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

Elisa Thoral, Damien Roussel, Christos Chinopoulos, Loïc Teulier, Karine Salin

► To cite this version:

Elisa Thoral, Damien Roussel, Christos Chinopoulos, Loïc Teulier, Karine Salin. Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Biology Letters, 2021, 17 (2), pp.20200759. 10.1098/rsbl.2020.0759. hal-03139752

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

Elisa Thoral, Damien Roussel, Christos Chinopoulos, Loïc Teulier, Karine Salin

► To cite this version:

Elisa Thoral, Damien Roussel, Christos Chinopoulos, Loïc Teulier, Karine Salin. Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Biology Letters, 2021, 17 (2), pp.20200759. 10.1098/rsbl.2020.0759. hal-03139752

HAL Id: hal-03139752

https://univ-lyon1.hal.science/hal-03139752v1

Submitted on 17 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Research

<figure><img src="image_2.png" /></figure>

Check for
updates

**Cite this article:** Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2021 Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. *Biol. Lett.* **17**: 20200759.

https://doi.org/10.1098/rsbl.2020.0759

Received: 23 October 2020

Accepted: 21 January 2021

**Subject Areas:**

cellular biology, biochemistry, environmental science

**Keywords:**

ATP/O ratio, mitochondrial respiratory capacities, dissolved oxygen, temperature

**Author for correspondence:**

Elisa Thoral

e-mail: elisathoral@gmail.com

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare. c.5289298.

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

Elisa Thoral<fnref n="1" />, Damien Roussel<fnref n="1" />, Christos Chinopoulos<fnref n="2" />, Loïc Teulier<fnref n="1" /> and Karine Salin<fnref n="3" />

<figure><img src="image_3.png" /></figure>

Aerobic metabolism of aquatic ectotherms is highly sensitive to fluctuating cli-
mates. 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 temp-
erature. 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. Predict-
tions on decline in performance of aquatic ectotherms owing to climate
change may not be accurate, since these predictions typically look at respirat-
ory capacity and ignore efficiency of ATP production.

**1. Introduction**

Temperature exerts profound effects on the performance of ectothermic
animals, partly through its effect on metabolism. The issue is particularly
severe for aquatic ectotherms since water warming also results in a decrease
in dissolved oxygen concentration [1]. Oxygen depletion is often more dramatic
for marine organisms that face exacerbated eutrophication-induced deoxygena-
tion in most estuarine and coastal habitats [2]. Rising temperature causes an
increase in aerobic metabolism but the lower oxygen availability might lead
to a point where the organism has insufficient oxygen to supply to mito-
chondria and meet metabolic demands [3–6]. In the long term, many species
can remodel their mitochondrial metabolism, and this acclimation response
can help to maintain cell energy homeostasis across environmental conditions
[7,8]. Generally, capacity for acclimation to mean warming and oxygen
depletion is considered as a key feature determining resilience to climate
change [9]. However, an increase in the frequency and magnitude of extreme
temperature events, such as heat waves [10], is likely to be biologically more
significant [11]. Because chemical reactions are inherently sensitive to tempera-
ture and substrate availability (e.g. oxygen) on an acute time scale, we contend

<footnote n="1">¹Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France</footnote>

<footnote n="2">²Department of Medical Biochemistry, Semmelweis University, Budapest, 1094, Hungary</footnote>

<footnote n="3">³Université de Brest, Ifremer, CNRS, IRD, Laboratory of Environmental Marine Sciences, Plouzané 29280, France</footnote>

<footnote>ID ET, 0000-0001-5218-9907; DR, 0000-0002-8865-5428; CC, 0000-0003-0183-4149; LT, 0000-0001-7779-7634; KS, 0000-0002-3368-9639</footnote>
Research

<figure><img src="image_2.png" /></figure>

Check for
updates

**Cite this article:** Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2021 Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. *Biol. Lett.* **17**: 20200759.

https://doi.org/10.1098/rsbl.2020.0759

Received: 23 October 2020

Accepted: 21 January 2021

**Subject Areas:**

cellular biology, biochemistry, environmental science

**Keywords:**

ATP/O ratio, mitochondrial respiratory capacities, dissolved oxygen, temperature

**Author for correspondence:**

Elisa Thoral

e-mail: elisathoral@gmail.com

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare. c.5289298.

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

Elisa Thoral<fnref n="1" />, Damien Roussel<fnref n="1" />, Christos Chinopoulos<fnref n="2" />, Loïc Teulier<fnref n="1" /> and Karine Salin<fnref n="3" />

<figure><img src="image_3.png" /></figure>

Aerobic metabolism of aquatic ectotherms is highly sensitive to fluctuating cli-
mates. 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 temp-
erature. 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. Predict-
tions on decline in performance of aquatic ectotherms owing to climate
change may not be accurate, since these predictions typically look at respirat-
ory capacity and ignore efficiency of ATP production.

**1. Introduction**

Temperature exerts profound effects on the performance of ectothermic
animals, partly through its effect on metabolism. The issue is particularly
severe for aquatic ectotherms since water warming also results in a decrease
in dissolved oxygen concentration [1]. Oxygen depletion is often more dramatic
for marine organisms that face exacerbated eutrophication-induced deoxygena-
tion in most estuarine and coastal habitats [2]. Rising temperature causes an
increase in aerobic metabolism but the lower oxygen availability might lead
to a point where the organism has insufficient oxygen to supply to mito-
chondria and meet metabolic demands [3–6]. In the long term, many species
can remodel their mitochondrial metabolism, and this acclimation response
can help to maintain cell energy homeostasis across environmental conditions
[7,8]. Generally, capacity for acclimation to mean warming and oxygen
depletion is considered as a key feature determining resilience to climate
change [9]. However, an increase in the frequency and magnitude of extreme
temperature events, such as heat waves [10], is likely to be biologically more
significant [11]. Because chemical reactions are inherently sensitive to tempera-
ture and substrate availability (e.g. oxygen) on an acute time scale, we contend

<footnote n="1">¹Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France</footnote>

<footnote n="2">²Department of Medical Biochemistry, Semmelweis University, Budapest, 1094, Hungary</footnote>

<footnote n="3">³Université de Brest, Ifremer, CNRS, IRD, Laboratory of Environmental Marine Sciences, Plouzané 29280, France</footnote>

<footnote>ID ET, 0000-0001-5218-9907; DR, 0000-0002-8865-5428; CC, 0000-0003-0183-4149; LT, 0000-0001-7779-7634; KS, 0000-0002-3368-9639</footnote>
that acute metabolic response on a short time scale is critical
for understanding the effects of climate extremes on the
performance of aquatic ectotherms [12].

