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Title: Comparing phenotypic effects and molecular correlates of developmental, gradual and rapid cold acclimation responses in Drosophila melanogaster

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Functional Ecology 2012, 26, 84–93

doi: 10.1111/j.1365-2435.2011.01898.x

Comparing phenotypic effects and molecular correlates
of developmental, gradual and rapid cold acclimation
responses in Drosophila melanogaster
Herve´ Colinet*,1,2,† and Ary A. Hoffmann2
Unite´ d’E´cologie et de Bioge´ographie, Biodiversity Research Centre, Universite´ catholique de
Louvain, Louvain-la-Neuve, Belgium; and 2Centre for Environmental Stress and Adaptation Research, Department of
Genetics, Bio21 Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
1

Summary
1. To cope with stressful environmental temperatures, organisms can enhance thermotolerance
when exposed to sub-lethal temperatures before thermal stress, a phenomenon referred to as
thermal acclimation. Acclimation includes different forms (developmental, gradual or rapid)
that vary in ecological importance depending on patterns of diurnal and seasonal thermal variation.
2. Here, we complete a comprehensive assessment of how the different forms of acclimation
based on simulated field temperatures affect cold tolerance in Drosophila melanogaster under different levels of cold stress ()4Æ5 C ⁄ 2 h and 0 C ⁄ 10 h).
3. We predict that (i) combinations of acclimation treatments may be particularly beneficial and
(ii) benefits of different acclimation types may differ for acute vs. chronic cold stress. We also
investigate whether distinct forms of acclimation promote differential molecular responses to
stress.
4. Acclimation treatments had very large effects on cold tolerance and resulted in phenotypes
ranging from sensitive to tolerant individuals within the specific cold stress applied ()4Æ5 C ⁄ 2 h
and 0 C ⁄ 10 h). Acclimation also influenced expression of several genes (Hsp23, Hsp70, Hsp40,
Hsp68, Starvin and Frost) during recovery from cold stress but effects depended on the nature of
the acclimation treatment.
5. Cumulative effects occurred between different forms of acclimation, and these as well as the
different molecular responses point to different underlying mechanisms.
6. These results highlight that combined acclimation treatments may strongly impact field stress
resistance.
Key-words: acclimation, phenotypic plasticity, cold stress, chilling injuries, Hsp, Frost, Starvin,
Drosophila melanogaster

Introduction
Organisms possess a diverse set of responses for dealing
with thermal extremes, and these may act on different
timescales, from long-term evolutionary adaptation to rapid
phenotypic adjustment (Fischer & Karl 2010). On a short
timescale, ectotherms have the capacity to enhance their

*Correspondence author. E-mail: herve.colinet@uclouvain.be

Present address: UMR CNRS 6553 Baˆt 14A, Universite´ de
Rennes1, 263 Avenue du Ge´ne´ral Leclerc CS 74205, 35042 Rennes
Cedex, France.

thermotolerance when exposed to sub-lethal temperature
before a thermal stress, a phenomenon referred to as
acclimation. Different forms of acclimatory responses exist
which differ in timing and the length of the pre-exposure
(Chown & Nicolson 2004; Angilletta 2009). Physiological
ecologists commonly distinguish between plastic responses
involving short-term vs. longer-term pre-exposure, referred
to as rapid hardening vs. acclimation, respectively (Hoffmann, Sørensen & Loeschcke 2003). Physiological adjustments also occur in response to developmental temperature
(traditionally called developmental plasticity) or in response
to environmental shifts during the later stages (Wilson &
Franklin 2002). In fact, all these phenotypic adjustments

