Multigenerational exposure to temperature influences mitochondrial oxygen fluxes in the Medaka fish (Oryzias latipes).

AIM: Thermal sensitivity of cellular metabolism is crucial for animal physiology and survival under climate change. Despite recent efforts, effects of multigenerational exposure to temperature on the metabolic functioning remain poorly understood. We aimed at determining whether multigenerational exposure to temperature modulate the mitochondrial respiratory response of Medaka fish. METHODS: We conducted a multigenerational exposure with Medaka fish reared multiple generations at 20 and 30°C (COLD and WARM fish, respectively). We then measured the oxygen consumption of tail muscle at two assay temperatures (20 and 30°C). Mitochondrial function was determined as the respiration supporting ATP synthesis (OXPHOS) and the respiration required to offset proton leak (LEAK(Omy)) in a full factorial design (COLD-20°C; COLD-30°C; WARM-20°C; WARM-30°C). RESULTS: We found that higher OXPHOS and LEAK fluxes at 30°C compared to 20°C assay temperature. At each assay temperature, WARM fish had lower tissue oxygen fluxes than COLD fish. Interestingly, we did not find significant differences in respiratory flux when mitochondria were assessed at the rearing temperature of the fish (i.e., COLD-20°C vs. WARM -30°C). CONCLUSION: The lower OXPHOS and LEAK capacities in warm fish are likely the result of the multigenerational exposure to warm temperature. This is consistent with a modulatory response of mitochondrial capacity to compensate for potential detrimental effects of warming on metabolism. Finally, the absence of significant differences in respiratory fluxes between COLD-20°C and WARM-30°C fish likely reflects an optimal respiration flux when organisms adapt to their thermal conditions.

Non-lethal sampling for assessment of mitochondrial function does not affect metabolic rate and swimming performance.

A fundamental issue in the metabolic field is whether it is possible to understand underlying mechanisms that characterize individual variation. Whole-animal performance relies on mitochondrial function as it produces energy for cellular processes. However, our lack of longitudinal measures to evaluate how mitochondrial function can change within and among individuals and with environmental context makes it difficult to assess individual variation in mitochondrial traits. The aims of this study were to test the repeatability of muscle mitochondrial metabolism by performing two biopsies of red muscle, and to evaluate the effects of biopsies on whole-animal performance in goldfish Carassius auratus. Our results show that basal mitochondrial respiration and net phosphorylation efficiency are repeatable at 14-day intervals. We also show that swimming performance (optimal cost of transport and critical swimming speed) was repeatable in biopsied fish, whereas the repeatability of individual oxygen consumption (standard and maximal metabolic rates) seemed unstable over time. However, we noted that the means of individual and mitochondrial traits did not change over time in biopsied fish. This study shows that muscle biopsies allow the measurement of mitochondrial metabolism without sacrificing animals and that two muscle biopsies 14 days apart affect the intraspecific variation in fish performance without affecting average performance of individuals. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Intertidal limits shape covariation between metabolic plasticity, oxidative stress and telomere dynamics in Pacific oyster (Crassostrea gigas).

In intertidal zones, species such as sessile shellfish exhibit extended phenotypic plasticity to face rapid environmental changes, but whether frequent exposure to intertidal limits of the distribution range impose physiological costs for the animal remains elusive. Here, we explored how phenotypic plasticity varied along foreshore range at multiple organization levels, from molecular to cellular and whole organism acclimatization, in the Pacific oyster (Crassostrea gigas). We exposed 7-month-old individuals for up to 16 months to three foreshore levels covering the vertical range for this species, representing 20, 50 and 80% of the time spent submerged monthly. Individuals at the upper range limit produced energy more efficiently, as seen by steeper metabolic reactive norms and unaltered ATP levels despite reduced mitochondrial density. By spending most of their time emerged, oysters mounted an antioxidant shielding concomitant with lower levels of pro-oxidant proteins and postponed age-related telomere attrition. Instead, individuals exposed at the lower limit range near subtidal conditions showed lower energy efficiencies, greater oxidative stress and shorter telomere length. These results unraveled the extended acclimatization strategies and the physiological costs of living too fast in subtidal conditions for an intertidal species.

Solving the conundrum of intra-specific variation in metabolic rate: A multidisciplinary conceptual and methodological toolkit: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species.

