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Title: Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress
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Insect Biochemistry and Molecular Biology 80 (2017) 52e60

Contents lists available at ScienceDirect

Insect Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/ibmb

Cold acclimation allows Drosophila flies to maintain mitochondrial
functioning under cold stress
Colinet a, *, David Renault a, Damien Roussel b

UMR CNRS 6553 ECOBIO, Universit
e de Rennes 1, 263 avenue du G
eral-Leclerc, 35042, Rennes, France
Laboratoire d'Ecologie des Hydrosystemes Naturels et Anthropises, UMR 5023, CNRS, Universite de Lyon 1, 69622, Villeurbanne, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 16 October 2016
Received in revised form
22 November 2016
Accepted 23 November 2016
Available online 27 November 2016

Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that
chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to
nonfreezing low temperatures presented conflicting results. In this study, we investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 C). We
assessed mitochondrial oxygen consumption while monitoring the rate of ATP synthesis at various times
(0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 C). We
compared organelle responses between cold-susceptible and cold-acclimated phenotypes. Continuous
exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and
ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically
uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP
synthesis rate was observed in control flies when they were assayed at low temperature. This change was
associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high
survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs).
Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in
isolated mitochondria. Our observations suggest that cold tolerance is linked to the ability to maintain
bioenergetics capacity under cold stress.
© 2016 Elsevier Ltd. All rights reserved.

Cold stress
Fruit flies


1. Introduction
The majority of insects are small ectotherms; as such, their body
temperature reflects environmental temperature. In temperate and
cold regions, temperature fluctuates daily and seasonally. Insects
may periodically face episodes of prolonged low temperature,
which can severely compromise metabolic and physiological activity (Hochachka and Somero, 2002; Colinet et al., 2015). Knowledge of insect physiological mechanisms responsible for coldinduced mortality remains incomplete, and the primary causes of
chilling injury are assumed to be numerous (reviewed by Teets and
Denlinger, 2013). Nonfreezing chilling injury is distinguished from
freezing injury, which is associated with nucleation or freezing of
body fluids. Nonfreezing chilling injury is divided into two subcategories, depending on the duration and intensity of exposure: (i)

^t 14A, Universite
de Ren* Corresponding author. UMR CNRS 6553 ECOBIO, Ba
n e
ral Leclerc CS 74205, 35042, Rennes, France.
nes1, 263 Avenue du Ge
E-mail address: herve.colinet@univ-rennes1.fr (H. Colinet).
0965-1748/© 2016 Elsevier Ltd. All rights reserved.

direct chilling injury, which results from rapid cooling (i.e., cold
shock) on the scale of minutes, and (ii) indirect chilling injury,
which follows long-term exposure on the scale of days to weeks
(Chown and Nicolson, 2004).
As is true for most environmental stresses, the primary effect of
cold stress is the disruption of molecular properties, which triggers
a cascade of secondary damage to biological structures and vital
biological functions, ultimately leading to death (Korsloot et al.,
2004). Chilling can induce separation of the phospholipid bilayer
and alter its permeability, possibly reducing the activity of
membrane-bound enzymes, including primary ion-pumping systems such as Naþ/Kþ-ATPase (Cossins, 1994; Hazel and Hazel, 1995;
MacMillan and Sinclair, 2011). As a result, ion homeostasis is
disturbed, and electrochemical membrane potentials partially or
completely dissipate, which leads to the rapid development of chill
l et al., 2004, 2006; MacMillan and
injury and mortality (Ko sta
Sinclair, 2011; MacMillan et al., 2012a). In addition, proteins can
presumably be denatured by cold, resulting in reduced function
(Franks and Hatley, 1991; Todgham et al., 2007). Low temperature
can theoretically disrupt the fine balance between substrates and

