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Comparative Biochemistry and Physiology, Part A 160 (2011) 63–67

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Disruption of ATP homeostasis during chronic cold stress and recovery in the chill
susceptible beetle (Alphitobius diaperinus)
H. Colinet ⁎
Earth and Life Institute ELI, Biodiversity Research Centre BDIV, Catholic University of Louvain; Croix du Sud 4-5, B-1348 Louvain-la-Neuve, Belgium
ECOBIO UMR CNRS 6553, Université de Rennes1, 263 Avenue du Général Leclerc CS 74205, 35042 Rennes Cedex, France

a r t i c l e

i n f o

Article history:
Received 2 March 2011
Received in revised form 4 May 2011
Accepted 4 May 2011
Available online 11 May 2011
Fluctuating thermal regime
Chilling injury

a b s t r a c t
This study examined the impact of fluctuating thermal regimes (FTRs) on cold tolerance of the polyphagous
beetle Alphitobius diaperinus. Daily pulses of elevated temperatures can provide breaks in chronic cold stress,
potentially allowing for physiological recovery and improving survival. Perturbations in central metabolism
appear to be a common physiological response in insects exposed to low temperatures. It has been suggested
that energy supplies, which may be depleted during cold exposure, can be regenerated during the warming
pulses of FTRs. This study tested the assumption that chronic cold stress may induce ATP depletion and that
recovery during FTR warming pulses may allow re-establishment of ATP supplies. In this study, A. diaperinus
were exposed to cold stress under different thermal regimes (constant or fluctuating). The results did not
confirm the aforementioned assumption. No cold-induced ATP depletion was observed. The lowest ATP levels
were repeatedly detected in the untreated controls. The data show that homoeostasis of ATP is lost when
adults A. diaperinus are exposed to cold stress, whatever thermal regime (constant or fluctuating). ATP
accumulation may be viewed as a symptom of a production/consumption imbalance under cold stress
conditions. Periodic short (2-h) warming pulses clearly improved cold survival. Cellular homeostasis,
however, probably requires a longer recovery period to be fully restored.
© 2011 Elsevier Inc. All rights reserved.

1. Introduction
Ectotherms are strongly affected by thermal variations, at every
level of their biological organization (Sinclair et al., 2003). Though
these organisms can tolerate thermal variation within certain limits,
in many situations, temperature can become incompatible with
optimal biological activity, being either too high or too low. Prolonged
exposure to low temperature (e.g. chronic cold stress) can severely
affect the survival of organisms. In insects, cold-shock induces direct
chilling injuries whose mechanisms are rather well characterized
(Ramløv, 2000; Chown and Nicolson, 2004). However, our understanding of the mechanisms underlying indirect chilling injuries that
results from chronic cold stress is limited (Chown and Nicolson, 2004;
Chown and Terblanche, 2006). Though it is known that prolonged
cold exposure induces loss of ion homeostasis in muscle and
hemolymph, together with water balance perturbation (Koštál et al.,
2004, 2006; MacMillan and Sinclair, 2011). In natural environments,
organisms may benefit from periodic opportunities to recover from
physiological stress during periods of optimal thermal conditions.
Several studies have tested the importance of this recovery effect on
⁎ Hervé Colinet, ECOBIO UMR CNRS 6553 Bât 14A, Université de Rennes1, 263
Avenue du Général Leclerc CS 74205, 35042 Rennes Cedex, France. Tel.: + 33 2 23 23 66
27; fax: + 33 2 23 23 50 26.
E-mail address: herve.colinet@uclouvain.be.
1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved.