Researchers from diverse disciplines, including organismal and cellular physiology, sports science, human nutrition, evolution and ecology, have sought to understand the causes and consequences of the surprising variation in metabolic rate found among and within individual animals of the same species. Research in this area has been hampered by differences in approach, terminology and methodology, and the context in which measurements are made. Recent advances provide important opportunities to identify and address the key questions in the field. By bringing together researchers from different areas of biology and biomedicine, we describe and evaluate these developments and the insights they could yield, highlighting the need for more standardisation across disciplines. We conclude with a list of important questions that can now be addressed by developing a common conceptual and methodological toolkit for studies on metabolic variation in animals.

The relationships between growth rate and mitochondrial metabolism varies over time.

Mitochondrial metabolism varies significantly between individuals of the same species and can influence animal performance, such as growth. However, growth rate is usually determined before the mitochondrial assay. The hypothesis that natural variation in mitochondrial metabolic traits is linked to differences in both previous and upcoming growth remains untested. Using biopsies to collect tissue in a non-lethal manner, we tested this hypothesis in a fish model (Dicentrarchus labrax) by monitoring individual growth rate, measuring mitochondrial metabolic traits in the red muscle, and monitoring the growth of the same individuals after the mitochondrial assay. Individual variation in growth rate was consistent before and after the mitochondrial assay; however, the mitochondrial traits that explained growth variation differed between the growth rates determined before and after the mitochondrial assay. While past growth was correlated with the activity of the cytochrome c oxidase, a measure of mitochondrial density, future growth was linked to mitochondrial proton leak respiration. This is the first report of temporal shift in the relationship between growth rate and mitochondrial metabolic traits, suggesting an among-individual variation in temporal changes in mitochondrial traits. Our results emphasize the need to evaluate whether mitochondrial metabolic traits of individuals can change over time.

Is mitochondrial reactive oxygen species production proportional to oxygen consumption? A theoretical consideration.

It has been assumed that at the whole organismal level, the mitochondrial reactive oxygen species (ROS) production is proportional to the oxygen consumption. Recently, a number of researchers have challenged this assumption, based on the observation that the ROS production per unit oxygen consumed in the resting state of mitochondrial respiration is much higher than that in the active state. Here, we develop a simple model to investigate the validity of the assumption and the challenge of it. The model highlights the significance of the time budget that mitochondria operate in the different respiration states. The model suggests that under three physiologically possible conditions, the difference in ROS production per unit oxygen consumed between the respiration states does not upset the proportionality between the whole animal ROS production and oxygen consumption. The model also shows that mitochondrial uncoupling generally enhances the proportionality.

Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish.

Aerobic metabolism of aquatic ectotherms is highly sensitive to fluctuating climates. Many mitochondrial traits exhibit phenotypic plasticity in response to acute variations in temperature and oxygen availability. These responses are critical for understanding the effects of environmental variations on aquatic ectotherms' performance. Using the European seabass, Dicentrarchus labrax, we determined the effects of acute warming and deoxygenation in vitro on mitochondrial respiratory capacities and mitochondrial efficiency to produce ATP (ATP/O ratio). We show that acute warming reduced ATP/O ratio but deoxygenation marginally raised ATP/O ratio, leading to a compensatory effect of low oxygen availability on mitochondrial ATP/O ratio at high temperature. The acute effect of warming and deoxygenation on mitochondrial efficiency might be related to the leak of protons across the mitochondrial inner membrane, as the mitochondrial respiration required to counteract the proton leak increased with warming and decreased with deoxygenation. Our study underlines the importance of integrating the combined effects of temperature and oxygen availability on mitochondrial metabolism. Predictions on decline in performance of aquatic ectotherms owing to climate change may not be accurate, since these predictions typically look at respiratory capacity and ignore efficiency of ATP production.

Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution.

Biologists have long appreciated the critical role that energy turnover plays in understanding variation in performance and fitness among individuals. Whole-organism metabolic studies have provided key insights into fundamental ecological and evolutionary processes. However, constraints operating at subcellular levels, such as those operating within the mitochondria, can also play important roles in optimizing metabolism over different energetic demands and time scales. Herein, we explore how mitochondrial aerobic metabolism influences different aspects of organismal performance, such as through changing adenosine triphosphate (ATP) and reactive oxygen species (ROS) production. We consider how such insights have advanced our understanding of the mechanisms underpinning key ecological and evolutionary processes, from variation in life-history traits to adaptation to changing thermal conditions, and we highlight key areas for future research.