H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

products because of the thermal dependence of various metabolic
pathways (Knight et al., 1986). Nonproportional decreases in
enzymatic reactions and transport can result in the accumulation of
potentially toxic metabolic substances, such as free radicals (Rojas
and Leopold, 1996; Grubor-Lajsic et al., 1997; Jing et al., 2005).
The nonproportional alteration of metabolic pathways could also
result in a shortage of ATP (Coulson et al., 1992; Dollo et al., 2010;
MacMillan et al., 2012b).
Little is currently known about the disruption of cellular bioenergetics in insects exposed to low temperatures that induce
stress (MacMillan et al., 2016). Studies have drawn mixed conclusions on the effect of nonfreezing low temperature on insect energy
production. Some studies reported an increase in ATP levels under
cold conditions (Pullin and Bale, 1988; Coulson et al., 1992; Colinet,
2011; MacMillan et al., 2012b, 2016; El-Shesheny et al., 2016),
whereas others reported a decrease (Pullin et al., 1990; Dollo et al.,
l et al., 2004). These in2010) or no effect on ATP levels (Ko sta
congruities may depend on the methodologies used to quantify ATP
(enzymatic assay, NMR, HPLC), the diversity and severity of cold
treatments applied, and the difficulty of robustly quantifying ATP
production without controlling the many potential moderators of
ATP levels at the organismal level.
As there is no consensus on the impact of low temperature on
the putative disruption of cellular bioenergetics in insects, we
decided to tackle this question using an experimental approach
focused on mitochondrial bioenergetics. The major metabolic
pathway responsible for ATP synthesis is cellular respiration within
mitochondria. Several approaches are currently available to evaluate mitochondrial function indirectly in vitro. However, investigating the functional properties of intact mitochondria isolated
directly from fresh tissues offers unmatched advantages, such as
the absence of interference from cytosolic factors and control over
testing conditions (Brand and Nicholls, 2011). Monitoring the rate
of ATP synthesis in freshly isolated mitochondria (ex vivo) allows
researchers to probe function at the major site of ATP production
directly (Lanza and Nair, 2009). Such information is crucial to
decipher whether the main site of ATP generation is critically
compromised at low stressing temperature.
Maintaining cellular energy homeostasis is a challenge for small
ectotherms such as insects, which are exposed to low stressful
temperatures in nature. During their lifetimes, insects have the
capacity to adjust their physiological mechanisms to promote cold
tolerance and cope with sublethal thermal conditions, a phenomenon referred to as thermal acclimation (Hoffmann et al., 2003;
Angilletta, 2009; Colinet and Hoffmann, 2012; Colinet et al.,
2013). Acclimation in Drosophila melanogaster causes thermal
compensation in various metabolic enzymes (Burnell et al., 1991)
and compensatory increases in metabolic rate (Berrigan, 1997;
Isobe et al., 2013). These changes likely help to maintain appropriate energy flux at low physiological temperature. Genetic cold
adaptation can also modify the properties of cellular bioenergetics.
For instance, ATP levels are typically higher in cold-adapted ectotherms (Napolitano and Shain, 2004). Similarly, cold-adapted
Drosophila flies maintained adenylate levels throughout cold
stress, while cold-susceptible flies experienced a drop during recovery (Williams et al., 2014) and this is associated with higher
rates of catabolism and anabolism (Williams et al., 2016a). One
mutation that promotes flies’ cold tolerance is also associated with
increased ATP levels and metabolic rates (Takeuchi et al., 2009).
Cold-hardy flies have greater metabolic plasticity than control or
susceptible lines (Williams et al., 2016b). Collectively, these observations suggest that cold tolerance, acquired via plastic or genetic
changes, is somewhat linked to the capacity to maintain bioenergetic capacity under cold stress.
In this study, we investigated the mitochondrial bioenergetics of


Drosophila adult flies exposed to chronic cold stress (at 4 C). We
monitored mitochondrial function (respiration and ATP synthesis
concurrently) at various times (three consecutive days) during
chronic cold stress and at two assay temperatures (25 and 4 C). We
compared mitochondrial responses between chill-susceptible
control and cold-acclimated phenotypes. We expected a decrease
in mitochondrial respiration and ATP synthesis at the lower assay
temperature. If impaired mitochondrial ATP supply is indeed
related to the development and progression of chilling injuries (e.g.,
membrane alteration, loss of ion/water homeostasis), signs of
mitochondrial dysfunction (reduced coupling or ATP production)
should arise in chill-susceptible flies. Finally, we predicted that cold
acclimation would promote cold tolerance, and that this plastic
change would be associated with maintained mitochondrial efficiency, particularly under cold stress.
2. Materials and methods
2.1. Fly culture
Flies from a laboratory population of D. melanogaster were used
for experiments. The population was founded from a large number
of individuals collected in October 2010 in Brittany, France. Flies
were maintained in the laboratory in 200-mL bottles at 25 ± 1 C
(light/dark: 12/12 h) on standard fly medium consisting of brewer
yeast (80 g/L), sucrose (50 g/L), agar (15 g/L), and Nipagin® (8 mL/L).
To generate flies for the experiments, groups of 15 mated females
were allowed to lay eggs in 200-mL rearing bottles during a
restricted period of at most 6 h under laboratory conditions. This
controlled procedure allowed larvae to develop under uncrowded
conditions. To avoid gender-induced variability, only females were
used in all experiments. Due to the high number of flies required, 2day-old flies were sexed using short CO2 exposure (<2 min).
Following anesthesia, females were left on food vials for 3 days to
allow complete recovery (Colinet and Renault, 2012).
2.2. Experimental design for acclimation and stress period
Five-day-old females were randomly exposed to gradual cold
acclimation (CA) or control conditions (CO) for five consecutive
days. The temperature was set to 15 or 25 C for CA or CO,
respectively, with a 12-h/12-h light/dark cycle (Fig. 1). Programmed
thermoregulated incubators (Model MIR-153, SANYO Electric Co.
Ltd, Munich, Germany) were used. Temperature was checked by
using automatic recorders (Hobo Data Logger, model U12-012,
accuracy ± 0.35 C, Onset Computer Corporation, Bourne, MA,
USA). Females were left on food vials during this period. After 5
days of thermal conditioning under CA and CO conditions, groups
of females were exposed to chronic cold stress at 4 C for three
consecutive days in food vials containing 50 individuals, which had
been placed in thermoregulated incubators. We selected this temperature and duration because preliminary experiments revealed
that early mortality (i.e., scored after 15-min recovery) increased
sharply after 3 days at 4 C (only in CO flies).
Poststress survival was scored based on a pool of 100 females for
each treatment (CO vs. CA) and duration (e.g., days 1, 2, and 3).
Females were removed from the cold incubator and placed at 25 C
in food vials (groups of 10). Survival of the 100 females was
monitored after 15 min, 4 h, and 24 h of recovery (Fig. 1). Individuals were categorized as dead (no apparent movement),
agonizing (movement but unable to stand on legs), or active (able
to crawl). We realize that assessing mortality 24 h after stress likely
estimates the detrimental effect of cold stress more effectively than
early mortality (after 15-min recovery). However, an early estimate
of survival was essential in evaluating whether isolated