Changes in mitochondrial metabolism may have
important consequences for an organism's ability to meet
metabolic demands during acute warming and a decline in
oxygen availability. This is because mitochondrial metab-
olism not only defines the cellular oxygen needs but also
determines cells' ability to produce ATP molecules that fuel
nearly all the important functions such as muscle contraction,
growth and reproduction. Oxygen consumption related to
ATP production (OXPHOS respiration) by mitochondria
exposed to acute warming *in vitro* increases exponentially
[13] (until mitochondria are no longer functional [14]). The
activities of enzymes associated with respiratory processes
are typically higher under acute warming conditions owing
to the thermodynamics of molecular movements [8]. For
example, cytochrome c oxidase (COX) activity, a measure of
the maximal oxidative capacities of the final electron accep-
tor, increases of 1.56-fold and 2-fold with a 7°C and a 10°C
temperature increase in the skeletal muscle mitochondria
of rat [15] and crocodile [16], respectively. Warming con-
ditions also lead to changes in other mitochondrial traits
affecting ATP production, including the proportion of
oxygen consumed to counteract the proton leakage across
the mitochondrial inner membrane (LEAK respiration) and
the proportion of oxygen consumed for ATP production.
During a transient temperature increase, such as episodic
heat waves, the increase in oxygen dissipated in proton
leak may lead to a decrease in ATP production, unless the
oxygen consumption related to ATP production increases
by an equivalent or greater amount [17]. Importantly, in
warming aquatic environments the ability to produce ATP
might no longer be compensated by the capacity to take up
higher amounts of oxygen, since there is likely not enough
dissolved oxygen available.

Shifts in the ability of mitochondria to make ATP
are another important mechanism for organisms to cover
their ATP needs. The number of ATP molecules produced
for each oxygen consumed by the mitochondria (termed the
ATP/O ratio), in addition to the proportion of oxygen con-
sumed for ATP production, can acutely vary [18,19].
Previous studies have demonstrated that the ATP/O ratio
*in vitro* decreases when the temperature is acutely raised
[20] and increases when oxygen levels decline [21]. While
these studies suggest that ATP/O ratio can be drastically
modulated with acute warming and oxygen depletion in
the opposite direction, combined effects of temperature and
oxygen remain largely unexplored although this would
have important implications for aquatic ectotherms in a
more thermally variable future.

Our aim was to investigate the acute effects of warming
and oxygen depletion on fish mitochondria *in vitro*. We
use a fully factorial approach to examine acute effects of
temperature and oxygen availability on the mitochondrial
respiratory capacities (OXPHOS and LEAK respirations,
and COX activity), ATP production and efficiency to produce
ATP (ATP/O ratio) of the red muscle of European seabass
*Dicentrarchus labrax*. We predict that low dissolved oxygen
concentration may have a compensatory effect on mitochon-
drial efficiency at high temperature, whereby the changes in
ATP/O ratio with warming are counteracted by changes
in ATP/O ratio with deoxygenation.

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Red muscle mitochondrial efficiency (ATP/O ratio) of European seabass (*Dicentrarchus labrax*) as a function of temperature and oxygen availability. Means ± s.e.m. are shown. *N* = 8 fish per condition. Letters represent a significant difference between testing conditions (*p* < 0.05).</figcaption></figure>

2. Material and methods

(a) Animals

Juvenile seabass were moved from the hatchery Les Poissons du Soleil (Balaruc-les-Bains, France) to Ifremer, Plouzané, in April 2017. The fish were kept in a communal tank, fed with commercial pellets (Le Gouessant, Lamballe), with a photoperiod of 12:12 and a water temperature following the daily mean natural temperature in Brest harbour. In April 2019, juvenile fish were culled (n = 8, body mass: mean ± s.e.m. = 163.3 ± 11.3 g) and samples of red muscle (13.4 ± 0.8 mg) were immediately collected and kept in ice-cold respirometry buffer for analysis of mitochondrial properties (see the electronic supplementary material for details on animal maintenance, culling of the fish and buffer composition).

(b) Mitochondrial respiratory capacities and efficiency

Muscle tissue was homogenized and replicate samples of
homogenate were then added to four chambers of high-resolution
respirometers equipped with fluorescent sensors (Oroboros
Instruments, Innsbruck, Austria). Oxygen and magnesium
green fluorescence were detected simultaneously as in [22,23].
Mitochondria were measured simultaneously for each fish in a
fully factorial acute test temperature and oxygen availability
design: 13°C/High [O₂] (552.4 ± 51.3 µM), 13°C/Low [O₂]
(288.3 ± 5.4 µM), 16°C/High [O₂] (557.7 ± 33.2 µM) and 16°C/
Low [O₂] (263.4 ± 7.6 µM). After addition of homogenate to respi-
rometers at 13°C or 16°C, pure oxygen gas was added into two
High [O₂] chambers to double the concentration compared to
Low [O₂] chambers. The 3°C warming of the mitochondrial
assay simulated the acute warming conditions juvenile seabass
would naturally experience at this life stage (3.06 ± 0.09°C [24]).
The 13°C experimental condition corresponded to the water
temperature of fish tanks at the time of the tissue sampling
(12.8°C ± 0.3°C) and to which mitochondria were acclimatized.
See electronic supplementary material for full details of the
experimental conditions of the mitochondrial assay.

The rates of OXPHOS and ATP production were assessed
by adding ADP to the chamber containing energy substrates
and magnesium green (see electronic supplementary material).
that acute metabolic response on a short time scale is critical for understanding the effects of climate extremes on the performance of aquatic ectotherms [12].