2011 The Authors. Functional Ecology 2011 British Ecological Society

Cold acclimation and stress response 85
are collectively referred to as thermal acclimation (Chown
& Nicolson 2004; Chown & Terblanche 2006; Angilletta
2009). In this study, we use the terms developmental acclimation (DA) in response to temperature experienced during
ontogeny, as well as gradual acclimation (GA) and rapid
acclimation (RA) in responses to respectively long-term
(days) and short-term (h) pre-exposures experienced in the
mature stage (see Angilletta 2009 for more details).
The nature of acclimatory responses is complex and differs among species, thermal treatments and timing of exposure (Rako & Hoffmann 2006; Terblanche, Marais &
Chown 2007; Marais & Chown 2008). Acclimation effects
have been widely investigated in the context of cold stress
(e.g. Hoffmann, Sørensen & Loeschcke 2003; Chown & Nicolson 2004; Fischer & Karl 2010; Lee & Denlinger 2010);
however, experimental designs frequently focused on only
one type, failed to separate the distinct forms, or confounded them (Wilson & Franklin 2002; Piersma & Drent
2003; Chown & Terblanche 2006; Angilletta 2009), or used
acclimatory conditions not likely to be encountered in the
field. So far no studies have simultaneously examined the
specific contributions of the three distinct forms of cold
acclimation (i.e. DA, GA and RA) despite their potential to
cumulatively contribute to fitness under cold conditions.
The acclimatory responses probably lie along a continuum
(Loeschcke & Sørensen 2005); however, the physiological
mechanisms underlying various forms of acclimation are
thought to be different (McDonald, Bale & Walters 1997;
Kristensen et al. 2008).
Here, we examine the specific benefits of the different
forms of cold acclimation on tolerance of Drosophila melanogaster adults to cold conditions. We predict that (i) the
combination of acclimatory treatments will show some
cumulative effects affecting survival, (ii) the benefit of each
type of acclimation will differ in magnitude and (iii) the
positive effects of acclimation forms will vary in response to
acute vs. chronic cold stress. To test these hypotheses, we
used a full-factorial design and acclimation treatments relevant to field conditions to investigate the way in which the
different forms of acclimation (i.e. DA, GA and RA) affect
tolerance to both acute and chronic cold stress.
We also compare molecular responses to the different
acclimation treatments. All acclimatory responses share
some common characteristics: the detection of environmental cues, the transduction of signals into a cellular response
and the activation of certain genes, proteins and metabolites
that cause phenotypic adjustments (Angilletta 2009). Genes
coding for heat shock proteins (Hsp) are prime candidates
for phenotypic adjustments because of their link with thermotolerance (Hoffmann, Sørensen & Loeschcke 2003;
Sørensen, Kristensen & Loeschcke 2003; Chown & Nicolson 2004). Except for Hsp70, not much is known about the
other Hsps and their regulation in response to thermal
extremes (Hoffmann, Sørensen & Loeschcke 2003; Sørensen, Kristensen & Loeschcke 2003). It has recently been
shown that several Hsp genes are expressed during the
recovery from cold stress (Colinet, Lee & Hoffmann 2010a;

Michaud & Denlinger 2010); and RNAi experiments have
demonstrated that their expression is essential for cold tolerance and recovery ability (Rinehart et al. 2007; Kosta´l &
Tollarova´-Borovanska´ 2009; Colinet, Lee & Hoffmann
2010b). Although it is recognized that these genes are coldresponsive, it is not known whether their expression is
plastic because of thermal acclimation. We thus tested if the
different types of acclimation are associated with some
changes in Hsps expression during recovery phase following
cold stress. In addition, we analysed expression of two other
genes involved in cold stress response in Drosophila: Starvin
(Stv) (Colinet & Hoffmann 2010) and Frost (Fst) (Colinet,
Lee & Hoffmann 2010c).

Materials and methods
FLY CULTURE

We conducted our experiments on a mass-bred D. melanogaster population derived from more than 100 individuals collected in Melbourne in 2009. This population was maintained in the laboratory by
rearing several hundred individuals in uncrowded conditions (by
restricting the oviposition period and density of adults) in 250 mL
bottles at 19 C and 70% relative humidity under continuous light on
standard fly medium consisting of sucrose, dried yeast and agar as
previously described (Colinet, Lee & Hoffmann 2010a).