H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

Fig. 1. Schematic representation of experimental design. Mature females were conditioned under two thermal regimes: cold acclimation at 15 C for five days (CA) or maintenance
at 25 C for 5 days (control, CO). Mitochondrial properties were monitored at the end of this conditioning period. Females from both groups were then exposed to cold stress at
constant 4 C for three consecutive days. At the end of each day, survival and mitochondrial function were analyzed. For each time-point: (i) survival was scored after 15 min, 4 h,
and 24 h of recovery poststress; and (ii) mitochondrial characteristics were simultaneously inspected at two assay temperatures: 4 C and 25 C. For each time-point and treatment,
100 females were used to assess survival. Six independent mitochondrial isolates (each consisting of 200 females) were used to evaluate mitochondrial function.

mitochondria would comprise dead individuals in samples. Proportions of dead, agonizing, and active individuals were compared
between phenotypic groups for each stress duration (days 1e3)
using chi-square contingency tests.
2.3. Mitochondrial isolation protocol
After thermal conditioning for 5 days, fresh mitochondria were
isolated from individuals that had been removed directly from their
respective temperatures (15 vs. 25 C). This first time-point allowed
us to assess mitochondrial function at the end of the conditioning
period (day 0), just before females were exposed to chronic cold
stress (4 C). Isolation was performed on six biological replicates,
each consisting of a pool of 200 females (i.e., 1200 females for CA
and CO at day 0 only). Mitochondrial isolation was then performed
on cold-stressed females. Six replicates of 200 females were
necessary for each period of cold stress (days 1e3) and treatment
(CA vs. CO) (see Fig. 1).
Drosophila mitochondria were isolated using standard differential centrifugation procedures (Miwa et al., 2003). Pools of 200
flies were homogenized with a PottereElvehjem homogenizer in
2 mL of ice-cold mitochondrial isolation buffer (250 mM sucrose,
5 mM Tris, 2 mM EGTA, 1% w/v bovine serum albumin, pH 7.4 at
4 C). Fly homogenate was centrifuged at 150 g for 3 min (4 C) to
remove major debris. The resulting supernatant was centrifuged at
9000g for 10 min (4 C) to pellet mitochondria. The pellet was
suspended in 2 mL of isolation buffer and centrifuged at 900g
(10 min, 4 C). The resulting supernatant was filtered through
cheesecloth and centrifuged at 9000g (10 min, 4 C). The pellet was
suspended and gently homogenized in 60 mL of ice-cold isolation
buffer. Protein concentration was spectrophotometrically
measured at 540 nm (VersaMax™ Microplate Reader, Molecular
Devise) using the Biuret method with bovine serum albumin as a
2.4. Oxygen consumption and ATP synthesis rates
Mitochondrial oxygen consumption rate (OCR) was measured in
a thermostatically controlled glass cell fitted with a Clark oxygen
electrode (Rank Brothers Ltd, Bottisham, UK). For each experimental condition, two oxygen electrodes were set up simultaneously. Each mitochondrial preparation was assessed at 4 and