Changes in mitochondrial metabolism may have important consequences for an organism's ability to meet metabolic demands during acute warming and a decline in oxygen availability. This is because mitochondrial metabolism not only defines the cellular oxygen needs but also determines cells' ability to produce ATP molecules that fuel nearly all the important functions such as muscle contraction, growth and reproduction. Oxygen consumption related to ATP production (OXPHOS respiration) by mitochondria exposed to acute warming *in vitro* increases exponentially [13] (until mitochondria are no longer functional [14]). The activities of enzymes associated with respiratory processes are typically higher under acute warming conditions owing to the thermodynamics of molecular movements [8]. For example, cytochrome c oxidase (COX) activity, a measure of the maximal oxidative capacities of the final electron acceptor, increases of 1.56-fold and 2-fold with a 7°C and a 10°C temperature increase in the skeletal muscle mitochondria of rat [15] and crocodile [16], respectively. Warming conditions also lead to changes in other mitochondrial traits affecting ATP production, including the proportion of oxygen consumed to counteract the proton leakage across the mitochondrial inner membrane (LEAK respiration) and the proportion of oxygen consumed for ATP production. During a transient temperature increase, such as episodic heat waves, the increase in oxygen dissipated in proton leak may lead to a decrease in ATP production, unless the oxygen consumption related to ATP production increases by an equivalent or greater amount [17]. Importantly, in warming aquatic environments the ability to produce ATP might no longer be compensated by the capacity to take up higher amounts of oxygen, since there is likely not enough dissolved oxygen available.

Shifts in the ability of mitochondria to make ATP are another important mechanism for organisms to cover their ATP needs. The number of ATP molecules produced for each oxygen consumed by the mitochondria (termed the ATP/O ratio), in addition to the proportion of oxygen consumed for ATP production, can acutely vary [18,19]. Previous studies have demonstrated that the ATP/O ratio *in vitro* decreases when the temperature is acutely raised [20] and increases when oxygen levels decline [21]. While these studies suggest that ATP/O ratio can be drastically modulated with acute warming and oxygen depletion in the opposite direction, combined effects of temperature and oxygen remain largely unexplored although this would have important implications for aquatic ectotherms in a more thermally variable future.

Our aim was to investigate the acute effects of warming and oxygen depletion on fish mitochondria *in vitro*. We use a fully factorial approach to examine acute effects of temperature and oxygen availability on the mitochondrial respiratory capacities (OXPHOS and LEAK respirations, and COX activity), ATP production and efficiency to produce ATP (ATP/O ratio) of the red muscle of European seabass *Dicentrarchus labrax*. We predict that low dissolved oxygen concentration may have a compensatory effect on mitochondrial efficiency at high temperature, whereby the changes in ATP/O ratio with warming are counteracted by changes in ATP/O ratio with deoxygenation.

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Red muscle mitochondrial efficiency (ATP/O ratio) of European seabass (*Dicentrarchus labrax*) as a function of temperature and oxygen availability. Means ± s.e.m. are shown. *N* = 8 fish per condition. Letters represent a significant difference between testing conditions (*p* < 0.05).</figcaption></figure>

## 2. Material and methods

### (a) Animals

Juvenile seabass were moved from the hatchery Les Poissons du Soleil (Balaruc-les-Bains, France) to Ifremer, Plouzané, in April 2017. The fish were kept in a communal tank, fed with commercial pellets (Le Gouessant, Lamballe), with a photoperiod of 12:12 and a water temperature following the daily mean natural temperature in Brest harbour. In April 2019, juvenile fish were culled (*n* = 8, body mass: mean ± s.e.m. = 163.3 ± 11.3 g) and samples of red muscle (13.4 ± 0.8 mg) were immediately collected and kept in ice-cold respirometry buffer for analysis of mitochondrial properties (see the electronic supplementary material for details on animal maintenance, culling of the fish and buffer composition).

### (b) Mitochondrial respiratory capacities and efficiency

Muscle tissue was homogenized and replicate samples of homogenate were then added to four chambers of high-resolution respirometers equipped with fluorescent sensors (Oroboros Instruments, Innsbruck, Austria). Oxygen and magnesium green fluorescence were detected simultaneously as in [22,23]. Mitochondria were measured simultaneously for each fish in a fully factorial acute test temperature and oxygen availability design: 13°C/High [O₂] (552.4 ± 51.3 µM), 13°C/Low [O₂] (288.3 ± 5.4 µM), 16°C/High [O₂] (557.7 ± 33.2 µM) and 16°C/Low [O₂] (263.4 ± 7.6 µM). After addition of homogenate to respirometers at 13°C or 16°C, pure oxygen gas was added into two High [O₂] chambers to double the concentration compared to Low [O₂] chambers. The 3°C warming of the mitochondrial assay simulated the acute warming conditions juvenile seabass would naturally experience at this life stage (3.06 ± 0.09°C [24]). The 13°C experimental condition corresponded to the water temperature of fish tanks at the time of the tissue sampling (12.8°C ± 0.3°C) and to which mitochondria were acclimatized. See electronic supplementary material for full details of the experimental conditions of the mitochondrial assay.

The rates of OXPHOS and ATP production were assessed by adding ADP to the chamber containing energy substrates and magnesium green (see electronic supplementary material).
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Effect of temperature and oxygen availability on red muscle mitochondrial properties of European seabass (*Dicentrarchus labrax*): (a) rate of oxygen consumption associated with ATP synthesis (OXPHOS respiration), (b) rate of ATP production, (c) rate of oxygen consumption to offset the proton leak (LEAK respiration) and (d) cytochrome c oxidase (COX) activity. Means ± s.e.m. are shown. *N* = 8 fish per condition. Letters represent a significant difference between testing conditions (*p* < 0.05).</figcaption></figure>

The rate of LEAK respiration was then measured by inhibiting ATP synthesis through the addition of carboxyatractyloside and COX activity was measured after addition of ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine. Finally, the ATP/O ratio was calculated as the ratio of ATP production to OXPHOS. See electronic supplementary material for full details of the protocol.

### (c) Statistical analysis

We analysed the effects of temperature and oxygen conditions on mitochondrial function using repeated-measures two-way ANOVAs and Tukey adjusted post hoc tests. The ANOVAs included mitochondrial properties (i.e. ATP/O ratio, OXPHOS respiration, ATP production, LEAK respiration and COX activity) as dependent variables, and temperature (13°C and 16°C) and oxygen availability (Low and High) as categorical predictors, and two-way interactions between temperature and oxygen levels. The level of significance was *p* < 0.05. All data are presented as means ± s.e.m. Statistical analyses were performed in *R* v. 3.6.1 with the packages *lme4* and *lsmeans*.