The relationship between membrane fatty acid content and mitochondrial efficiency differs within- and between- omega-3 dietary treatments.

An important, but underappreciated, consequence of climate change is the reduction in crucial nutrient production at the base of the marine food chain: the long-chain omega-3 highly unsaturated fatty acids (n-3 HUFA). This can have dramatic consequences on consumers, such as fish as they have limited capacity to synthesise n-3 HUFA de novo. The n-3 HUFA, such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), are critical for the structure and function of all biological membranes. There is increasing evidence that fish will be badly affected by reductions in n-3 HUFA dietary availability, however the underlying mechanisms remain obscure. Hypotheses for how mitochondrial function should change with dietary n-3 HUFA availability have generally ignored ATP production, despite its importance to a cell's total energetics capacity, and in turn, whole-animal performance. Here we (i) quantified individual variation in mitochondrial efficiency (ATP/O ratio) of muscle and (ii) examined its relationship with content in EPA and DHA in muscle membrane of a primary consumer fish, the golden grey mullet Chelon auratus, receiving either a high or low n-3 HUFA diet. Mitochondria of fish fed on the low n-3 HUFA diet had higher ATP/O ratio than those of fish maintained on the high n-3 HUFA diet. Yet, mitochondrial efficiency varied up about 2-fold among individuals on the same dietary treatment, resulting in some fish consuming half the oxygen and energy substrate to produce the similar amount of ATP than conspecific on similar diet. This variation in mitochondrial efficiency among individuals from the same diet treatment was related to individual differences in fatty acid composition of the membranes: a high ATP/O ratio was associated with a high content in EPA and DHA in biological membranes. Our results highlight the existence of interindividual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in response to food chain changes.

Differences in mitochondrial efficiency explain individual variation in growth performance.

The physiological causes of intraspecific differences in fitness components such as growth rate are currently a source of debate. It has been suggested that differences in energy metabolism may drive variation in growth, but it remains unclear whether covariation between growth rates and energy metabolism is: (i) a result of certain individuals acquiring and consequently allocating more resources to growth, and/or is (ii) determined by variation in the efficiency with which those resources are transformed into growth. Studies of individually housed animals under standardized nutritional conditions can help shed light on this debate. Here we quantify individual variation in metabolic efficiency in terms of the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by liver and muscle mitochondria and examine its effects, both on the rate of protein synthesis within these tissues and on the rate of whole-body growth of individually fed juvenile brown trout (Salmo trutta) receiving either a high or low food ration. As expected, fish on the high ration on average gained more in body mass and protein content than those maintained on the low ration. Yet, growth performance varied more than 10-fold among individuals on the same ration, resulting in some fish on low rations growing faster than others on the high ration. This variation in growth for a given ration was related to individual differences in mitochondrial properties: a high whole-body growth performance was associated with high mitochondrial efficiency of ATP production in the liver. Our results show for the first time, to our knowledge, that among-individual variation in the efficiency with which substrates are converted into ATP can help explain marked variation in growth performance, independent of food intake. This study highlights the existence of inter-individual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in whole-animal performance.

Decreased mitochondrial metabolic requirements in fasting animals carry an oxidative cost.

Many animals experience periods of food shortage in their natural environment. It has been hypothesised that the metabolic responses of animals to naturally-occurring periods of food deprivation may have long-term negative impacts on their subsequent life-history.In particular, reductions in energy requirements in response to fasting may help preserve limited resources but potentially come at a cost of increased oxidative stress. However, little is known about this trade-off since studies of energy metabolism are generally conducted separately from those of oxidative stress.Using a novel approach that combines measurements of mitochondrial function with in vivo levels of hydrogen peroxide (H2O2) in brown trout (Salmo trutta), we show here that fasting induces energy savings in a highly metabolically active organ (the liver) but at the cost of a significant increase in H2O2, an important form of reactive oxygen species (ROS).After a 2-week period of fasting, brown trout reduced their whole-liver mitochondrial respiratory capacities (state 3, state 4 and cytochrome c oxidase activity), mainly due to reductions in liver size (and hence the total mitochondrial content). This was compensated for at the level of the mitochondrion, with an increase in state 3 respiration combined with a decrease in state 4 respiration, suggesting a selective increase in the capacity to produce ATP without a concomitant increase in energy dissipated through proton leakage. However, the reduction in total hepatic metabolic capacity in fasted fish was associated with an almost two-fold increase in in vivo mitochondrial H2O2 levels (as measured by the MitoB probe).The resulting increase in mitochondrial ROS, and hence potential risk of oxidative damage, provides mechanistic insight into the trade-off between the short-term energetic benefits of reducing metabolism in response to fasting and the potential long-term costs to subsequent life-history traits.