25 C with constant stirring (see Fig. 1). The glass cell was filled with
500 mL of air-saturated respiratory reaction medium (5 mM
KH2PO4, 120 mM KCl, 1 mM EGTA, 2 mM MgCl2, 1.6 U/mL hexokinase, 20 mM glucose, 3 mM HEPES, pH 7.4), with a final fatty acidfree bovine serum albumin concentration of 3 mg/mL (0.3% w/v),
supplemented with 10e15 mL of mitochondrial suspension (1e2 mg
A mixture of 5 mM pyruvate and 5 mM proline was used as
respiratory substrate to initiate the reaction. ADP (500 mM) was
added to start State 3 respiration, which reflects the oxygen
consumed by mitochondria to synthesize ATP from ADP and
phosphate. Mitochondrial ATP synthesis was followed by glucose6-phosphate accumulation using an ADP-regenerating system
(hexokinase plus glucose). After recording OCR State 3, four aliquots
of 100 mL each of mitochondrial suspension were withdrawn from
the chamber at 2-min intervals and immediately quenched in
100 mL of perchloric acid solution (10% HClO4 and 25 mM EDTA).
After centrifugation of the denatured protein (15,000g for 5 min),
supernatants were neutralized with a KOH solution containing
0.2 M KOH and 0.3 M MOPS. The glucose-6-phosphate content of
the samples was measured by spectrophotometry at 340 nm in an
assay medium consisting of triethanolamine-HCl (50 mM), MgCl2
(7.5 mM), and EDTA (3.75 mM) adjusted to pH 7.4 at room temperature, supplemented with NAD (0.5 mM) and glucose-6phosphate dehydrogenase from Leuconostoc mesenteroides (0.5 U)
(Salin et al., 2012). The rate of mitochondrial ATP production was
calculated from the slope of the linear accumulation of glucose-6phosphate over the sampling time interval (6 min). The linearity
of glucose-6-phosphate accumulation allowed us to confirm that
the system maintained a steady state.
We ensured that the measured rates were specific to mitochondrial ATP synthase activity by determining basal nonphosphorylating oxygen consumption (State 4oligo) and ATP
synthesis rates in the presence of 5 mg/mL oligomycin (an inhibitor
of mitochondrial ATP synthase). Respiratory control ratios (RCRs)
were calculated as the ratio of OCR State 3 to OCR State 4oligo. RCR
provides an indication of the degree of uncoupling in the mitochondria. Low RCR values (<4) generally suggest poorly coupled
mitochondria. An RCR value of 1 indicates fully uncoupled mitochondria (i.e., mitochondria that actively consume oxygen with no
ATP synthesis) (Guderley and St Pierre, 1996). The mitochondrialspecific rate of ATP synthesis was calculated by subtracting the

H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

resting rate of ATP synthesis in the presence of oligomycin from the
global rate of ATP synthesis (shown in Fig. S1). The ATP/O ratio was
calculated by dividing the mitochondrial-specific rate of ATP synthesis by OCR State 3. This ratio represents the amount of ATP
generated per molecule of oxygen consumed by mitochondria and
measures mitochondrial oxidative phosphorylation efficiency
(Salin et al., 2015).
Data related to OCR and ATP synthesis were analyzed by using
linear models with the following predictors: time (cold stress
duration from 0 to 3 days); acclimation (CA vs. CO); assay temperature (4 vs. 25 C); and secondary interactions. Regression lines
were drawn from raw individual data. All analyses were conducted
using R 3.0.3 statistical software (R Core Team, 2013). We ran the
Anova function in the “car” package to analyze the effect of each
variable in the GLM models through the table of deviance (summarized in Table 1). Model outputs (coefficient estimates and
model fit parameters) are provided in the supplementary materials
(Table S1).
3. Results
3.1. Cold tolerance
As expected, cold acclimation had a profound effect on cold
tolerance in D. melanogaster females (Fig. 2). The proportion among
classes (active, agonizing, and dead) differed markedly between CO
and CA females. CA flies were mainly active after each period of cold
stress. In CO flies, the proportion of dead individuals increased
strikingly with both the duration of cold stress and sampling time.
When flies were scored after 15 min of recovery (Fig. 2A), nearly all
CA flies were active for all exposure durations, whereas the proportion of agonizing CO flies increased with the duration of cold
stress. The difference between CO and CA flies was significant only
for stress durations of 2 or 3 days (Chi2 ¼ 35.17, P < 0.001;
Chi2 ¼ 141.6, P < 0.001, respectively). After 4 h of recovery (Fig. 2B),
proportions among classes were different between CO and CA flies
for stress durations of 2 and 3 days (Chi2 ¼ 42.13, P < 0.001;
Chi2 ¼ 145.2, P < 0.001, respectively). Finally, when flies were
observed 24 h after cold stress (Fig. 2C), CA flies still appeared
weakly affected by cold stress, whereas the mortality of CO flies
increased dramatically with the duration of cold stress. The difference between CO and CA flies was significant at all stress durations (day 1: Chi2 ¼ 9.42, P ¼ 0.009; day 2: Chi2 ¼ 82.12, P < 0.001;


