DawsonNJ (2021) Avian red blood cell mitochondria produce more heat in winter than in autumn. FASEB J 35 : e21490. 10.1096/fj.202100107R.33829547
Orr WC , SohalRS (1992) The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic drosophila melanogaster. Arch Biochem Biophys 297 : 35–41. 10.1016/0003-9861(92)90637-C.1379030
Palmeira CM , MorenoAJ (2018) MitochondrialBioenergetics: Methods and Protocols. PalmeiraCM, OliveiraPJ, eds. Springer International Publishing, London UK
Parry HA , YapKN, HillGE, HoodWR, GladdenLB, EddyM, KavazisAN (2021) Development of a mobile mitochondrial physiology laboratory for measuring mitochondrial energetics in the field. J Vis Exp 174 : e62956. 10.3791/62956.
Patil YN , MardenB, BrandMD, HandSC (2013) Metabolic downregulation and inhibition of carbohydrate catabolism during diapause in embryos of Artemia franciscana. Physiol Biochem Zool 86 : 106–118. 10.1086/667808.23303325
Pelletier D , BlierP, VézinaF, GuillemetteM (2023a) Good times bad times—unfavorable breeding conditions, more than divorce, lead to increased parental effort and reduced physiological condition of northern gannets. Front Ecol Evol 11 : 1108293. 10.3389/fevo.2023.1108293.
Pelletier D , BlierPU, VézinaF, DufresneF, PaquinF, ChristenF, GuillemetteM (2023b) Under pressure—exploring partner changes, physiological responses and telomere dynamics in northern gannets across varying breeding conditions. PeerJ 11 : e16457. 10.7717/peerj.16457.38054014
Pelletier D , DutilJD, BlierP, GuderleyH (1994) Relation between growth rate and metabolic organization of white muscle, liver and digestive tract in cod, Gadus morhua. J Comp Physiol B 164 : 179–190. 10.1007/BF00354078.
Pendergrass W , WolfN, PootM (2004) Efficacy of mitotracker green and cmxrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A 61A : 162–169. 10.1002/cyto.a.20033.
Perkins G , HsiaoY-H, YinS, TjongJ, TranMT, LauJ, XueJ, LiuS, EllismanMH, ZhouD (2012) Ultrastructural modifications in the mitochondria of hypoxia-adapted drosophila melanogaster. PloS One 7 : e45344. 10.1371/journal.pone.0045344.23028948
Pettersen AK , MarshallDJ, WhiteCR (2018) Understanding variation in metabolic rate. J Exp Biol 221 : jeb166876. 10.1242/jeb.166876.29326115
Pichaud N , ChatelainEH, BallardJWO, TanguayR, MorrowG, BlierPU (2010) Thermal sensitivity of mitochondrial metabolism in two distinct mitotypes of drosophila simulans: evaluation of mitochondrial plasticity. J Exp Biol 213 : 1665–1675. 10.1242/jeb.040261.20435817
Pichaud N , EkströmA, HellgrenK, SandblomE (2017) Dynamic changes in cardiac mitochondrial metabolism during warm acclimation in rainbow trout. J Exp Biol 220 : 1674–1683. 10.1242/jeb.152421.28202582
Pörtner HO , FarrellAP (2008) Physiology and climate change. Science 322 : 690–692. 10.1126/science.1163156.18974339
Pouliot-Drouin A , NiaisonT, BretonS, BettinazziS (2024) Investigating the role of mitochondrial membrane potential in paternal inheritance of mitochondria. Biol J Linn Soc . 10.1093/biolinnean/blae050.