insect cold tolerance using fluctuating temperature regimes (FTRs),
applied either once within a prolonged cold stress (Chen and
Denlinger, 1992; Dollo et al., 2010) or repeatedly on daily basis
(Nedvěd et al., 1998; Renault et al., 2004; Colinet et al., 2006; Koštál
et al., 2007; Colinet and Hance, 2010). Under fluctuating conditions,
cold exposure alternates with periodic short pulses of optimal
temperatures, allowing a physiological recovery that reduces coldinduced mortality. Although our understanding of the mechanisms
underlying the positive impact of FTRs is partial, several studies have
contributed to the understanding of this phenomenon.
The recovery process during optimal temperature pulses involves
(1) the re-establishment of ion gradient homeostasis (Koštál et al.,
2007), (2) the anabolism or catabolism of cryoprotectants (Wang
et al., 2006; Lalouette et al., 2007), (3) the consumption of amino acid
pools (Colinet et al., 2007a; Lalouette et al., 2007), (4) the expression
of heat shock proteins (Hsps) at the transcript level (Wang et al.,
2006; Tollarová-Borovanská et al., 2009; Colinet et al., 2010) and at
the protein level (Colinet et al., 2007b), (5) the activation of antioxidant system to counter reactive oxygen species production (Lalouette
et al., 2011), and (6) the upregulation of various proteins that play key
roles in proteolysis, cytoskeleton and energy metabolism (Colinet
et al., 2007b). It has been suggested that the energy supplies, likely
depleted during prolonged cold exposure, may be regenerated during
the pulses of warm temperature (Chen and Denlinger, 1992). The
upregulation of energy production pathways during recovery periods

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H. Colinet / Comparative Biochemistry and Physiology, Part A 160 (2011) 63–67

might be important for providing the energy requirements for repair
processes (Colinet et al., 2007b). The activities of ion pumps and
Hsps, proteolysis, and synthesis of cryoprotectants (e.g. glycerol) are
ATP-dependent (Storey and Storey, 1996; Hersko and Ciechanover,
1998; Pratt and Toft, 2003; Koštál et al., 2006).
Environmental stress generally disturbs cellular homeostastis
(Korsloot et al., 2004). Heat shock, for example, disrupts the ability
of mitochondria to phosphorylate ATP's diphosphate precursor,
resulting in ATP depletion (Corton et al., 1994; Korsloot et al.,
2004). This study tests the assumption that (i) cold stress may induce
ATP depletion and that (ii) recovery during high temperature pulses
may allow the reestablishment of ATP supplies. The polyphagous
beetle Alphitobius diaperinus (Coleoptera: Tenebrionidae), commonly
known as the lesser meal worm, was chosen as a study model.
Because of its invasive status, the thermobiology of this tropical
species has been studied extensively in recent years (e.g. Renault et
al., 1999, 2003), and ample data have shown that FTRs increase the
cold survival of A. diaperinus (e.g. Renault et al., 2004, Lalouette et al.,
2007; Renault, 2011).
2. Materials and methods
2.1. Rearing
Adult A. diaperinus beetles were collected from poultry house litter
in the Treffendel commune (Brittany, France, 2°0'0''W, 48°2'24''N) in
October 2010. The insects were reared in complete darkness under
standard rearing conditions (20 ± 1 °C, ±60% RH) and supplied with
water and food ad libitum (moistened bran and dry dog food). All of
the insects examined in the experiment were adults of less than one
month of age. Individuals were chosen randomly for survival and
biochemical assays.
2.2. Survival assays
Adult A. diaperinus were cold-stressed at 3 °C, a temperature
known to induce chill-coma (Renault et al., 1999). Groups of 10
beetles were transferred to Petri dishes with water supply and placed
inside thermo-regulated incubators (Model SANYO MIR-153) in
complete darkness. Temperatures were checked using automatic
recorders (Hobo® data logger, model U12-012, Onset Computer
Corporation). Under FTR, the incubators were programmed to
fluctuate between 3 and 20 °C. The actual temperature changed
across the different steps at a rate of about 0.7–0.8 °C/min. Beetles
were randomly assigned to one of two groups: constant low
temperature (Cst: continuous exposure at 3 °C) or FTR (exposure at
3 °C interspersed with daily shifts to 20 °C for 2 h). A 2-h warming
pulse has been proven to be an optimal recovery duration in several
models (e.g. Nedvěd et al., 1998; Renault et al., 2004; Colinet et al.,
2006; Koštál et al., 2007). A short pre-acclimation at 15 °C increases
the cold survival of A. diaperinus (Renault et al., 2004; Renault Pers.
Comm.). Therefore to avoid the risk of early mortality and ensure that
ATP levels would be assessed on stressed but living individuals,
insects were acclimated at 15 °C for 24 h before being transferred to
incubators for survival and ATP assays. As a control (Co), groups of
beetles were kept under standard rearing conditions (20 ± 1 °C, ±60%
RH). Survival was assessed in order to (i) confirm the beneficial
impact of FTR and (ii) establish the cold exposure duration where
mortality starts to be observed under chosen conditions. From each of
the two experimental conditions, Cst and FTR, one Petri dish
containing 10 beetles was removed from cold incubators and
transferred to standard rearing conditions at daily intervals. Survival
was scored as the proportion of walking beetles after 24 h. Time to
50% lethality (LT50) was computed for each condition using Probit
analysis and survival curves were compared using Mantel-Cox
analysis. Statistical analyses were conducted using MINITAB Statistical

