How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution

Abstract

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

Oxidative stress in aspic vipers facing pregnancy and water constraints

“…Additionally, recent work suggests that changes in mitochondrial bioenergetics and ROS production during reproduction are potential key mechanisms underlying the costs of reproduction (Zhang and Hood, 2016). Non-mammalian vertebrates like the aspic viper will offer great opportunities in the future to study such questions in a minimally invasive manner as their RBCs possess functional mitochondria, unlike those of mammalian species (Stier et al, 2013(Stier et al, , 2015a(Stier et al, , 2017. Finally, it will be important to evaluate the combined effect of water restriction and reproduction on oxidative stress levels in other species, especially in species that are less tolerant to drought than the aspic viper.…”

“…RBCs in non-mammalian vertebrates are fully functional (i.e. possess a nucleus and mitochondria, in contrast to mammalian RBCs) and can be used to gather information on several physiological mechanisms including cellular oxidative stress and mitochondrial function (Stier et al, 2013(Stier et al, , 2015a(Stier et al, , 2017. Oxidative stress markers measured in RBC lysate were normalised by the protein content of the sample, because of the difficulty of standardising the amount of RBCs being used.…”

Do carotenoid‐based ornaments entail resource trade‐offs? An evaluation of theory and data

“…A challenge central to any studies examining oxidative stress parameters is that the interplay between pro‐oxidants and antioxidants causes complex and variable fluctuation in levels of antioxidants, pro‐oxidants and oxidative damage (Costantini, ; Hõrak & Cohen, ; Monaghan, Metcalfe, & Torres, ), and such levels may vary between different locations in an individual’s body. As in many tests of immune function, studies on live animals are restricted to assessing oxidative stress from blood samples to avoid terminal experimentation (Cohen, de Magalhães, & Gohil, ), although levels of oxidative damage markers or antioxidant capacity in the blood may have limited relevance to oxidative stress in other organs (Santos, ; but see Stier et al., ). Nonetheless, given that blood is a main location of carotenoid storage and transport, measuring blood‐based oxidative stress parameters may be considered relevant to gauging carotenoid antioxidant activity.…”

“…As in many tests of immune function, studies on live animals are restricted to assessing oxidative stress from blood samples to avoid terminal experimentation (Cohen, de Magalhães, & Gohil, 2010), although levels of oxidative damage markers or antioxidant capacity in the blood may have limited relevance to oxidative stress in other organs (Santos, 2017; but see Stier et al, 2017). Nonetheless, given that blood is a main location of carotenoid storage and transport, measuring blood-based oxidative stress parameters may be considered relevant to gauging carotenoid antioxidant activity.…”

Plastic but repeatable: rapid adjustments of mitochondrial function and density during reproduction in a wild bird species

“…To' fill' this' knowledge' gap,' we' repeatedly' measured' mitochondrial' function' and' density' from' blood' cells' of' free1living' female' pied' flycatchers' (Ficedula* hypoleuca)' at' the' incubation' and' chick1rearing' stages. ' We' previously' demonstrated' that' birds' possess' functional'mitochondria'in'their'red'blood'cells'[9],'and'thus'that'mitochondrial'function'can' be' repeatedly' measured' from' the' same' individuals' using' a' minimally' invasive' repeated' blood' sampling' approach' [10]. ' Hence,' our' design' allowed' testing' whether' mitochondrial' traits' are' repeatable' within' individuals,' with' for' instance' some' individuals' having' consistently' more' mitochondria' with' greater' respiration' rates' or' efficiencies' than' others,' which' is' an' underlying' assumption' of' the' individual' quality' hypothesis.'…”