## 3. Results

The ATP/O ratio significantly decreased with acute warming (*F*<sub>1,21</sub> = 11.7, *p* = 0.003) and increased slightly—but not significantly—with acute deoxygenation (*F*<sub>1,21</sub> = 4.1, *p* = 0.057). As a result, mitochondria tested at 13°C/High [O<sub>2</sub>] and 16°C/Low [O<sub>2</sub>] did not differ with respect to ATP/O ratio (Tukey adjusted post hoc test: *p* = 0.759, figure 1).

OXPHOS respiration (*F*<sub>1,21</sub> = 40.1, *p* < 0.001), ATP production (*F*<sub>1,21</sub> = 8.6, *p* = 0.008), LEAK respiration (*F*<sub>1,21</sub> = 29.1, *p* < 0.001) and COX activity (*F*<sub>1,21</sub> = 35.5, *p* < 0.001, figure 2) increased with temperature. However, the effects of deoxygenation and combined warming and deoxygenation on mitochondrial responses differed among traits. OXPHOS respiration did not change with oxygen levels (*F*<sub>1,21</sub> = 1.7, *p* = 0.205), ATP production tended to increase with deoxygenation, but this difference was marginally not significant (*F*<sub>1,21</sub> = 3.9, *p* = 0.061, figure 2b), LEAK respiration and COX activity decreased with oxygen depletion (LEAK: *F*<sub>1,21</sub> = 11.9, *p* = 0.002; COX activity: *F*<sub>1,21</sub> = 85.9, *p* < 0.001). Finally,
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Effect of temperature and oxygen availability on red muscle mitochondrial properties of European seabass (*Dicentrarchus labrax*): (a) rate of oxygen consumption associated with ATP synthesis (OXPHOS respiration), (b) rate of ATP production, (c) rate of oxygen consumption to offset the proton leak (LEAK respiration) and (d) cytochrome c oxidase (COX) activity. Means ± s.e.m. are shown. *N* = 8 fish per condition. Letters represent a significant difference between testing conditions (*p* < 0.05).</figcaption></figure>

The rate of LEAK respiration was then measured by inhibiting
ATP synthesis through the addition of carboxyatractyloside
and COX activity was measured after addition of ascorbate
and N,N,N',N'-tetramethyl-p-phenylenediamine. Finally, the
ATP/O ratio was calculated as the ratio of ATP production to
OXPHOS. See electronic supplementary material for full details
of the protocol.

(c) Statistical analysis

We analysed the effects of temperature and oxygen conditions on
mitochondrial function using repeated-measures two-way
ANOVAs and Tukey adjusted post hoc tests. The ANOVAs
included mitochondrial properties (i.e. ATP/O ratio, OXPHOS
respiration, ATP production, LEAK respiration and COX activity)
as dependant variables, and temperature (13°C and 16°C) and
oxygen availability (Low and High) as categorical predictors,
and two-way interactions between temperature and oxygen
levels. The level of significance was *p* < 0.05. All data are pre-
sented as means ± s.e.m. Statistical analyses were performed in
*R* v. 3.6.1 with the packages *lme4* and *lsmeans*.

3. Results

The ATP/O ratio significantly decreased with acute warming (*F*<sub>1,21</sub> = 11.7, *p* = 0.003) and increased slightly—but not significantly—with acute deoxygenation (*F*<sub>1,21</sub> = 4.1, *p* = 0.057). As a result, mitochondria tested at 13°C/High [O<sub>2</sub>] and 16°C/Low [O<sub>2</sub>] did not differ with respect to ATP/O ratio (Tukey adjusted post hoc test: *p* = 0.759, figure 1).

OXPHOS respiration (*F*<sub>1,21</sub> = 40.1, *p* < 0.001), ATP pro-
duction (*F*<sub>1,21</sub> = 8.6, *p* = 0.008), LEAK respiration (*F*<sub>1,21</sub> = 29.1,
*p* < 0.001) and COX activity (*F*<sub>1,21</sub> = 35.5, *p* < 0.001, figure 2)
increased with temperature. However, the effects of deoxy-
genation and combined warming and deoxygenation on
mitochondrial responses differed among traits. OXPHOS
respiration did not change with oxygen levels (*F*<sub>1,21</sub> = 1.7,
*p* = 0.205), ATP production tended to increase with deoxy-
genation, but this difference was marginally not significant
(*F*<sub>1,21</sub> = 3.9, *p* = 0.061, figure 2b), LEAK respiration and COX
activity decreased with oxygen depletion (LEAK: *F*<sub>1,21</sub> =
11.9, *p* = 0.002; COX activity: *F*<sub>1,21</sub> = 85.9, *p* < 0.001). Finally,
OXPHOS respiration and ATP production in mitochondria tested at 16°C/Low [O₂] were significantly higher than those in mitochondria tested at 13°C/High [O₂] (Tukey adjusted post hoc test: OXPHOS respiration: *p* < 0.001; ATP production: *p* = 0.011, figure 2a,b), while there was no difference in LEAK respiration and COX activity between mitochondria tested at 16°C/Low [O₂] and 13°C/High [O₂] (Tukey adjusted post hoc test: LEAK: *p* = 0.53; COX activity: *p* = 0.12, figure 2c,d).

## 4. Discussion

The key result from our study is that plasticity of the mitochondrial metabolism has the capacity to compensate for the acute warming effect on ATP/O ratio when oxygen availability declines. Acute warming decreased ATP/O ratio but deoxygenation marginally raised ATP/O ratio. As a result, mitochondria under high temperature and low oxygen availability operate as efficiently as mitochondria under low temperature and high oxygen availability. The predicted decline in the performance of aquatic ectotherms owing to acute warming and oxygen depletion may be not as severe, since the predictions typically look at respiratory capacity and ignore ATP synthesis capacities and efficiencies ([7,25] but see [17]).