ACCLIMATION TREATMENTS

Flies were acclimated using different treatment combinations organized in a fully crossed design (see Fig. 1). To generate flies for the
experiments, groups of fifty 6-day-old females were allowed to lay
eggs in 250 mL bottles containing medium during a restricted period of 12 h at 19 C. This semi-controlled procedure allows the
flies to develop under uncrowded conditions. Females were then
removed, and bottles with eggs were randomly placed under either
cool or intermediate (i.e. moderately warm) conditions to continue
development until adult eclosion (effect of DA, Fig. 1). Programmed thermo-regulated incubators (Model IM1000R; Clayson
Laboratory Apparatus Pty Ltd, Narangba, Queensland, Australia)
were used for maintaining the flies in each experimental thermal
condition. Development proceeded under daily thermoperiods simulating either summer (i.e. moderately warm, mid 20 C, Fig. 1) or
early spring (i.e. cool, mid 15 C, Fig. 1) conditions in Melbourne
(Bureau of Meteorology: http://www.bom.gov.au). In the intermediate cycle, the temperature fluctuated as follows: 17 C from 0.00
to 04.00, 20 C from 04.00 to 08.00, 25 C from 08.00 to 11.00,
27 C from 11.00 to 13.00, 25 C from 13.00 to 16.00, 20 C from
16.00 to 20.00, 17 C from 20.00 to 24.00; the mid temperature
was 20 C, and a 14L:10D cycle starting at 05.00 was used. Flies
developing under these moderately warm conditions took 12–
13 days to emerge. In the cool thermoperiod, the temperature was
cycled as follows: 10 C from 0.00 to 04.00, 15 C from 04.00 to
08.00, 20 C from 08.00 to 11.00, 22 C from 11.00 to 13.00, 20 C
from 13.00 to 16.00, 15 C from 16.00 to 20.00, 10 C from 20.00
to 24.00; the mid temperature was 15 C and a 10L:14D cycle
starting at 07.00 was used. Flies reared under these cool conditions
took 22–23 days to emerge. Temperature variations inside incubators were recorded with Datalogger (One-Wire , model DS2422;
Thermodata Pty Ltd, South Yarra, Victoria, Australia) and actual

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

86 H. Colinet and A. A. Hoffmann
Developmental
acclimation

Gradual
acclimation

Mid temp. 15 °C
(15-15)
Mid temp. 15 °C
(15)
Mid temp. 20 °C
(15-20)

Mid ttemp. 15 °C
(20-15)

Rapid
acclimation
0 ° C for 2 h
(15-15-0)
Mid temp. 15 °C
(15-15-15)
0 °C for 2 h
(15-20-0)
Mid temp. 20 °C
(15-20-20)
0 °C for 2 h
((20-15-0))
Mid temp. 15 °C
(15-15-15)

Mid temp. 20 °C
(20)

0 °C for 2 h
(20-20-0)
Mid temp. 20 °C
(20-20)

Mid temp. 20 °C
(20-20-20)

Fig. 1. Schematic diagram of the experimental design used to investigate effects of developmental acclimation (DA), gradual acclimation
(GA), rapid acclimation (RA) and the resulting treatment combinations. Cool (mid 15 C) vs. intermediate (mid 20 C) thermoperiods
were used to acclimate Drosophila flies either during development
(DA) or in adult stage (GA). RA involved a 2 h preconditioning at
0 C applied on adults. Numbers inside parentheses represent the successive acclimation treatments received (DA-GA-RA): 15 for mid
15 C thermoperiod, 20 for mid 20 C thermoperiod and 0 for preconditioning at 0 C.

temperature changed between the different steps at a rate of about
0Æ15–0Æ2 C min)1.
After development, newly hatched adults were exposed to either
intermediate or cool thermoperiods (as described earlier) for equivalent physiological durations (effect of GA, Fig. 1). Flies were left
3 days under the intermediate thermoperiod, corresponding to 5 days
under the cool thermoperiod on a physiological time base (i.e. degreeday) (Taylor 1981) using 8Æ2 C as the base temperature (Petavy et al.
2001). Finally, adults resulting from these treatment combinations
were, or were not, rapidly acclimated (effect of RA, Fig. 1) through a
pre-exposure to cold treatment. Treated individuals were chilled at
0 C for 2 h in an ice-box, a treatment known to trigger rapid cold
hardening response (Rako & Hoffmann 2006). In contrast untreated
individuals were directly cold-stressed without RA preconditioning.
Flies were tested for cold tolerance at the end of a thermoperiod cycle
(i.e. when temperature was at a minimum in a treatment).