day 3: Chi2 ¼ 141.4, P < 0.001).
3.2. Mitochondrial characteristics
The OCRs (States 3 and 4oligo) and both ATP production rates
(mitochondrial and resting) were significantly affected by the time
spent under cold stress (Figs. 3 and 4 and Table 1). In all cases, rates
decreased with increasing durations of cold stress (Figs. 3 and 4 and
Table S1). RCRs and ATP/O ratios were not affected by the duration
of cold exposure (Table 1). OCRs (States 3 and 4oligo) were affected
by temperature (Table 1 and Fig. 3). OCRs were much higher when
mitochondria were assayed at 25 vs. 4 C (Table S1). RCRs were also
affected by assay temperature and were globally higher at 25 vs.
4 C (Fig. 3, Tables 1 and S1). Both ATP production rates (mitochondrial and resting) and ATP/O decreased significantly with
lower assay temperature (Fig. 4, Tables 1 and S1). The temperature
predictor accounted for the largest part of the variance in all models
(Table S1).
Concerning the acclimation effect, the models showed that ATP
synthesis rates were affected by acclimation (Table 1 and Fig. 4).
They were higher in mitochondria from CA compared to CO flies
(Fig. 4, Tables 1 and S1). ATP/O was not affected by acclimation
(Table 1). OCRs (States 3 and 4oligo) were independent of acclimation treatment, although RCRs were globally higher in CA vs. CO
flies (Fig. 3, Tables 1 and S1). Temporal decreases were not specific
to acclimation treatment for any metric examined (no
Time Acclim interaction, Table 1). Mitochondria from both phenotypes were similarly affected by assay temperature (no
Acclim Temp interaction, Table 1). Time Temp interactions
were significant for OCR State 3, RCR, mitochondrial ATP synthesis,
and ATP/O; meaning that with these metrics, temporal decreases
were dependent on assay temperature (Figs. 3 and 4; Tables 1 and
S1). For OCR State 3 and mitochondrial ATP synthesis, the temporal
decreases were slower at lower assay temperatures. For RCR and
ATP/O, there was a temporal decrease at 25 C. At 4 C, the ratio did
not change or had a tendency to increase with time in CA flies.
4. Discussion
Most biological (enzymatic) processes exhibit a thermal dependence that follows Arrhenius law. Therefore, it is often assumed that
prolonged exposure to low temperature stress should reduce aerobic
metabolism and, consequently, decrease the availability of energy

Table 1
Analysis of variance from linear models showing the significance of the following variables: Time (duration of cold stress), Acclim (CA vs. CO flies) and Temp (temperature of the
assay: 4 vs. 25 C), as well as the secondary interactions. Significant values are highlighted in bold. Summary of estimated coefficients, linear model fits (coefficients, r2), and
measures of relative importance for each variable included in the models are provided in Table S1.








OCR state 3

Time Acclim
Acclim Temp
Time Temp
Time Acclim
Acclim Temp
Time Temp
Time Acclim
Acclim Temp
Time Temp



Mitochondrial ATP

Time Acclim
Acclim Temp
Time Temp
Time Acclim
Acclim Temp
Time Temp
Time Acclim
Acclim Temp
Time Temp



OCR state 4oligo


Non-mitochondrial ATP



H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

Fig. 2. Proportion of active (white), agonizing (gray), and dead (black) D. melanogaster females after chronic cold stress at 4 C for three consecutive days in the control (CO, red) and
cold-acclimated (CA, blue) phenotypes. Stress duration (in days) and treatment are indicated on the x-axis. After cold stress, individuals were returned to 25 C. Their status (active,
agonizing, or dead) was scored after 15 min (A), 4 h (B), or 24 h (C) of recovery (n ¼ 100 flies for each experimental treatment). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

