Summary 221. Mitochondria are the powerhouse of animal cells. They produce through oxidative 23 phosphorylation more than 90% of the cellular energy (ATP) required for organism's 24 growth, reproduction and maintenance. Hence, information on mitochondrial 25 function is expected to bring important insights in animal ecology and evolution. 26Unfortunately, the invasiveness of the procedures required to measure 27 mitochondrial function (e.g. sampling of liver or muscles) has limited its study in wild 28 vertebrate populations so far. Here, we capitalize on the fact that bird red blood cells 29 (RBCs) possess functional mitochondria to describe a minimally--invasive approach to 30 study mitochondrial function using blood samples. 31 2. In the king penguin, we present a protocol using a high--resolution respirometry 32 system and specific agonists and antagonists enabling the assessment of 33 mitochondrial function in RBCs. We evaluated the inter--assay repeatability of our 34 measures of mitochondrial function, and tested the influence of sample storage and 35 bird handling time on these measures. We also compared measures of mitochondrial 36 function in RBCs and in the pectoral muscle obtained from the same individuals. opens new opportunities to study mitochondrial function in free--living animals and 48 could bring knowledge gains in ecology and evolution. Fish, amphibians and reptiles 49 also possess mitochondria in their RBCs, and the approach presented here could also 50 be applicable to these taxa. 51 52

Metabolic activity sets the rates of individual resource uptake from the environment and resource allocations. For this reason, the relationship with body size has been heavily documented from ecosystems to cells. Until now, most of the studies used the fluxes of oxygen as a proxy of energy output without knowledge of the efficiency of biological systems to convert oxygen into ATP. The aim of this study was to examine the allometry of coupling efficiency (ATP/O) of skeletal muscle mitochondria isolated from 12 mammal species ranging from 6 g to 550 kg. Mitochondrial efficiencies were measured at different steady states of phosphorylation. The efficiencies increased sharply at higher metabolic rates. We have shown that body mass dependence of mitochondrial efficiency depends on metabolic intensity in skeletal muscles of mammals. Mitochondrial efficiency positively depends on body mass when mitochondria are close to the basal metabolic rate; however, the efficiency is independent of body mass at the maximum metabolic rate. As a result, it follows that large mammals exhibit a faster dynamic increase in ATP/O than small species when mitochondria shift from basal to maximal activities. Finally, the invariant value of maximal coupling efficiency across mammal species could partly explain why scaling exponent values are very close to 1 at maximal metabolic rates.

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