The vast majority of the ATP present in organisms is generated by mitochondria and can only be measured through *in vitro* assays of the rate of ATP production ([22] but see [26]). However, these assays measure mitochondrial ATP generated under highly controlled conditions. Thus, there are benefits and limitations to phenotype mitochondrial metabolism *in vitro*. On the one hand, while previous studies generally describe the response of mitochondrial properties to very high change in assay temperature, the 3°C warming used in our study might be more biologically relevant acute warming in relation to the seabass environment. This warming was defined based on recorded extremes of daily thermal range over the past decade in Brest Harbour [24], where juvenile seabass naturally live. Because variation in the daily thermal ranges is predicted to increase in both frequency and intensity as a result of climate change [10], quantifying mitochondrial responses at extended thermal range may also be warranted. On the other hand, assessing mitochondrial properties in response to the warming-induced decline in oxygen availability would require partial pressures of oxygen set to *in vivo* levels at 13°C and 16°C during measurement of mitochondrial properties. These considerations can quickly become logistically challenging as the difference in cellular oxygen levels *in vivo* for a 3°C warming is difficult to reach with the equipment suitable for ATP/O ratio assay in fish mitochondria. There is a need to develop more sophisticated techniques for measuring mitochondrial metabolism to extrapolate *in vitro* results to the *in vivo* situation.

It has recently become apparent that mitochondrial efficiency has a strong positive effect on organism performance, such as locomotor ability [27], feeding efficiency [28] and growth performance [29,30]. Understanding the scope for mitochondrial plasticity has then become critical for predicting how populations can respond to environmental change [31,32]. Our study is the first to indicate compensation of the combined effects of deoxygenation and warming on mitochondrial ATP/O ratio. The decrease in the ATP/O ratio in response to warming and the increase in ATP/O ratio in response to deoxygenation have previously been observed independently in mitochondria from birds [33] and rats [21]. Our study therefore underlines the importance of integrating the combined effects of temperature and oxygen availability on mitochondrial metabolism. Interestingly, trends for increase in ATP/O ratio in response to deoxygenation suggest that mitochondrial efficiency in the red muscle of seabass is controlled by a corresponding decline in LEAK respiration. An increase in ATP/O ratio with decreasing LEAK respiration may therefore represent a decrease in the proportion on energy dissipated through proton leakage across the inner mitochondrial membrane, and in turn increasing that energy in ATP synthesis [18]. These *in vitro* measurements nonetheless are likely to indicate the direction of the mitochondrial responses in the live animal [17]. This is particularly relevant in ectotherms where the temperature and oxygen conditions that the mitochondria are exposed to can vary within the animal on a short time scale. Our results suggest that animal performance under ongoing climate change will likely depend on their plasticity in the aerobic metabolism, especially the change in the efficiency of their mitochondria in producing ATP.

**Ethics.** Use of animals was in accordance with directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. All procedures were carried out under the jurisdiction of the French Department of Veterinary Services (license no. B 29-212-05).

**Data accessibility.** Data are available via Dryad Digital Repository: https://doi.org/10.5061/dryad.dfn2z34zk. [34]. Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2020 Data from: Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Dryad Digital Repository. (doi:10.5061/dryad.dfn2z34zk)

**Authors' contributions.** E.T. and K.S. conceived the study and conducted the experiment; all authors analysed and interpreted the data. E.T. and K.S. led the writing of the manuscript. L.T., D.R. and C.C. revised the manuscript and added comments; all authors approved the final version of the manuscript and agree to be held accountable for the work performed.

**Competing interests.** We declare we have no competing interests.

**Funding.** This work was supported by the French Government (to E.T., PhD grant—2018–2021) and by the Research Federation ‘Bio-Environnement et Santé’ (Université Claude Bernard Lyon 1) with the mobility grant that enabled E.T. to travel to Brittany for the project.

**Acknowledgements.** We thank M. Hervy and S. Collet for the initial raising of the fish used in this experiment.

## References

1. Pörtner H. 2001 Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. *Naturwissenschaften* **88**, 137–146. (doi:10.1007/s001140100216)

2. Diaz RJ, Rosenberg R. 2008 Spreading dead zones and consequences for marine ecosystems. *Science* **321**, 926–929. (doi:10.1126/science.1156401)

3. Tattersall GJ, Sinclair BJ, Withers PC, Fields PA, Seebacher F, Cooper CE, Maloney SK. 2012 Coping with thermal challenges: physiological adaptations to environmental temperatures.
OXPHOS respiration and ATP production in mitochondria tested at 16°C/Low [O₂] were significantly higher than those in mitochondria tested at 13°C/High [O₂] (Tukey adjusted post hoc test: OXPHOS respiration: *p* < 0.001; ATP production: *p* = 0.011, figure 2a,b), while there was no difference in LEAK respiration and COX activity between mitochondria tested at 16°C/Low [O₂] and 13°C/High [O₂] (Tukey adjusted post hoc test: LEAK: *p* = 0.53; COX activity: *p* = 0.12, figure 2c,d).

## 4. Discussion

The key result from our study is that plasticity of the mitochondrial metabolism has the capacity to compensate for the acute warming effect on ATP/O ratio when oxygen availability declines. Acute warming decreased ATP/O ratio but deoxygenation marginally raised ATP/O ratio. As a result, mitochondria under high temperature and low oxygen availability operate as efficiently as mitochondria under low temperature and high oxygen availability. The predicted decline in the performance of aquatic ectotherms owing to acute warming and oxygen depletion may be not as severe, since the predictions typically look at respiratory capacity and ignore ATP synthesis capacities and efficiencies ([7,25] but see [17]).

The vast majority of the ATP present in organisms is generated by mitochondria and can only be measured through *in vitro* assays of the rate of ATP production ([22] but see [26]). However, these assays measure mitochondrial ATP generated under highly controlled conditions. Thus, there are benefits and limitations to phenotype mitochondrial metabolism *in vitro*. On the one hand, while previous studies generally describe the response of mitochondrial properties to very high change in assay temperature, the 3°C warming used in our study might be more biologically relevant acute warming in relation to the seabass environment. This warming was defined based on recorded extremes of daily thermal range over the past decade in Brest Harbour [24], where juvenile seabass naturally live. Because variation in the daily thermal ranges is predicted to increase in both frequency and intensity as a result of climate change [10], quantifying mitochondrial responses at extended thermal range may also be warranted. On the other hand, assessing mitochondrial properties in response to the warming-induced decline in oxygen availability would require partial pressures of oxygen set to *in vivo* levels at 13°C and 16°C during measurement of mitochondrial properties. These considerations can quickly become logistically challenging as the difference in cellular oxygen levels *in vivo* for a 3°C warming is difficult to reach with the equipment suitable for ATP/O ratio assay in fish mitochondria. There is a need to develop more sophisticated techniques for measuring mitochondrial metabolism to extrapolate *in vitro* results to the *in vivo* situation.