COLD TOLERANCE ASSESSMENT

The nature of chilling injuries derived from acute and chronic cold
stress is different (see Chown & Nicolson 2004; Lee & Denlinger 2010
for review); thus, we assumed that flies exposed to different forms of
acclimation may differentially respond to the two types of cold stress.
Therefore, tolerance to both acute and chronic cold stress was
assessed. Tolerance to acute cold stress was scored by measuring mortality 24 h after a 2 h exposure at )4Æ5 C. Most mortality in D. melanogaster adults happens within 24 h after cold stress (Rako &
Hoffmann 2006), and we therefore did not consider a longer period.
The )4Æ5 C temperature was chosen based on preliminary tests
which revealed 90% mortality in nonacclimated flies. For each treatment combination (i.e. DA · PA · RA), 15 different replicated
groups of 25 flies were placed in 42 mL glass vials immersed in a gly-

col solution cooled to )4Æ5 C for 2 h. After the stress, the flies were
returned to 25 C for recovery with food and the mortality was scored
after 24 h. The assay was performed using synchronized 5-day-old
females, sexed visually without CO2 anaesthesia using an aspirator. A
total of 375 flies was monitored for each treatment (n = 15 groups of
25 flies). A three-way ANOVA was used to investigate three main effects
(DA, PA and RA) and their interactions. Every factor had two levels:
with or without cold acclimation. Proportion dead (mortality) was
arcsin-transformed, although log-transformed data were also tested
(Promislow et al. 1996) and led to similar conclusions. The model was
refitted without the 3-way interaction because inclusion of this interaction did not improve the model. The analysis was conducted using
PASW, RELEASE 18 (SPSS Inc.).
To assess chronic cold stress tolerance, recovery time following a
nonlethal stress was measured as previously described (Colinet, Lee &
Hoffmann 2010a). Briefly, for each treatment sixty 5-day-old females
were exposed to 0 C for 10 h by immersing vials in a glycol solution,
then flies were allowed to recover at 25 C and recovery times were
individually recorded. Flies were considered recovered from chill
coma when they stood up (Hoffmann, Anderson & Hallas 2002).
Data were used to generate temporal recovery curves which were
compared among treatments with Mantel–Cox analysis using PRISM
V 5.01 (GraphPad software, Inc.) (Colinet, Lee & Hoffmann
2010a).We did not assess the effect of RA on recovery time because
total stress periods at 0 C, and consequently recovery times, were
not strictly identical between the two conditions. RA involved 2 h
preconditioning at 0 C, therefore total duration at 0 C was
2 + 10 h for treated flies vs. 10 h for untreated flies. Comparisons
were only performed among treatments resulting from the combination of DA · GA (see Fig. 1).

RNA EXTRACTION AND REAL-TIME PCR

To examine whether the distinct forms of cold acclimation affected
molecular responses to stress, we examined the expression of the following responsive genes: Hsp22, Hsp23, Hsp26, Hsp27, Hsp40,
Hsp68, Hsp70, Hsp83, Fst and Stv. Preliminary experiments revealed
that expression of Hsp60, Hsp67 and Hsc70 was not modulated after
both chronic and acute cold stress, so these were not considered. The
responsive genes show expression peaks after 2 h of recovery (Colinet
& Hoffmann 2010; Colinet, Lee & Hoffmann 2010a,c); therefore, all
flies were allowed to recover for 2 h before they were snap-frozen in
liquid nitrogen and stored at )80 C until RNA extraction. If there is
no shift in Hsp expression during recovery from cold stress among the
different phenotypes, the molecular response to stress would either be
independent of the environment experienced prior to the stress (i.e.
thermal signals) or be independent of the phenotypic adjustments
derived from these signals (i.e. increased cold tolerance). On the other
hand, differential expression of stress response would indicate acclimation-related plasticity.
RNA extractions were performed with the RNeasy RNA extraction kit and the RNase-Free DNase Set (Qiagen, Doncaster, Vic.,
Australia) as previously described (Colinet, Lee & Hoffmann 2010a).
The cDNA was synthesized using the Superscript III First-Strand
Synthesis System (Invitrogen, Mulgrave, Vic., Australia), according
to manufacturer’s instructions. Real-time PCRs were performed on
the LightCycler 480 system (Roche Diagnostics, Castle Hill, New
South Wales, Australia) following the method previously described
(Colinet, Lee & Hoffmann 2010a). The ratio of the target gene (i.e.
fold change) was expressed in treated samples vs. an untreated control
(calibrator) and normalized using the housekeeping reference gene,