Putnam JG , SteinerJN, RichardJC, LeisE, GoldbergTL, DunnCD, AgbalogR, KnowlesS, WallerDL (2023) Mussel mass mortality in the Clinch river, USA: metabolomics detects affected pathways and biomarkers of stress. Conserv Physiol 11 : coad074. 10.1093/conphys/coad074.37680611
Quéméneur J-B , DanionM, CabonJ, ColletS, Zambonino-InfanteJ-L, SalinK (2022) The relationships between growth rate and mitochondrial metabolism varies over time. Sci Rep 12 : 16066. 10.1038/s41598-022-20428-9.36167968
Rank NE , MardulynP, HeidlSJ, RobertsKT, ZavalaNA, SmileyJT, DahlhoffEP (2020) Mitonuclear mismatch alters performance and reproductive success in naturally introgressed populations of a montane leaf beetle. Evolution 74 : 1724–1740. 10.1111/evo.13962.32246837
Rauhamäki V , WolframJ, JokitaloE, HanskiI, DahlhoffEP (2014) Differences in the aerobic capacity of flight muscles between butterfly populations and species with dissimilar flight abilities. PloS One 9 : e78069. 10.1371/journal.pone.0078069.24416122
Rhodes EM , YapKN, MesquitaPHC, ParryHA, KavazisAN, KrauseJS, HillGE, HoodWR (2024) Flexibility underlies differences in mitochondrial respiratory performance between migratory and non-migratory white-crowned sparrows (Zonotrichia leucophrys). Sci Rep 14 : 9456. 10.1038/s41598-024-59715-y.38658588
Rissoli RZ , daSilvaVE, RantinFT, KalininAL (2017) Effects of exercise training on excitation-contraction coupling, calcium dynamics and protein expression in the heart of the neotropical fish Brycon amazonicus. Comp Biochem Physiol A Mol Integr Physiol 214 : 85–93. 10.1016/j.cbpa.2017.09.016.28966144
Rodríguez E , BettinazziS, InwongwanS, CamusMF, LaneN (2023) Harmonising protocols to measure drosophila respiratory function in mitochondrial preparations. Bioenerg Commun 2023 : 3. 10.26124/bec:2023-0003
Rodríguez E , DégletagneC, HagenTM, AbeleD, BlierPU (2019) Mitochondrial traits previously associated with species maximum lifespan do not correlate with longevity across populations of the bivalve Arctica islandica. Front Physiol 10 : 946. 10.3389/fphys.2019.00946.31404340
Rodríguez E , Grover ThomasF, CamusMF, LaneN (2021) Mitonuclear interactions produce diverging responses to mild stress in drosophila larvae. Front Genet 12 : 734255. 10.3389/fgene.2021.734255.34603395
Rodríguez E , HakkouM, HagenTM, LemieuxH, BlierPU (2020) Divergences in the control of mitochondrial respiration are associated with life-span variation in marine bivalves. J Gerontol A Biol Sci Med Sci 76 : 796–804. 10.1093/gerona/glaa301.
Rogelj J , MeinshausenM, KnuttiR (2012) Global warming under old and new scenarios using Ipcc climate sensitivity range estimates. Nat Clim Change 2 : 248–253. 10.1038/nclimate1385.
Roussel D , BoëlM, RomestaingC (2018) Fasting enhances mitochondrial efficiency in duckling skeletal muscle by acting on the substrate oxidation system. J Exp Biol 221 : jeb172213. 10.1242/jeb.172213.29361595
Salin K , AuerSK, AndersonGJ, SelmanC, MetcalfeNB (2016a) Inadequate food intake at high temperatures is related to depressed mitochondrial respiratory capacity. J Exp Biol 219 : 1356–1362. 10.1242/jeb.133025.26944497
Salin K , VillasevilEM, AndersonGJ, LamarreSG, MelansonCA, McCarthyI, SelmanC, MetcalfeNB (2019) Differences in mitochondrial efficiency explain individual variation in growth performance. Proc R Soc B Biol Sci 286 : 20191466. 10.1098/rspb.2019.1466.
Salin K , VillasevilEM, AuerSK, AndersonGJ, SelmanC, MetcalfeNB, ChinopoulosC (2016b) Simultaneous measurement of mitochondrial respiration and atp production in tissue homogenates and calculation of effective p/o ratios. Physiol Rep 4 : e13007. 10.14814/phy2.13007.27798358
Salmón P , MilletC, SelmanC, MonaghanP, DawsonNJ (2023) Tissue-specific reductions in mitochondrial efficiency and increased ros release rates during ageing in zebra finches, Taeniopygia guttata. GeroScience 45 : 265–276. 10.1007/s11357-022-00624-1.35986126
Sappal R , FastM, StevensD, KibengeF, SiahA, KamundeC (2015) Effects of copper, hypoxia and acute temperature shifts on mitochondrial oxidation in rainbow trout (Oncorhynchus mykiss) acclimated to warm temperature. Aquat Toxicol 169 : 46–57. 10.1016/j.aquatox.2015.10.006.26513222
Schell ER , McCrackenKG, ScottGR, WhiteJ, LavretskyP, DawsonNJ (2023) Consistent changes in muscle metabolism underlie dive performance across multiple lineages of diving ducks. Proc R Soc B Biol Sci 290 : 20231466. 10.1098/rspb.2023.1466.