It has recently become apparent that mitochondrial efficiency has a strong positive effect on organism performance, such as locomotor ability [27], feeding efficiency [28] and growth performance [29,30]. Understanding the scope for mitochondrial plasticity has then become critical for predicting how populations can respond to environmental change [31,32]. Our study is the first to indicate compensation of the combined effects of deoxygenation and warming on mitochondrial ATP/O ratio. The decrease in the ATP/O ratio in response to warming and the increase in ATP/O ratio in response to deoxygenation have previously been observed independently in mitochondria from birds [33] and rats [21]. Our study therefore underlines the importance of integrating the combined effects of temperature and oxygen availability on mitochondrial metabolism. Interestingly, trends for increase in ATP/O ratio in response to deoxygenation suggest that mitochondrial efficiency in the red muscle of seabass is controlled by a corresponding decline in LEAK respiration. An increase in ATP/O ratio with decreasing LEAK respiration may therefore represent a decrease in the proportion on energy dissipated through proton leakage across the inner mitochondrial membrane, and in turn increasing that energy in ATP synthesis [18]. These *in vitro* measurements nonetheless are likely to indicate the direction of the mitochondrial responses in the live animal [17]. This is particularly relevant in ectotherms where the temperature and oxygen conditions that the mitochondria are exposed to can vary within the animal on a short time scale. Our results suggest that animal performance under ongoing climate change will likely depend on their plasticity in the aerobic metabolism, especially the change in the efficiency of their mitochondria in producing ATP.

**Ethics.** Use of animals was in accordance with directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. All procedures were carried out under the jurisdiction of the French Department of Veterinary Services (license no. B 29-212-05).

**Data accessibility.** Data are available via Dryad Digital Repository: https://doi.org/10.5061/dryad.dfn2z34zk. [34]. Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2020 Data from: Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Dryad Digital Repository. (doi:10.5061/dryad.dfn2z34zk)

**Authors' contributions.** E.T. and K.S. conceived the study and conducted the experiment; all authors analysed and interpreted the data. E.T. and K.S. led the writing of the manuscript. L.T., D.R. and C.C. revised the manuscript and added comments; all authors approved the final version of the manuscript and agree to be held accountable for the work performed.

**Competing interests.** We declare we have no competing interests.

**Funding.** This work was supported by the French Government (to E.T., PhD grant—2018–2021) and by the Research Federation ‘Bio-Environnement et Santé’ (Université Claude Bernard Lyon 1) with the mobility grant that enabled E.T. to travel to Brittany for the project.

**Acknowledgements.** We thank M. Hervy and S. Collet for the initial raising of the fish used in this experiment.

## References

1. Pörtner H. 2001 Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. *Naturwissenschaften* **88**, 137–146. (doi:10.1007/s001140100216)

2. Diaz RJ, Rosenberg R. 2008 Spreading dead zones and consequences for marine ecosystems. *Science* **321**, 926–929. (doi:10.1126/science.1156401)

3. Tattersall GJ, Sinclair BJ, Withers PC, Fields PA, Seebacher F, Cooper CE, Maloney SK. 2012 Coping with thermal challenges: physiological adaptations to environmental temperatures.
Compr. Physiol. **2**, 2151–2202. (doi:10.1002/cphy-c.110055)

4. Abe TK, Kitagawa T, Makiguchi Y, Sato K. 2019 Chum salmon migrating upriver adjust to environmental temperatures through metabolic compensation. *J. Exp. Biol.* **222**, jeb186189. (doi:10.1242/jeb.186189)

5. Richards JG. 2011 Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. *J. Exp. Biol.* **214**, 191–199. (doi:10.1242/jeb.047951)

6. Zhou BS, Wu RSS, Randall DJ, Lam PKS, Ip YK, Chew SF. 2000 Metabolic adjustments in the common carp during prolonged hypoxia. *J. Fish Biol.* **57**, 1160–1171. (doi:10.1006/jfbi.2000.1382)

7. Khan JR, Iftikar FI, Herbert NA, Gnaiger E, Hickey AJR. 2014 Thermal plasticity of skeletal muscle mitochondrial activity and whole animal respiration in a common intertidal triplefin fish, *Forsterygion lapillum* (Family: Tripterygiidae). *J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.* **184**, 991–1001. (doi:10.1007/s00360-014-0861-9)

8. 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. (doi:10.1242/jeb.118851)

9. Iftikar FI, MacDonald JR, Baker DW, Renshaw GMC, Hickey AJR. 2014 Could thermal sensitivity of mitochondria determine species distribution in a changing climate? *J. Exp. Biol.* **217**, 2348–2357. (doi:10.1242/jeb.098798)

10. Thompson RM, Beardall J, Beringer J, Grace M, Sardina P. 2013 Means and extremes: building variability into community-level climate change experiments. *Ecol. Lett.* **16**, 799–806. (doi:10.1111/ele.12095)

11. Butcher JB, Covington S. 1995 Dissolved-oxygen analysis with temperature dependence. *J. Environ. Eng. (United States)* **121**, 756–759. (doi:10.1061/(ASCE)0733-9372(1995)121:10(756)

12. Havird JC, Neuwald JL, Shah AA, Mauro A, Marshall CA, Ghalambor CK. 2020 Distinguishing between active plasticity due to thermal acclimation and passive plasticity due to Q₁₀ effects: why methodology matters. *Funct. Ecol.* **34**, 1015–1028. (doi:10.1111/1365-2435.13534)

13. Hilton Z, Clements KD, Hickey AJR. 2010 Temperature sensitivity of cardiac mitochondria in intertidal and subtidal triplefin fishes. *J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.* **180**, 979–990. (doi:10.1007/s00360-010-0477-7)

14. Schulte PM, Healy TM, Fangue NA. 2011 Thermal performance curves, phenotypic plasticity, and the time scales of temperature exposure. *Integr. Comp. Biol.* **51**, 691–702. (doi:10.1093/icb/icr097)

15. Jarmuszkiewicz W, Woyda-Ploszczyca A, Koziel A, Majerczak J, Zoladz JA. 2015 Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria. *Free Radic. Biol. Med.* **83**, 12–20. (doi:10.1016/j.freeradbiomed.2015.02.012)