Fig. 3. Oxygen consumption rate (OCR) during maximal ADP-stimulated State 3 (from A to D). OCR during State 4oligo (basal nonphosphorylating respiration in the presence of
oligomycin) (from E to H). Respiratory control ratio (RCR), corresponding to OCR State 3/OCR State 4oligo ratio (from I to L). All respiration parameters were monitored according to
cold stress duration (x-axis) in isolated mitochondria coming from control flies [CO] (boxplots in red: A, C, E, G, I, K) or cold-acclimated flies [CA] (boxplots in blue: B, D, F, H, J, L). For
both phenotypes, mitochondria were concurrently assayed at two temperatures: 25 C (A, B, E, F, I, J) and 4 C (C, D, G, H, K, L). Mean and median are indicated within each box by a
black dot and horizontal line, respectively (based on six independent replicates). Colored lines depict regressions from linear models based on raw data. Scales differ for assays at 4
and 25 C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

substrate (ATP) (Coulson et al., 1992; Dollo et al., 2010; MacMillan
et al., 2012b). Surprisingly little is currently known about the
disruption of cellular bioenergetics in insects exposed to low
stressing temperatures, particularly with regard to mitochondrial

physiology. Insect studies have drawn mixed conclusions on the effect of nonfreezing low temperature on ATP supply, So that it is
unclear whether ATP deficiency is a genuine cause of chilling injury
(Teets and Denlinger, 2013; MacMillan et al., 2016).

H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60


Fig. 4. Mitochondrial-specific rate of ATP synthesis (from A to D). Resting rate of ATP synthesis in the presence of oligomycin (i.e., nonmitochondrial ATP synthesis) (from E to H).
ATP/O ratio corresponding to the mitochondrial ATP synthesis/OCR State 3 ratio (from I to L). Global rate of ATP synthesis (mitochondrial plus resting) is shown in Fig. S1. ATP
synthesis parameters were monitored according to stress duration (x-axis) in isolated mitochondria from either control flies [CO] (boxplots in red: A, C, E, G, I, K) or cold-acclimated
flies [CA] (boxplots in blue: B, D, F, H, J, L). For both phenotypes, mitochondria were assayed at two temperatures: 25 C (A, B, E, F, I, J) and 4 C (C, D, G, H, K, L). Mean and median are
indicated within each box by a black dot and horizontal line, respectively, and are based on six independent replicates. Lines depict regressions from linear models based on raw
data. Scales for assays at 4 and 25 C are different. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

As there is no consensus with regard to the impact of prolonged
exposure to cold stress on the disruption of cellular bioenergetics in
insects, we decided to tackle this issue using a hypothesis-driven
approach focused on the major site of ATP synthesis: the mitochondrion. We assessed mitochondrial functions (respiration and
ATP synthesis concurrently) at different time intervals during
chronic cold stress. If the progressive failure of ATP production is
causally linked to the development of chilling injuries, we should
expect to see clear signs of mitochondrial dysfunction (e.g.,
decreased RCR or ATP synthesis). We compared the characteristics
of freshly isolated mitochondria from cold-susceptible vs. coldacclimated phenotypes. We hypothesized that acclimation would
foster cold tolerance, and that this phenotypic change would
translate into improved mitochondrial efficiency under cold stress.
Chilling injuries accumulated particularly in CO flies exposed to
4 C, as attested by the increased mortality observed in this
phenotypic group. The survival data at 15-min recovery indicated
that flies were either fit or agonizing, thus ensuring that mitochondrial samples were prepared from live individuals. In CO flies,
mortality quickly built up during the poststress recovery period,
particularly after 3 days of stress. This mortality pattern differed
substantially from CA flies, which were marginally affected by cold

stress (24 h mortality < 10%), confirming the beneficial effect of cold
To examine the impact of prolonged cold stress on mitochondrial function, we evaluated the respiratory function of isolated
mitochondria by monitoring OCR while providing flies with substrates in the presence of ADP (State 3) or while inhibiting ATP
synthase with oligomycin (State 4oligo). We used half of the fresh
mitochondrial suspension at two different assay temperatures (4
and 25 C) using two oxygen electrodes simultaneously. As expected, both OCRs (States 3 and 4oligo) decreased with increasing
durations of cold stress and with decreases in assay temperature.
Mitochondria from both phenotypes reacted similarly to thermal
effects. The OCR during ADP phosphorylation (State 3) reflects the
maximum oxidative capacity, whereas the OCR in the presence of
oligomycin (State 4oligo) can be used as a proxy for the proton leak
across the mitochondrial membrane (Brand and Nicholls, 2011).
Respiration States 3 and 4oligo are both temperature dependent
€rtner, 2004; Fangue et al.,
(Danks and Tribe, 1979; Sommer and Po
2009); therefore, reduced rates at low assay temperature (4 C)
are reasonable. Our findings show that the maximum oxidative
capacity and proton leakage capacity decreased as the duration of
cold stress increased in both phenotypes. A temporal reduction in