Schulte PM (2015) The effects of temperature on aerobic metabolism: towards a mechanistic understanding of the responses of ectotherms to a changing environment. J Exp Biol 218 : 1856–1866. 10.1242/jeb.118851.26085663
Scott GR , GuoKH, DawsonNJ (2018) The mitochondrial basis for adaptive variation in aerobic performance in high-altitude deer mice. Integr Comp Biol 58 : 506–518. 10.1093/icb/icy056.29873740
Scott GR , SchultePM, EggintonS, ScottALM, RichardsJG, MilsomWK (2010) Molecular evolution of cytochrome c oxidase underlies high-altitude adaptation in the bar-headed goose. Mol Biol Evol 28 : 351–363. 10.1093/molbev/msq205.20685719
Seebacher F , FranklinCE (2012) Determining environmental causes of biological effects: the need for a mechanistic physiological dimension in conservation biology. Philos Trans R Soc B Biol Sci 367 : 1607–1614. 10.1098/rstb.2012.0036.
Seebacher F , JamesRS (2008) Plasticity of muscle function in a thermoregulating ectotherm (Crocodylus porosus): biomechanics and metabolism. Am J Physiol Regul Integr Comp Physiol 294 : R1024–R1032. 10.1152/ajpregu.00755.2007.18199589
Smith S , TurbillC, SuchentrunkF (2010) Introducing mother’s curse: low male fertility associated with an imported mtdna haplotype in a captive colony of brown hares. Mol Ecol 19 : 36–43. 10.1111/j.1365-294X.2009.04444.x.19943893
Sokolova I (2018) Mitochondrial adaptations to variable environments and their role in animals’ stress tolerance. Integr Comp Biol 58 : 519–531. 10.1093/icb/icy017.29701785
Somero GN (2005) Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Front Zool 2 : 1. 10.1186/1742-9994-2-1.15679952
Somero GN (2010) The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine ‘winners’ and ‘losers’. J Exp Biol 213 : 912–920. 10.1242/jeb.037473.20190116
Spinazzi M , CasarinA, PertegatoV, SalviatiL, AngeliniC (2012) Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 7 : 1235–1246. 10.1038/nprot.2012.058.22653162
Steffen JBM , SokolovEP, BockC, SokolovaIM (2023) Combined effects of salinity and intermittent hypoxia on mitochondrial capacity and reactive oxygen species efflux in the pacific oyster, Crassostrea gigas. J Exp Biol 226 : jeb246164. 10.1242/jeb.246164.37470191
Stier A , HsuB-Y, Cossin-SevrinN, GarcinN, RuuskanenS (2021) From climate warming to accelerated cellular ageing: an experimental study in wild birds. bioRxiv . 10.1101/2021.12.21.473625.
Stier A , MonaghanP, MetcalfeNB (2022) Experimental demonstration of prenatal programming of mitochondrial aerobic metabolism lasting until adulthood. Proc Biol Sci 289 : 20212679. 10.1098/rspb.2021.2679.35232239
Stier A , RomestaingC, SchullQ, LefolE, RobinJP, RousselD, BizeP (2017b) How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution. Methods Ecol Evol 8 : 1172–1182. 10.1111/2041-210X.12724.