Les restrictions alimentaires sont courantes dans le milieu naturel et peuvent impacter de nombreux animaux. Il a été émis l'hypothèse que les animaux, face à ces épisodes de restriction alimentaire, mettaient en place des réponses métaboliques pouvant affecter leurs histoires de vie future.En particulier, si une diminution des besoins énergétiques lors du jeûne peut contribuer à préserver les réserves de l'animal cela peut néanmoins entraîner une augmentation du stress oxydant. Ce type de compromis n'a toutefois pas encore été démontré car l'étude du métabolisme énergétique est généralement réalisée séparément de celle du stress oxydant.Par une nouvelle approche combinant des mesures du fonctionnement mitochondrial et des niveaux in vivo de peroxyde d'hydrogène (H2O2) chez la truite commune (Salmo trutta), nous montrons ici que le jeûne entraîne une économie d'énergie dans un tissu métaboliquement très actif tel que le foie, mais au coût d'une augmentation significative en H2O2, une forme majeure des espèces réactives de l'oxygène.Après deux semaines de jeûne, les truites communes ont réduit leurs capacités respiratoires mitochondriales (état 3, état 4 et l'activité de la cytochrome c oxydase) principalement du fait d'une réduction de la taille du foie (et donc du nombre total de mitochondries). Une compensation a été observée au niveau de la mitochondrie. Cela se traduit par une augmentation de la respiration en état 3 et une diminution concomitante de celle en état 4, suggérant une augmentation sélective des capacités de production de l'ATP sans augmentation parallèle de l'énergie dissipée par la fuite de protons. La diminution des capacités métaboliques du foie chez les poissons à jeun était associée in vivo à des niveaux quasiment doubles de H2O2 mitochondriaux (mesurés par la sonde MitoB).Cette augmentation en espèces réactives de l'oxygène dans les mitochondries, avec son risque inhérent de dommages oxydatifs, apporte une vision mécanistique du compromis entre les bénéfices énergétiques à court terme d'une réduction métabolique en réponse au jeûne et les possibles coûts à long terme sur leurs traits histoires de vie futurs. A http://onlinelibrary.wiley.com/doi/10.1111/1365-2435.13125/suppinfo is available for this article.

The Mitochondrial Contribution to Animal Performance, Adaptation, and Life-History Variation.

Animals display tremendous variation in their rates of growth, reproductive output, and longevity. While the physiological and molecular mechanisms that underlie this variation remain poorly understood, the performance of the mitochondrion has emerged as a key player. Mitochondria not only impact the performance of eukaryotes via their capacity to produce ATP, but they also play a role in producing heat and reactive oxygen species and function as a major signaling hub for the cell. The papers included in this special issue emerged from a symposium titled "Inside the Black Box: The Mitochondrial Basis of Life-history Variation and Animal Performance." Based on studies of diverse animal taxa, three distinct themes emerged from these papers. (1) When linking mitochondrial function to components of fitness, it is crucial that mitochondrial assays are performed in conditions as close as the intracellular conditions experienced by the mitochondria in vivo. (2) Functional plasticity allows mitochondria to retain their performance, as well as that of their host, over a range of exogenous conditions, and selection on mitochondrial and nuclear-derived proteins can optimize the match between the environment and the bioenergetic capacity of the mitochondrion. Finally, (3) studies of wild and wild-derived animals suggest that mitochondria play a central role in animal performance and life history strategy. Taken as a whole, we hope that these papers will foster discussion and inspire new hypotheses and innovations that will further our understanding of the mitochondrial processes that underlie variation in life history traits and animal performance.

The RCR and ATP/O Indices Can Give Contradictory Messages about Mitochondrial Efficiency.