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

Cold acclimation and stress response 87
RpS20 (see Colinet, Lee & Hoffmann 2010a). Expression of RpS20
did not show any modulation under any conditions tested (data not
shown), ensuring that this gene was an appropriate control. Normally, three replicates are recommended when designing expression
experiments (Lee et al. 2000). In this study, we used four true biological replicates consisting of 20 females. Hsps, Stv, Fst and RpS20 primers were the same as previously described (Colinet & Hoffmann 2010;
Colinet, Lee & Hoffmann 2010a,c). For acute cold stress, flies derived
from rearing at mid 20 C showed high and early mortality (observed
directly by the end of the stress); therefore, we did not measure gene
expression in these treatments. On the other hand, flies that developed
at mid 15 C were still alive by the end of cold stress and even after
2 h (as ascertained under a stereomicroscope); mortality occurred
later in these flies. Therefore, expression levels during recovery from
acute cold stress were only measured in flies derived from rearing at
mid 15 C (i.e. 15-15-0, 15-15-15, 15-20-0 & 15-20-20, see Fig. 1).
Expression levels were compared among treatments using ANOVA with
two fixed factors: GA and RA for acute cold stress and DA and GA
for chronic cold stress.

Results
ACUTE COLD STRESS

Acute cold tolerance varied considerably according to acclimation treatments. Mortality ranged from 87Æ7% in nonacclimated flies (20-20-20) to only 3Æ5% in flies that received
multiple acclimation treatments (15-15-0) (Fig. 2). The
analysis revealed that the main effects (i.e. DA, GA & RA)
were all significant (Table 1, Fig. 3). The strong impact of
GA on acute cold tolerance was manifested by a high proportion of total variation explained by this factor (i.e.
effect-size, g2) (Table 1). The benefit of acclimation was
detectable even when only one treatment was applied (2020-20 vs. 20-20-0 or 20-15-15 or 15-20-20). Combinations of
two acclimation treatments (20-15-0 or 15-20-0 or 15-15-15)
further decreased mortality compared with a single acclima-

tion treatment (20-20-0 or 20-15-15 or 15-20-20) (Fig. 2).
Absence of DA · GA interaction (Table 1, Fig. 3) indicates
that the positive effect of GA was similar whatever the
developmental temperature. The significant DA · RA and
GA · RA interactions (Table 1, Fig. 3) resulted from
stronger effects of RA in flies that had not been previously
acclimated.

CHRONIC COLD STRESS

For this stress, all flies recovered within a period ranging
from 9 to 43 min (Fig. 4). The kinetics of recovery varied
significantly according to GA. Flies recovered faster when
they had been exposed to a few days of thermoperiodic
acclimation as adults (20-20 vs. 20-15 & 15-20 vs. 15-15;
Table 1, Fig. 4). On the other hand, we did not observe any
effect of developmental temperature (DA) on the recovery
patterns (20-20 vs. 15-20 & 20-15 vs. 15-15; Table 1, Fig. 4).
Finally, comparisons across treatments indicated that GA
treatment resulted in faster recovery whatever the developmental temperature (20-15 vs. 15-20 & 20-20 vs. 15-15;
Table 1, Fig. 4).

MOLECULAR RESPONSE TO COLD STRESS

We focused on 10 genes involved in cold stress response
and tested whether they exhibited acclimation-related plasticity of expression during the recovery from cold stress.
In the flies recovering from acute cold stress, six genes
showed significant variation of expression according to

Table 1. Analyses of cold responses. A three-way ANOVA assessed the
effects of developmental acclimation (DA), gradual acclimation (GA)
and rapid acclimation (RA) on tolerance to acute cold stress ()4Æ5 C
for 2 h). The eta-squared (g2) describes the proportion of the
variance that is attributable to each effect. Concerning chronic cold
tolerance, temporal recovery curves were compared among the
treatments resulting from the combination of DA · GA, using
Mantel–Cox analysis. Cool (mid 15 C) or intermediate (mid 20 C)
thermoperiods were used to acclimate the flies (refer to Fig. 1 for
details on acclimation treatment codes used).
Acute cold stress

Fig. 2. Percent mortality of Drosophila melanogaster adults exposed
to acute cold stress ()4Æ5 C) for 2 h. Mortality was scored 24 h after
acute cold stress. Bars represent mean percentage (+SE) derived
from 375 females for each treatment combination (n = 15 groups of
25 flies). Codes for every acclimation treatment combinations are provided along the x-axis (refer to Fig. 1 for details).