Stier A , SchullQ, BizeP, LefolE, HaussmannM, RousselD, RobinJ-P, ViblancVA (2019) Oxidative stress and mitochondrial responses to stress exposure suggest that king penguins are naturally equipped to resist stress. Sci Rep 9 : 8545. 10.1038/s41598-019-44990-x.31189949
Sussarellu R , DudognonT, FabiouxC, SoudantP, MoragaD, KraffeE (2013) Rapid mitochondrial adjustments in response to short-term hypoxia and re-oxygenation in the pacific oyster, Crassostrea gigas. J Exp Biol 216 : 1561–1569. 10.1242/jeb.075879.23307802
Teulier L , ThoralE, QueirosQ, MckenzieDJ, RousselD, DuttoG, GassetE, BourjeaJ, SarauxC (2019) Muscle bioenergetics of two emblematic mediterranean fish species: Sardina pilchardus and Sparus aurata. Comp Biochem Physiol A Mol Integr Physiol 235 : 174–179. 10.1016/j.cbpa.2019.06.008.31220620
Thibault M , BlierPU, GuderleyH (1997) Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 16 : 139–155. 10.1007/BF00004671.
Thoral E , DargèreL, Medina-SuárezI, ClairA, AvertyL, SigaudJ, MoralesA, SalinK, TeulierL (2024a) Non-lethal sampling for assessment of mitochondrial function does not affect metabolic rate and swimming performance. Philos Trans R Soc B Biol Sci 379 : 20220483. 10.1098/rstb.2022.0483.
Thoral E , FarhatE, RousselD, ChengH, GuillardL, PamenterME, WeberJM, TeulierL (2022a) Different patterns of chronic hypoxia lead to hierarchical adaptive mechanisms in goldfish metabolism. J Exp Biol 225 : jeb243194. 10.1242/jeb.243194.34881781
Thoral E , García-DíazCC, PerssonE, ChamkhaI, ElmérE, RuuskanenS, NordA (2024b) The relationship between mitochondrial respiration, resting metabolic rate and blood cell count in great tits. Biol Open 13 : bio060302. 10.1242/bio.060302.38385271
Thoral E , RousselD, ChinopoulosC, TeulierL, SalinK (2021) Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Biol Lett 17 : 20200759. 10.1098/rsbl.2020.0759.33563134
Thoral E , RousselD, QuispeL, VoituronY, TeulierL (2022b) Absence of mitochondrial responses in muscles of zebrafish exposed to several heat waves. Comp Biochem Physiol A Mol Integr Physiol 274 : 111299. 10.1016/j.cbpa.2022.111299.36031060
Thorstensen MJ , TurkoAJ, HeathDD, JeffriesKM, PitcherTE (2022) Acute thermal stress elicits interactions between gene expression and alternative splicing in a fish of conservation concern. J Exp Biol 225 : jeb244162. 10.1242/jeb.244162.35673877
Timonin ME , CarrièreCJ, DudychAD, LatimerJGW, UnruhST, WillisCKR (2011) Individual differences in the behavioural responses of meadow voles to an unfamiliar environment are not correlated with variation in resting metabolic rate. J Zool 284 : 198–205. 10.1111/j.1469-7998.2011.00792.x.
Van Heerwaarden B , SgròCM (2021) Male fertility thermal limits predict vulnerability to climate warming. Nat Commun 12 : 2214. 10.1038/s41467-021-22546-w.33850157
Vandenberg GG , DawsonNJ, HeadA, ScottGR, ScottAL (2021) Astrocyte-mediated disruption of ros homeostasis in fragile x mouse model. Neurochem Int 146 : 105036. 10.1016/j.neuint.2021.105036.33785420
Wagner S , SteinbeckJ, FuchsP, LichtenauerS, ElsässerM, SchippersJHM, NietzelT, RubertiC, Van AkenO, MeyerAJet al. (2019) Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport. New Phytol 224 : 1668–1684. 10.1111/nph.16093.31386759
Wallace DC , FanW (2010) Energetics, epigenetics, mitochondrial genetics. Mitochondrion 10 : 12–31. 10.1016/j.mito.2009.09.006.19796712
Waller D , PutnamJ, SteinerJN, FisherB, BurchamGN, OliverJ, SmithSB, EricksonR, RemekA, BodoekerN (2023) Targeted metabolomics characterizes metabolite occurrence and variability in stable freshwater mussel populations. Conserv Physiol 11 : coad040. 10.1093/conphys/coad040.37701372
Závorka L , CrespelA, DawsonNJ, PapatheodoulouM, KillenSS, KainzMJ (2021) Climate change-induced deprivation of dietary essential fatty acids can reduce growth and mitochondrial efficiency of wild juvenile salmon. Funct Ecol 35 : 1960–1971. 10.1111/1365-2435.13860.