Mitochondrial efficiency is typically taken to represent an animal's capacity to convert its resources into ATP. However, the term mitochondrial efficiency, as currently used in the literature, can be calculated as either the respiratory control ratio, RCR (ratio of mitochondrial respiration supporting ATP synthesis to that required to offset the proton leak) or as the amount of ATP generated per unit of oxygen consumed, ATP/O ratio. The question of how flexibility in mitochondrial energy properties (i.e., in rates of respiration to support ATP synthesis and offset proton leak, and in the rate of ATP synthesis) affects these indices of mitochondrial efficiency has tended to be overlooked. Furthermore, little is known of whether the RCR and ATP/O ratio vary in parallel, either among individuals or in response to environmental conditions. Using data from brown trout Salmo trutta we show that experimental conditions affect mitochondrial efficiency, but the apparent direction of change depends on the index chosen: a reduction in food availability was associated with an increased RCR (i.e., increased efficiency) but a decreased ATP/O ratio (decreased efficiency) in liver mitochondria. Moreover, there was a negative correlation across individuals held in identical conditions between their RCR and their ATP/O ratio. These results show that the choice of index of mitochondrial efficiency can produce different, even opposing, conclusions about the capacity of the mitochondria to produce ATP. Neither ratio is necessarily a complete measure of efficiency of ATP production in the living animal (RCR because it contains no assessment of ATP production, and ATP/O because it contains no assessment of respiration to offset the proton leak). Consequently, we suggest that a measure of mitochondrial efficiency obtained nearer to conditions where respiration simultaneously offsets the proton leak and produce ATP would be sensitive to changes in both proton leakage and ATP production, and is thus likely to be more representative of the state of the mitochondria in vivo.

Individuals exhibit consistent differences in their metabolic rates across changing thermal conditions.

Metabolic rate has been linked to growth, reproduction, and survival at the individual level and is thought to have far reaching consequences for the ecology and evolution of organisms. However, metabolic rates must be consistent (i.e. repeatable) over at least some portion of the lifetime in order to predict their longer-term effects on population dynamics and how they will respond to selection. Previous studies demonstrate that metabolic rates are repeatable under constant conditions but potentially less so in more variable environments. We measured the standard (=minimum) metabolic rate, maximum metabolic rate, and aerobic scope (=interval between standard and maximum rates) in juvenile brown trout (Salmo trutta) after 5weeks acclimation to each of three consecutive test temperatures (10, 13, and then 16°C) that simulated the warming conditions experienced throughout their first summer of growth. We found that metabolic rates are repeatable over a period of months under changing thermal conditions: individual trout exhibited consistent differences in all three metabolic traits across increasing temperatures. Initial among-individual differences in metabolism are thus likely to have significant consequences for fitness-related traits over key periods of their life history.

Nutrients from salmon parents alter selection pressures on their offspring.

Organisms can modify their surrounding environment, but whether these changes are large enough to feed back and alter their evolutionary trajectories is not well understood, particularly in wild populations. Here we show that nutrient pulses from decomposing Atlantic salmon (Salmo salar) parents alter selection pressures on their offspring with important consequences for their phenotypic and genetic diversity. We found a strong survival advantage to larger eggs and faster juvenile metabolic rates in streams lacking carcasses but not in streams containing this parental nutrient input. Differences in selection intensities led to significant phenotypic divergence in these two traits among stream types. Stronger selection in streams with low parental nutrient input also decreased the number of surviving families compared to streams with high parental nutrient levels. Observed effects of parent-derived nutrients on selection pressures provide experimental evidence for key components of eco-evolutionary feedbacks in wild populations.

Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress.

In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H2O2) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.

Disentangling the effect of dietary restriction on mitochondrial function using recombinant inbred mice.