Factors

d.f.

DA
GA
RA
DA · GA
DA · RA
GA · RA
Error

1
3Æ80
112Æ18
1
7Æ47
220Æ37
1
1Æ77
52Æ36
1
0Æ086
2Æ52
1
0Æ144
4Æ24
1
0Æ199
5Æ85
119
17Æ31
Chronic cold stress

Curve comparisons

d.f.

20-20 vs. 20-15
20-20 vs. 15-20
20-20 vs. 15-15
15-20 vs. 15-15
15-20 vs. 20-15
20-15 vs. 15-15

1
1
1
1
1
1

SS

F

v2

P

33Æ03
0Æ397
34Æ63
39Æ38
36Æ43
0Æ002

<0Æ001
0Æ528
<0Æ001
<0Æ001
<0Æ001
0Æ968

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

P

g2

<0Æ001
<0Æ001
<0Æ001
0Æ115
0Æ042
0Æ017

21Æ97
43Æ16
10Æ25
0Æ49
0Æ83
1Æ14
22Æ13

88 H. Colinet and A. A. Hoffmann

Fig. 3. Main effect plots describing average outcome for each type of acclimation (DA, developmental acclimation; GA, gradual acclimation;
RA, rapid acclimation) on acute cold tolerance of Drosophila melanogaster. Marginal means ± SE of arcsin-square root transformed proportion
mortality are shown. Interaction plots are also shown and illustrate the relationships between the three main factors (refer to Table 1 for full
results of the general linear model on acute cold tolerance).

Fig. 4. Temporal recovery curves of Drosophila melanogaster adults exposed to
chronic cold stress (0 C) for 10 h. Each dot
represents the mean proportion (+SE) of
recovering flies in relation to time after cold
stress. Sixty females were tested per condition. Each acclimation treatment combination is indicated using different symbols and
codes (refer to Fig. 1 for code details).

acclimation treatment (Fig. 5). Hsp23 was highly up-regulated in every condition but expression levels were significantly higher in flies that received GA treatment (15-15-15
& 15-15-0) compared with flies without GA treatment
(15-20-20 & 15-20-0) (Fig. 5, Table S1, Supporting
Information). Hsp40, Hsp68, Hsp70 and Stv were also
up-regulated during the recovery but their expression were
significantly higher in flies that did not receive GA
treatment (i.e. less cold-tolerant phenotypes: 15-20-20 &
15-20-0) compared with flies with GA treatment (15-15-15
& 15-15-0) (Fig. 5, Table S1, Supporting Information).
ANOVAs revealed a significant effect of GA for all these
genes but no effect of RA and no interaction (Table S1,
Supporting Information). Fst was the only gene whose

expression varied significantly depending on both the GA
and RA treatments. There was also an interaction between
these two factors (Table S1, Supporting Information).
Expression of Hsp22, Hsp26, Hsp27 and Hsp83 did not
vary according to any acclimation treatment (Fig. 5,
Table S1, Supporting Information).
In the flies recovering from chronic cold stress, Hsp23
was highly up-regulated and its expression was again significantly higher in flies that received GA treatment (20-15 &
15-15) compared with flies without GA treatment (20-20 &
15-20) (Fig. 6, Table S1, Supporting Information). In contrast, expression of Hsp40, Hsp70, Fst and Stv was significantly higher in flies that did not receive GA treatment (i.e.
less cold-tolerant phenotypes: 20-20 & 15-20) (Fig. 6,

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

Cold acclimation and stress response 89

Fig. 5. Acute cold stress: the mRNA expression of the assayed genes in Drosophila melanogaster females recovering for 2 h after being exposed
to )4Æ5 C for 2 h. The mRNA level (i.e. fold change) of the target genes is expressed relative to control and normalized against the housekeeping
reference gene, RpS20. Bars represent mean expression (+SE) derived from four independent samples. Each acclimation treatment combination
is indicated using different bar colours and codes on the x-axis (refer to Fig. 1 for code details). The symbol (*) indicates when acclimation (GA
or RA) significantly affected gene expression.