Software Release 13 (MINITAB Inc., State College, Pennsylvania) and
Prism V5.01 (GraphPad Software Inc., San Diego, California).
2.3. Treatments for biochemical assays
Survival tests revealed that mortality began after four days of
continuous cold exposure. Therefore, ATP assays were performed on
adults cold-stressed for one, two and three consecutive days. Several
samples from different time points were analyzed each day (see Fig. 1
for design):
– Co: Control treatment, adults kept at rearing temperature (20 °C)
– Cst: Constant treatment, beetles continuously exposed to 3 °C.
– Fb30: Fluctuating treatment, 30 min before recovery (temperature
was still 3 °C).
– F30: Fluctuating treatment, 30 min of recovery at 20 °C
– F1: Fluctuating treatment, 1 h of recovery at 20 °C
– F2: Fluctuating treatment, 2 h of recovery at 20 °C
– Fa30: Fluctuating treatment, 30 min after recovery (temperature
returned to 3 °C).
– Fa1: Fluctuating treatment, 1 h after recovery (temperature
returned to 3 °C).
For each specific time point, several pools of three insects were
quickly removed from incubators, placed in 1.5-mL tubes, and frozen
in liquid nitrogen. They were stored at−80 °C until ATP assays were
performed. This procedure was repeated on days 1–3. For each
specific time point and day (see Fig. 1), the ATP assays were
performed on eight different pools of beetles (i.e. eight true biological
replicates, n = 8).
2.4. Sample preparation and ATP assays
The beetles were weighed (fresh mass) using a Mettler® microbalance accurate to 0.01 mg prior to ATP extraction. ATP was
extracted using the trichloroacetic acid (TCA) method as described
previously (Lundin and Thore, 1975; Larsson and Olsson, 1979).
Inactivation and precipitation of proteins were achieved by pouring
230 mL of cold (0 °C) TCA (0.51 M) containing EDTA (3 mM) into 1.5mL tubes containing beetles with two tungsten beads on ice. Samples
were homogenized with a bead beater for 1.5 min, and then
centrifuged for 5 min (15,000×g and 4 °C) to pellet insoluble
materials, and 170 mL of clear supernatant was then collected. The
TCA was removed by extraction with ice-cold water-saturated

Fig. 1. Schematic diagram of the experimental design used to investigate the effect of
thermal regimens on the ATP levels. Samples were obtained from (i) individuals kept
under standard conditions at 20 °C (control, Co), (ii) beetles cold stressed under a
constant 3 °C exposure (Cst), and (iii) individuals exposed to fluctuating thermal
regimes (FTR), where the 3 °C exposure was interrupted by a daily transfer to 20 °C for
2 h. Under FTR, several samples were obtained from different time points: 30 min
before the recovery (Fb30), 30 min of recovery (F30), 1 and 2 h of recovery (F1 and F2),
30 min and 1 h after the recovery (Fa30 and Fa1). ATP levels were compared among
these eight treatments on three consecutive days.