H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

leak rate, even modest, may indicate a change in mitochondrial
membrane conductance (Brand and Nicholls, 2011). This feature
could result from the direct effect of cold, which is known to induce
a decrease in mitochondrial proton leak activity (Chamberlin, 2004;
Chung and Schulte, 2015).
When looking at mitochondrial respiration, we found no clear
indication that acclimation affected OCR. Increased mitochondrial
respiration (State 3) has been observed in response to acclimation
in ectothermic animals (Danks and Tribe, 1979; Guderley and St
Pierre, 1996; Rogers et al., 2007), but this effect is not a general
€ rtner, 2004; Fangue et al., 2009). We found,
rule (Sommer and Po
however, that the RCR was significantly higher in CA flies at both
assay temperatures. The RCR is considered to be the most useful
measure of function in isolated mitochondria (Brand and Nicholls,
2011). Values obtained in our work (ranging 4e6) are consistent
with values reported for Drosophila species (Pichaud et al., 2010;
Correa et al., 2012). Low RCR values (<4) generally suggest poorly
coupled mitochondria. An RCR value of 1 indicates fully uncoupled
mitochondria (i.e., mitochondria that actively consume oxygen with
no ATP synthesis) (Guderley and St Pierre, 1996).
In our study, the RCR values (>1) showed that mitochondria
were still functional in both fly phenotypes, even after 3 days of
cold stress. A general effect of acclimation was observed, in that
mitochondria from cold-acclimated flies showed higher RCR values
than did mitochondria from CO flies. Data analysis indicated that
the higher RCR in CA flies primarily reflected slightly higher
mitochondrial oxidative phosphorylation activity (State 3),
whereas resting respiration (State 4oligo) remained identical in both
phenotypes. Similarly, in the marsh frog, RCR was superior in coldacclimated vs. control individuals, due to a change in State 3, while
State 4 was not affected by acclimation (Rogers et al., 2007). Hence,
the present data imply that mitochondria from CA flies have a
higher capacity for substrate oxidative phosphorylation than do
mitochondria from CO flies.
Lipid membranes of cells and organelles are cold-sensitive
macromolecular structures. Chilling can alter the permeability of
biological membranes and reduce the activity of membrane-bound
enzymes, such as Naþ/Kþ-ATPase (Cossins, 1994; Hazel and Hazel,
l, 2010; MacMillan et al., 2015). This effect, in turn,
1995; Ko sta
can lead to a loss of ion/water homeostasis across membranes
l et al., 2004, 2006; MacMillan et al. 2012a) and membrane
(Ko sta
depolarization (Hosler et al., 2000; MacMillan et al., 2014). Inhibition of Naþ/Kþ-ATPase adversely affects mitochondrial energetics
(Li et al., 2014). Under continuous exposure to 4 C, a temperature
that provoked chill coma in our flies, these changes may contribute
to the progressive reduction in mitochondrial activity. This
assumption is supported by the temporal reduction in ATP synthesis rates measured in both cold-exposed phenotypes. Nonetheless, ATP production rates (mitochondrial and resting) were
globally higher in CA flies. This result would provide more energyequivalent to CA flies during periods of stress. Even if acclimation
did not prevent the temporal decline in ATP synthesis rates during
cold exposure, it mitigated this process. Analogously, Coello
Alvarado et al. (2015) showed that a progressive cold-induced loss
of ion/water homeostasis in crickets was not prevented (but was
lowered) by cold acclimation.
Interestingly, we observed a sharp decline (>50%) of mitochondrial ATP synthesis in samples from CO flies exposed to 4 C for
three consecutive days, which were assayed at the same temperature (Fig. 4C). Such a decline was not observed in mitochondria
from CA flies, which produced about 70% more ATP per milligram of
mitochondrial protein than CO flies under the same restrictive
experimental conditions (Fig. 4D). After 3 days of stress, the RCR
value of CO flies at 4 C was the lowest recorded throughout the
entire experiment (Fig. 3K). This finding may reflect the onset of