16. Seebacher F, James RS. 2008 Plasticity of muscle function in a thermoregulating ectotherm (*Crocodylus porosus*): biomechanics and metabolism. *Am. J. Physiol. - Regul. Integr. Comp. Physiol.* **294**, 1024–1032. (doi:10.1152/ajpregu.00755.2007)

17. Iftikar FI, Hickey AJR. 2013 Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in *Notolabrus celidotus*. *PLoS ONE* **8**, e64120. (doi:10.1371/journal.pone.0064120)

18. Brand M. 2005 The efficiency and plasticity of mitochondrial energy transduction. *Biochem. Soc. Trans.* **33**, 897–904. (doi:10.1042/BST20050897)

19. Salin K, Auer SK, Rey B, Selman C, Metcalfe NB. 2015 Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance. *Proc. R. Soc. B* **282**, 20151028. (doi:10.1098/rspb.2015.1028)

20. Pörtner HO, Hardewig I, Peck LS. 1999 Mitochondrial function and critical temperature in the Antarctic bivalve, *Laternula elliptica*. *Comp. Biochem. Physiol. A: Mol. Integr. Physiol.* **124**, 179–189. (doi:10.1016/S1095-6433(99)00105-1)

21. Gnaiger E, Méndez G, Hand SC. 2000 High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. *Proc. Natl Acad. Sci. USA* **97**, 11 080–11 085. (doi:10.1073/pnas.97.20.11080)

22. Salin K, Villasevil EM, Auer SK, Anderson GJ, Selman C, Metcalfe NB, Chinopoulos C. 2016 Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios. *Physiol. Rep.* **4**, 1–9. (doi:10.14814/phy2.13007)

23. Salin K, Auer SK, Rudolf AM, Anderson GJ, Selman C, Metcalfe NB. 2016 Variation in metabolic rate among individuals is related to tissue-specific differences in mitochondrial leak respiration. *Physiol. Biochem. Zool.* **89**, 511–523. (doi:10.1086/688769)

24. Petton S, Le Roy V, Bellec G, Queau I, Le Souchu P, Pouvreau S. 2020 Marine environmental station database of Daoulas bay. *SEANOE*. (doi:10.17882/42493)

25. Fangue NA, Richards JG, Schulte PM. 2009 Do mitochondrial properties explain intraspecific variation in thermal tolerance? *J. Exp. Biol.* **212**, 514–522. (doi:10.1242/jeb.024034)

26. Stubbs M, Van Den Boogaart A, Bashford CL, Miranda PMC, Rodrigues LM, Howe FA, Griffiths JR. 1996 ³¹P-Magnetic resonance spectroscopy studies of nucleated and non-nucleated erythrocytes; time domain data analysis (VARPRO) incorporating prior knowledge can give information on the binding of ADP. *Biochim. Biophys. Acta - Gen. Subj.* **1291**, 143–148. (doi:10.1016/0304-4165(96)00058-X)

27. Conley KE. 2016 Mitochondria to motion: optimizing oxidative phosphorylation to improve exercise performance. *J. Exp. Biol.* **219**, 243–249. (doi:10.1242/jeb.126623)

28. Bottje WG, Carstens GE. 2009 Association of mitochondrial function and feed efficiency in poultry and livestock species. *J. Anim. Sci.* **87**, E48–E63. (doi:10.2527/jas.2008-1379)

29. Salin K, Villasevil EM, Anderson GJ, Lamarre SG, Melanson CA, McCarthy I, Selman C, Metcalfe NB. 2019 Differences in mitochondrial efficiency explain individual variation in growth performance. *Proc. R. Soc. B* **286**, 20191466. (doi:10.1098/rspb.2019.1466)

30. Salin K, Luquet E, Rey B, Roussel D, Voituron Y. 2012 Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (*Rana temporaria*) tadpoles. *J. Exp. Biol.* **215**, 863–869. (doi:10.1242/jeb.062745)

31. Seebacher F, White CR, Franklin CE. 2015 Physiological plasticity increases resilience of ectothermic animals to climate change. *Nat. Clim. Chang.* **5**, 61–66. (doi:10.1038/nclimate2457)

32. Hood WR et al. 2018 The mitochondrial contribution to animal performance, adaptation, and life-history variation. *Integr. Comp. Biol.* **58**, 480–485. (doi:10.1093/icb/icy089)

33. Monternier PA, Marmillot V, Rouanet JL, Roussel D. 2014 Mitochondrial phenotypic flexibility enhances energy savings during winter fast in king penguin chicks. *J. Exp. Biol.* **217**, 2691–2697. (doi:10.1242/jeb.104505)

34. Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2021 Data from: Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. Dryad Digital Repository. (doi:10.5061/dryad.dfn2z34zk)
*Compr. Physiol.* **2**, 2151–2202. (doi:10.1002/cphy-c110055)

4. Abe TK, Kitagawa T, Makiguchi Y, Sato K. 2019 Chum salmon migrating upriver adjust to environmental temperatures through metabolic compensation. *J. Exp. Biol.* **222**, jeb186189. (doi:10.1242/jeb.186189)

5. Richards JG. 2011 Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. *J. Exp. Biol.* **214**, 191–199. (doi:10.1242/jeb.047951)

6. Zhou BS, Wu RSS, Randall DJ, Lam PKS, Ip YK, Chew SF. 2000 Metabolic adjustments in the common carp during prolonged hypoxia. *J. Fish Biol.* **57**, 1160–1171. (doi:10.1006/jfbi.2000.1382)

7. Khan JR, Iftikar FI, Herbert NA, Gnaiger E, Hickey AJR. 2014 Thermal plasticity of skeletal muscle mitochondrial activity and whole animal respiration in a common intertidal triplefin fish, *Forsterygion lapillum* (Family: Tripterygiidae). *J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.* **184**, 991–1001. (doi:10.1007/s00360-014-0861-9)

8. 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. (doi:10.1242/jeb.118851)

9. Iftikar FI, MacDonald JR, Baker DW, Renshaw GMC, Hickey AJR. 2014 Could thermal sensitivity of mitochondria determine species distribution in a changing climate? *J. Exp. Biol.* **217**, 2348–2357. (doi:10.1242/jeb.098798)

10. Thompson RM, Beardall J, Beringer J, Grace M, Sardina P. 2013 Means and extremes: building variability into community-level climate change experiments. *Ecol. Lett.* **16**, 799–806. (doi:10.1111/ele.12095)