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H. Colinet / Comparative Biochemistry and Physiology, Part A 160 (2011) 63–67

diethylether (3 × 170 mL). After the last ether extraction, watersaturated N2 gas was bubbled through the solution for 5–10 min to
remove the solubilized ether. Finally, the ATP level was measured
using an ATP colorimetric/fluorometric assay kit (Biovision, Catalog
#K354-100) according to the manufacturer's instructions. Briefly,
40 μL of sample, 10 mL of assay buffer, and 50 μL of ATP reaction mix
were added to each well in a 96-well plate. After 45 min of incubation
at 27 °C in darkness, the absorbance was measured at 570 nm
(spectrophotometer VERSAmax, Molecular Devices, USA). For every
sample, a background control was performed by replacing the ATP
converter enzyme with the ATP assay buffer in the reaction mix (as
recommended by the manufacturer). The resulting background was
then subtracted from the ATP reading. The concentration of ATP was
calculated using a standard curve and expressed in nmol/mg fresh
mass. Mean ATP levels were compared using a general linear model
with different treatments (see Fig. 1) as factor, and duration as
covariate. ATP levels were also compared within each duration using
Student-Newman–Keuls comparison tests. The analysis was conducted using PASW, Release 18 (SPSS Inc., 2009).
3. Results
Cold survival was significantly affected by temperature regimen
(Fig. 2). Under continuous cold exposure, mortality occurred rapidly
with an LT50 of only 6.3 ± 0.3 days (100% mortality after 10 days).
When daily transfers from 3 °C to 20 °C were applied, mortality
decreased markedly. The LT50 under the fluctuating condition was
11.5 ± 0.7 days (100% mortality after 14 days). Comparison of survival
curves indicated a significant difference between the Cst and FTR
groups (Mantel–Cox: χ 2 = 158.5, d.f. = 1, P b 0.001). There was no
mortality before days 4 and 8 under the Cst and FTR conditions,
respectively, ensuring that ATP levels were measured on cold-stressed
but living individuals.
ATP levels varied significantly among the treatment groups
(F = 17.28, d.f. = 7, P b 0.001) (Fig. 3). On day 1, the difference was
mostly attributable to the Co value being significantly lower than the
experimental treatment values (SNK tests, P b 0.05). On day 2, ATP
levels were lowest in beetles in the Co condition, followed by the Cst
condition, wherein beetles had an intermediate value. The ATP level in
the Fa1 treatment was the highest (SNK tests, P b 0.05). The pattern of
ATP levels on day 3 was similar to that observed on day 2, with the Co
value being the lowest, followed by an intermediate Cst value; ATP

Fig. 2. Development of chill-injury in the adults of A. diaperinus exposed to a constant
low temperature of 3 °C (Cst) or a fluctuating thermal regime (FTR) (i.e. the 3 °C
exposure was interrupted by a daily transfer to 20 °C for 2 h). For each treatment 10
beetles were daily checked for survival, scored as the proportion of walking beetles
after 24 h. Lethal time LT50 was obtained from Probit analysis and temporal survival
curves were obtained from Mantel–Cox analysis. Each data point represents a mean
survival proportion (± SE).