Dietary restriction (DR) extends lifespan and healthspan in many species, but precisely how it elicits its beneficial effects is unclear. We investigated the impact of DR on mitochondrial function within liver and skeletal muscle of female ILSXISS mice that exhibit strain-specific variation in lifespan under 40% DR. Strains TejJ89 (lifespan increased under DR), TejJ48 (lifespan unaffected by DR) and TejJ114 (lifespan decreased under DR) were studied following 10 months of 40% DR (13 months of age). Oxygen consumption rates (OCR) within isolated liver mitochondria were unaffected by DR in TejJ89 and TejJ48, but decreased by DR in TejJ114. DR had no effect on hepatic protein levels of PGC-1a, TFAM, and OXPHOS complexes IV. Mitonuclear protein imbalance (nDNA:mtDNA ratio) was unaffected by DR, but HSP90 protein levels were reduced in TejJ114 under DR. Surprisingly hepatic mitochondrial hydrogen peroxide (H2O2) production was elevated by DR in TejJ89, with total superoxide dismutase activity and protein carbonyls increased by DR in both TejJ89 and TejJ114. In skeletal muscle, DR had no effect on mitochondrial OCR, OXPHOS complexes or mitonuclear protein imbalance, but H2O2 production was decreased in TejJ114 and nuclear PGC-1a increased in TejJ89 under DR. Our findings indicate that hepatic mitochondrial dysfunction associated with reduced lifespan of TejJ114 mice under 40% DR, but similar dysfunction was not apparent in skeletal muscle mitochondria. We highlight tissue-specific differences in the mitochondrial response in ILSXISS mice to DR, and underline the importance and challenges of exploiting genetic heterogeneity to help understand mechanisms of ageing.

Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios.

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of "uncorrected" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.

Variation in Metabolic Rate among Individuals Is Related to Tissue-Specific Differences in Mitochondrial Leak Respiration.

Standard metabolic rate (SMR) and maximum metabolic rate (MMR) typically vary two- or threefold among conspecifics, with both traits assumed to significantly impact fitness. However, the underlying mechanisms that determine such intraspecific variation are not well understood. We examined the influence of mitochondrial properties on intraspecific variation in SMR and MMR and hypothesized that if SMR supports the cost of maintaining the metabolic machinery required for MMR, then the mitochondrial properties underlying these traits should be shared. Mitochondrial respiratory capacity (leak and phosphorylating respiration) and mitochondrial content (cytochrome c oxidase activity) were determined in the liver and white muscle of brown trout Salmo trutta of similar age and maintenance conditions. SMR and MMR were uncorrelated across individuals and were not associated with the same mitochondrial properties, suggesting that they are under the control of separate physiological processes. Moreover, tissue-specific relationships between mitochondrial properties and whole-organism metabolic traits were observed. Specifically, SMR was positively associated with leak respiration in liver mitochondria, while MMR was positively associated with muscle mitochondrial leak respiration and mitochondrial content. These results suggest that a high SMR or MMR, rather than signaling a higher ability for respiration-driven ATP synthesis, may actually reflect greater dissipation of energy, driven by proton leak across the mitochondrial inner membrane. Knowledge of these links should aid interpretation of the potential fitness consequences of such variation in metabolism, given the importance of mitochondria in the utilization of resources and their allocation to performance.

Male sexually coercive behaviour drives increased swimming efficiency in female guppies.

Sexual coercion of females by males is widespread across sexually reproducing species. It stems from a conflict of interest over reproduction and exerts selective pressure on both sexes. For females, there is often a significant energetic cost of exposure to male sexually coercive behaviours.Our understanding of the efficiency of female resistance to male sexually coercive behaviour is key to understanding how sexual conflict contributes to population level dynamics and ultimately to the evolution of sexually antagonistic traits.Overlooked within this context are plastic physiological responses of traits within the lifetime of females that could moderate the energetic cost imposed by coercive males. Here, we examined whether conflict over the frequency and timing of mating between male and female guppies Poecilia reticulata can induce changes in swimming performance and aerobic capacity in females as they work to escape harassment by males.Females exposed to higher levels of harassment over a 5-month period used less oxygen to swim at a given speed, but displayed no difference in resting metabolic rate, maximal metabolic rate, maximal sustained swimming speed or aerobic scope compared to females receiving lower levels of harassment.The observed increase in swimming efficiency is at least partially related to differences in swimming mechanics, likely brought on by a training effect of increased activity, as highly harassed females spent less time performing pectoral fin-assisted swimming.Sexual conflict results in sexually antagonistic traits that impose a variety of costs, but our results show that females can reduce costs through phenotypic plasticity. It is also possible that phenotypic plasticity in swimming physiology or mechanics in response to sexual coercion can potentially give females more control over matings and affect which male traits are under selection.