11. Butcher JB, Covington S. 1995 Dissolved-oxygen analysis with temperature dependence. *J. Environ. Eng. (United States)* **121**, 756–759. (doi:10.1061/(ASCE)0733-9372(1995)121:10(756)

12. Havird JC, Neuwald JL, Shah AA, Mauro A, Marshall CA, Ghalambor CK. 2020 Distinguishing between active plasticity due to thermal acclimation and passive plasticity due to Q₁₀ effects: why methodology matters. *Funct. Ecol.* **34**, 1015–1028. (doi:10.1111/1365-2435.13534)

13. Hilton Z, Clements KD, Hickey AJR. 2010 Temperature sensitivity of cardiac mitochondria in intertidal and subtidal triplefin fishes. *J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.* **180**, 979–990. (doi:10.1007/s00360-010-0477-7)

14. Schulte PM, Healy TM, Fangue NA. 2011 Thermal performance curves, phenotypic plasticity, and the time scales of temperature exposure. *Integr. Comp. Biol.* **51**, 691–702. (doi:10.1093/icb/icr097)

15. Jarmuszkiewicz W, Woyda-Ploszczyca A, Koziel A, Majerczak J, Zoladz JA. 2015 Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria. *Free Radic. Biol. Med.* **83**, 12–20. (doi:10.1016/j.freeradbiomed.2015.02.012)

16. Seebacher F, James RS. 2008 Plasticity of muscle function in a thermoregulating ectotherm (*Crocodylus porosus*): biomechanics and metabolism. *Am. J. Physiol. - Regul. Integr. Comp. Physiol.* **294**, 1024–1032. (doi:10.1152/ajpregu.00755.2007)

17. Iftikar FI, Hickey AJR. 2013 Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in *Notolabrus celidotus*. *PLoS ONE* **8**, e64120. (doi:10.1371/journal.pone.0064120)

18. Brand M. 2005 The efficiency and plasticity of mitochondrial energy transduction. *Biochem. Soc. Trans.* **33**, 897–904. (doi:10.1042/BST20050897)

19. Salin K, Auer SK, Rey B, Selman C, Metcalfe NB. 2015 Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance. *Proc. R. Soc. B* **282**, 20151028. (doi:10.1098/rspb.2015.1028)

20. Pörtner HO, Hardewig I, Peck LS. 1999 Mitochondrial function and critical temperature in the Antarctic bivalve, *Laternula elliptica*. *Comp. Biochem. Physiol. A: Mol. Integr. Physiol.* **124**, 179–189. (doi:10.1016/S1095-6433(99)00105-1)

21. Gnaiger E, Méndez G, Hand SC. 2000 High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. *Proc. Natl Acad. Sci. USA* **97**, 11 080–11 085. (doi:10.1073/pnas.97.20.11080)

22. Salin K, Villasevil EM, Auer SK, Anderson GJ, Selman C, Metcalfe NB, Chinopoulos C. 2016 Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios. *Physiol. Rep.* **4**, 1–9. (doi:10.14814/phy2.13007)

23. Salin K, Auer SK, Rudolf AM, Anderson GJ, Selman C, Metcalfe NB. 2016 Variation in metabolic rate among individuals is related to tissue-specific differences in mitochondrial leak respiration. *Physiol. Biochem. Zool.* **89**, 511–523. (doi:10.1086/688769)

24. Petton S, Le Roy V, Bellec G, Queau I, Le Souchu P, Pouvreau S. 2020 Marine environmental station database of Daoulas bay. *SEANOE*. (doi:10.17882/42493)

25. Fangue NA, Richards JG, Schulte PM. 2009 Do mitochondrial properties explain intraspecific variation in thermal tolerance? *J. Exp. Biol.* **212**, 514–522. (doi:10.1242/jeb.024034)

26. Stubbs M, Van Den Boogaart A, Bashford CL, Miranda PMC, Rodrigues LM, Howe FA, Griffiths JR. 1996 ³¹P-Magnetic resonance spectroscopy studies of nucleated and non-nucleated erythrocytes; time domain data analysis (VARPRO) incorporating prior knowledge can give information on the binding of ADP. *Biochim. Biophys. Acta - Gen. Subj.* **1291**, 143–148. (doi:10.1016/0304-4165(96)00058-X)

27. Conley KE. 2016 Mitochondria to motion: optimizing oxidative phosphorylation to improve exercise performance. *J. Exp. Biol.* **219**, 243–249. (doi:10.1242/jeb.126623)

28. Bottje WG, Carstens GE. 2009 Association of mitochondrial function and feed efficiency in poultry and livestock species. *J. Anim. Sci.* **87**, E48–E63. (doi:10.2527/jas.2008-1379)

29. Salin K, Villasevil EM, Anderson GJ, Lamarre SG, Melanson CA, McCarthy I, Selman C, Metcalfe NB. 2019 Differences in mitochondrial efficiency explain individual variation in growth performance. *Proc. R. Soc. B* **286**, 20191466. (doi:10.1098/rspb.2019.1466)

30. Salin K, Luquet E, Rey B, Roussel D, Voituron Y. 2012 Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (*Rana temporaria*) tadpoles. *J. Exp. Biol.* **215**, 863–869. (doi:10.1242/jeb.062745)

31. Seebacher F, White CR, Franklin CE. 2015 Physiological plasticity increases resilience of ectothermic animals to climate change. *Nat. Clim. Chang.* **5**, 61–66. (doi:10.1038/nclimate2457)

32. Hood WR et al. 2018 The mitochondrial contribution to animal performance, adaptation, and life-history variation. *Integr. Comp. Biol.* **58**, 480–485. (doi:10.1093/icb/icy089)

33. Monternier PA, Marmillot V, Rouanet JL, Roussel D. 2014 Mitochondrial phenotypic flexibility enhances energy savings during winter fast in king penguin chicks. *J. Exp. Biol.* **217**, 2691–2697. (doi:10.1242/jeb.104505)

34. Thoral E, Roussel D, Chinopoulos C, Teulier L, Salin K. 2021 Data from: Low oxygen levels can help to prevent the detrimental effect of acute warming on mitochondrial efficiency in fish. *Dryad Digital Repository*. (doi:10.5061/dryad.dfn2z34zk)