mitochondrial dysfunction. More broadly, it may show early signs
of a complete system breakdown in CO flies, as suggested by survival data (i.e., high late mortality in CO flies after three days).
Complex changes in mitochondrial structure and function,
including the disorganization of membranes and decreased activity
of bound enzymes under cold conditions, could cause a sharp
decrease in ATP production in CO samples. Cold acclimation prevented such mitochondrial dysfunction. It is also noteworthy that at
4 C, the ATP/O ratio was constant in CO flies. Therefore, in these
flies, the decreases in OCR and ATP synthesis with cold treatment
were not compensated by any improvement in mitochondrial
oxidative phosphorylation efficiency (ATP/O). In CA flies, however,
the ATP/O ratio tended to increase at 4 C; this increase may partly
explain the reduced impact on ATP production.
At present, we have no available data to bear on a mechanistic
basis for the protective effect of cold acclimation with respect to
mitochondrial function. An obvious candidate mechanism is the
effect of acclimation on mitochondrial membranes and enzymatic
capacities. Adaptive homeoviscous adaptation of mitochondrial
membranes and thermal compensation of mitochondrial enzymes
are typical responses to cold acclimation (e.g., reviewed by
Guderley and St Pierre, 1996). These responses may allow mitochondria to function at relatively lower temperature (or function
longer under stress). Along these lines, mitochondria from heatacclimated fish can function at higher temperatures than can
those from cold-acclimated counterparts (Fangue et al., 2009).
The sharp decline in ATP synthesis at 4 C in CO samples after 3
days of stress could have dramatic consequences for organismal
physiology and survival. Mitochondria are key organelles in
determining the interplay between apoptosis and necrosis. At a
certain level, a reduction in intracellular ATP levels switches the cell
from apoptosis, which requires ATP, to necrosis (Eguchi et al., 1997).
The clear ATP decline in CO flies could be causally related to
lethality. At this point, however, it would be premature to conclude
as much without further confirmation. Alternatively, this alteration
may simply reflect a byproduct of cold stress, acting more directly
on related biological processes. Whatever the direction (cause or
consequence), there seems to be little doubt that CA flies can
maintain more coupled mitochondria and sustain higher ATP production for vital functions under thermal conditions that eventually become lethal to CO flies. Consequently, our observations add
new pieces to our understanding of the cold stress physiology and
reinforce the notion that cold tolerance is linked to maintenance of
bioenergetic capacity under conditions of low temperature
(Takeuchi et al., 2009; Williams et al., 2014, 2016b; MacMillan et al.,
Previous studies have reported various patterns of ATP levels in
cold-exposed insects. Increased ATP levels have regularly been reported under such conditions (Pullin and Bale, 1988; Coulson et al.,
1992; Colinet, 2011; MacMillan et al., 2012b, 2016; El-Shesheny
et al., 2016). This result may occur if the relative rate of ATP synthesis exceeds that of ATP consumption, although both rates
decline at low temperatures (Napolitano and Shain, 2004; present
study). A primary consumer of ATP in most cells is the ATPdependent transport of ions across membranes (up to 40%)
(Clausen, 1986; McMullen and Storey, 2008). A progressive and
extensive reduction in the activity of various ATPases occurs under
chilling conditions (Jing et al., 2005; MacMillan et al., 2015). Such a
reduction in ATPase activity is considered to be causatively linked
to increased organismal ATP levels (El-Shesheny et al., 2016;
MacMillan et al., 2016).
Here, we show that ATP synthesis in isolated mitochondria is
strongly reduced at 4 C and is progressively lowered as the duration of cold exposure increases. The cells could partially compensate for the reduction in mitochondrial ATP supply through

H. Colinet et al. / Insect Biochemistry and Molecular Biology 80 (2017) 52e60

nonmitochondrial ATP generation (e.g., via adenylate kinase or the
phosphagen system). Under our experimental conditions, nonmitochondrial ATP synthesis created about 25% of the global pool of
ATP (shown in Fig. S1). Even if the slope of temporal reduction in
nonmitochondrial ATP synthesis was much more gradual than that
of mitochondrial ATP production, there was no evident sign of
In conclusion, we confirm that low temperature causes a decline
in the ability of the mitochondria to produce ATP. Prolonged
exposure to 4 C provoked a gradual reduction in mitochondrial
ATP synthesis and mitochondrial respiration rates in both phenotypes. These progressive reductions may not directly trigger mortality, at least in CA flies. ATP synthesis rates and mitochondrial
coupling were globally lower in chill-susceptible CO flies compared
to CA flies. The former group exhibited a sharp decline in mitochondrial ATP production after 3 days of stress at 4 C, with reduced
survival capacity. RCR values suggested that global mitochondrial
integrity was not compromised, but our data suggest that mitochondrial bioenergetics start to become dysfunctional beyond
certain limits. To our knowledge, this study is the first to examine
the impact of prolonged cold stress on mitochondrial function, by
comparing phenotypes of contrasted thermotolerance. Our reductionist approach utilizing isolated mitochondria allowed us to
probe organelle function directly. Many mitochondrial aspects
remain to be explored, such as the properties of mitochondrial
membranes and the expression/activity of membrane-bound enzymes over the course of cold stress, in phenotypes adapted or
acclimated to various thermal environments.
We thank Paysaclim team (Ecobio) for contributing to travel
costs. This study was supported by CNRS and SUZUKILL project
[ANR-15-CE21-0017-01 and FWF I 2604-B25].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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