levels in the F30, Fa30, and Fa1 assessments were highest (SNK tests,
P b 0.05). Though small variations occurred over the three sampling
days, there was no evidence of significant time-dependent variation
(F = 1.760, d.f. = 1, P = 0.186).
4. Discussion
The mechanisms underlying indirect chilling injury are not fully
understood (Chown and Terblanche, 2006). Indirect chilling injury
appears to be caused, at least in part, by alteration of selective
membrane permeability and ion homeostasis (Koštál et al., 2004,
2006; Lee, 2010; MacMillan and Sinclair, 2011). Indirect chilling
injury may also result from complex metabolic disorders (Jing et al.,
2005) with cellular respiration enzymes being particularly targeted
(Michaud and Denlinger, 2010). Mortality induced by chronic cold
stress is markedly reduced by periodic short pulses during which
insects are exposed to temperatures within an optimal range (Nedvěd
et al., 1998; Renault et al., 2004; Colinet et al., 2006; Colinet and
Hance, 2010). In the present study, the LT50 nearly doubled under the
FTR condition, relative to the Cst condition, confirming the beneficial
impact of daily warming pulses on cold survival. The recovery (repair)
processes during warming pulses involve a wealth of biological
functions (as described in the introduction) that underlie the
complexity of the nature of indirect chilling injury.
The ATP levels differed significantly across different conditions.
Contrary to the prediction, no cold-induced ATP depletion was
observed. In fact, the opposite trend was noticed in that the lowest
ATP levels were repeatedly detected in non-stressed controls. The
concentrations observed (1–2 nmol/mg) are in the range of values
generally observed in insects (e.g. Pullin and Bale, 1988; Kölsch et al.,
2002; Koštál et al., 2004). The data suggest that in beetles exposed to
cold stress, regardless of the thermal regime administered (constant
or fluctuating), metabolic pathways are disrupted and concentrations
of metabolites are thus affected resulting in modification of ATP levels.
The absence of cold-induced ATP depletion indicates that the beetles'
mitochondrial membranes were not damaged by cold exposure, at
least not to an extent that compromised ATP synthesis. Previous
studies on insects did not detect ATP depletion as a result of low
temperature exposure (Pullin and Bale, 1988; Pullin et al., 1990;
Koštál et al., 2004). On the contrary, Pullin and Bale (1988) observed
that chilled aphids accumulated high levels of ATP (up to 50% above
the level of untreated controls) 30 min after the start of chilling before
any substantial mortality occurred. Along the same line, Coulson et al.
(1992) reported a clear increase in ATP NMR signal following cold
exposure in domestic flies. An increase in the ATP pool during chilling
has also been documented in plants (Graham and Patterson, 1982;
Kuzma et al., 1995) and this occurs in both chill-resistant and chillsensitive tissues (Graham and Patterson, 1982). A modification of ATP
level could result from mismatch among various metabolic pathways
(Knight et al., 1986). For example, an increase in the ATP level can
occur when the relative rate of ATP synthesis exceeds that of ATP
consumption, though both rates are likely to decline at low
temperatures (i.e. in parallel with declining respiratory rates)
(Napolitano and Shain, 2004). The high ATP levels observed here
suggest that the respiratory apparatus of beetles (i.e. catabolic
respiratory process) was not critically damaged by low temperature,
such that ATP continued to be processed. A substantial decrease in
ATP level was reported in aphids two days after cold stress. However,
this decrease was a consequence of an increasing proportion of dead
aphids in the groups (Pullin and Bale, 1988). Dollo et al. (2010)
hypothesized that chilling injury is linked to a shortage of ATP
reserves. In their study, a decline in ATP levels was documented in
flesh flies exposed to constant 0 °C for 20 days, a treatment that was
associated with an extremely high (99%) mortality. This ATP decline
likely resulted from increasing cell death, which induces rapid
degradation of ATP by intracellular enzymes (Olejnik et al., 2004).

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H. Colinet / Comparative Biochemistry and Physiology, Part A 160 (2011) 63–67

Fig. 3. ATP levels measured in (i) individuals kept under standard condition at 20 °C (Control, Co), (ii) beetles cold stressed under a constant 3 °C exposure (Cst), and (iii) individuals
exposed to fluctuating thermal regimes (FTRs) (refer to Fig. 1 for code details). Each bar represents a mean concentration of ATP (± SE) obtained from eight true biological replicates.
White bars for non-stressed controls, gray bars for treatments at 3 °C (constant or fluctuating), and black bars for treatments at 20 °C during the fluctuation. ATP levels were
compared between the eight different treatments on day one (A), two (B) and three (C). Different letters indicate significant difference between groups within each day (SNK tests;
P b 0.05).

In the present study, high ATP levels were detected in cold-exposed
beetles during a sampling period in which no mortality occurred.
Therefore, ATP accumulation may be viewed as a symptom of an ATP
production/consumption imbalance under cold stress. A primary
consumer of energy in most cells is the ATP-dependent transport of
ions across membranes (McMullen and Storey, 2008). The reduced
activity of the ATP-consuming pumping system at low temperature
(Jing et al., 2005; Koštál et al., 2007) may partially underlie such an
imbalance. This hypothesis needs further testing.
Cold-induced mortality was strongly affected by temperature
regimens and was markedly reduced by periodic short warming
pulses. It has been suggested that energy supplies, possibly depleted
during cold exposure, could be regenerated during the warming
pulses (Chen and Denlinger, 1992). This assumption was attractive as
perturbations of the energy metabolism appear to be a common
physiological response exhibited by insects exposed to low temperatures (e.g. Colinet et al., 2007b; Overgaard et al., 2007; Ellers et al.,
2008; Michaud and Denlinger, 2010). Insects were exposed to low
temperatures in both the Cst and FTR groups in this study, and both
groups had higher ATP levels than non-stressed controls. However, no
significant increase in ATP levels was observed at any time point
within the recovery (Cst vs. F30, F1, F2).A small but significant
increase in ATP level was noted following the temperature fluctuation
when the insects were returned the 3 °C condition (i.e. Fa30, Fa1).
These data do not corroborate the assumption that ATP supplies can
be replenished during warming pulses. In flesh flies, pupae exposed to
constant cold (0 °C for 20 days) suffered higher mortality than those
exposed to cold interrupted by a unique 24 h pulse at 15 or 20 °C
(Dollo et al., 2010), confirming the positive impact of FTRs on survival.
Contrary to the present data, an increase in ATP level was noted
during the warming pulse at 20 °C (Dollo et al., 2010). However, there
was a noteworthy discrepancy between survival and ATP data. That is,
flesh flies that received a 15 °C pulse had lower mortality in the
absence of any measurable ATP accumulation during the fluctuation.
Under these conditions (i.e. 15 °C pulse), the ATP level differed from
the constant cold treatment only on day 20. The cascade of recovery
mechanisms that occur during a unique long pulse (24 h) must be
somewhat different from those that occur during short (2 h) warming
pulses applied on a daily basis. Homeostatic perturbations following
thermal stress (heat or cold) show considerable variation within a few
hours following stress (Malmendal et al., 2006; Overgaard et al.,
2007). In the present study, periodic 2-h warming pulses were clearly
beneficial to the beetles. However, an overshoot in ATP production
was not detected when compared to constant cold conditions. It is
possible that a 2-h period was too short to produce significant changes
in ATP levels.
Although rates of catabolism and anabolism were not quantified,
the results suggest that low temperature exposure has less of an effect
on the production of ATP than on its utilization, resulting in a net ATP

accumulation. After thermal stress, restoration of metabolite homeostasis takes place over a relatively long period (several hours or days)
(Malmendal et al., 2006; Overgaard et al., 2007). Therefore a 2-h
warming pulse is likely too short to reset the ATP balance completely.
Even if survival time was longer under the FTR than the Cst condition,
the beetles were deeply stressed under FTR as well, as evidenced by
the observations that beetles in the FTR and Cst groups generally
survived for less than a couple of weeks—a much shorter life span than
the average 400-day life span exhibited by beetles living under
optimal conditions (Preiss and Davidson, 1971). Abiotic stressors
generally disturb metabolic pathways, perturbing concentrations of
metabolites. The homeostatic response to these perturbations involves a fine regulation of intra- and extra-cellular environments. The
perturbation and subsequent response likely result in modifications of
metabolite compositions, such as ATP (Korsloot et al., 2004). The
findings of this study suggest that disruption of ATP homeostasis may
be viewed as a symptom of cold stress. Although periodic short
warming pulses clearly improved cold survival, cellular homeostasis
appears to require longer recovery periods to be fully restored.
Except in flesh flies for which beneficial effects of warming pulses
only operate at 15 °C (not at 5, 10, 20, 25, and 30 °C) (Chen and
Denlinger, 1992), in most species improvement of cold survival under
alternating temperatures generally occurs within a broad range of
temperatures (5–30 °C) (e.g. Nedvěd et al., 1998; Hanč and Nedvĕd,
1999; Jing et al., 2005). There might be some species-specificity in the
way recovery processes are achieved among different organisms.
Further, eco-physiological studies are needed to determine the
mechanisms underlying both the ability to recover and the speed by
which species are able to recover from chilling injuries.
I am grateful to David Renault for comments on early version of the
manuscript and Vanessa Larvor for technical assistance. This study
was supported by Fonds de la Recherche Scientifique — FNRS in
Belgium. This paper is number BRC197 of the Biodiversity Research
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