Fig. 6. Chronic cold stress: the mRNA expression of the assayed genes in Drosophila melanogaster females recovering for 2 h after being
exposed to 0 C for 10 h. The mRNA level (i.e. fold change) of the target genes is expressed relative to control and normalized against the housekeeping reference gene, RpS20. Bars represent mean expression (+SE) derived from four independent samples. Each acclimation treatment combination is indicated using different bar colours and codes on the x-axis (refer to Fig. 1 for code details). The symbol (*) indicates when
acclimation (developmental acclimation or gradual acclimation) significantly affected gene expression.

Table S1, Supporting Information). ANOVAs revealed significant effect of GA for these five genes but no effect of DA
and no interaction (Table S1, Supporting Information).

Expression of Hsp22, Hsp26, Hsp27, Hsp68 and Hsp83 did
not vary after any of the acclimation treatments (Fig. 6,
Table S1, Supporting Information).

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

90 H. Colinet and A. A. Hoffmann

Discussion
To understand how D. melanogaster copes with low temperatures, we investigate phenotypic plasticity of cold tolerance
in response to three distinct forms of cold acclimation:
developmental, gradual and rapid. Whereas many studies
have not distinguished between these distinct forms and
tend to use constant temperatures for acclimation, we used
thermoperiodic acclimation regimes relevant to thermal
conditions experienced in nature. To investigate environmental effects on cold tolerance plasticity, mortality after
acute cold stress and recovery after chronic cold stress were
analysed; both indices have been linked to adaptive patterns
that match expectations based on climatic conditions (Watson & Hoffmann 1996; Hoffmann, Anderson & Hallas
2002; Ayrinhac et al. 2004).
Flies suffered high mortality when they were not previously acclimated either during development or as an adult.
Every acclimation type individually promoted acute cold
tolerance, although with varying magnitude. Thermoperiodic acclimation of only a few days (GA) induced a strong benefit, confirming previous observations that GA markedly
increases acute cold tolerance (Chen & Walker 1994; Kristensen et al. 2008). If it is assumed that overall dynamics of
poststress mortality is similar between phenotypic groups,
GA treatment appeared to have the largest effect on mortality. In the monarch butterfly, long-term acclimation (GA)
promotes cold tolerance to a greater extent than RA (Larsen
& Lee 1994). Interestingly, the magnitude of LT50 reduction
associated with RA is considerably smaller than that
induced by GA in Drosophila, suggesting a different and less
effective cold protection mechanism (Sinclair & Roberts
2005). The cold protection acquired during natural thermoperiod is cumulative (Kelty & Lee 2001), so the level of cold
tolerance could progressively increase with multiple thermoperiods experienced during GA. On the other hand, RA
response is known to be transient and to be rapidly lost on
return to optimal temperatures (McDonald, Bale & Walters
1997; Li, Gong & Park 1999). The benefit of RA was more
evident in flies that had not been previously acclimated. Similarly, the beneficial effect of RA is greater in aphids maintained at 20 C compared with aphids acclimated at 10 C
(Powell & Bale 2006). Terblanche & Chown (2006) also
reported a differential RA response, where summer-acclimated kelp fly larvae showed a RA response unlike winteracclimated individuals. This suggests that the magnitude of
RA response depends on basal thermotolerance previously
acquired and explains the interactions observed. The fact
that ability to rapidly cold-harden is maintained in flies that
have been previously cold acclimated also suggests that the
two processes are different and can function in combination.
Our data also confirm the prediction that combinations
of acclimatory treatments result in cumulative effects. Such
effects between two acclimation treatments have been noted
in other species (McDonald, Bale & Walters 1997; Powell &
Bale 2005; Shintani & Ishikawa 2007). This suggests that

underpinning mechanisms of the different acclimation
forms are at least partially independent. However, additive
response is not a general rule. Rajamohan & Sinclair (2009)
reported an antagonistic interaction between GA with RA.
Their study was performed on developing stages (larvae),
and increased mortality may have been caused by the cost
associated with the RA treatment itself on this cold-susceptible stage (Jensen, Overgaard & Sørensen 2007). Overall, a
striking feature of the current study is the high level of variability of acute cold tolerance because of plastic responses.
Mortality of flies experiencing multiple acclimation treatments was negligible, suggesting tolerance to the specific
cold stress applied, whereas almost all nonacclimated flies
died. This massive environment-dependent variability corroborates previous observations (Ayrinhac et al. 2004;
Hoffmann, Shirriffs & Scott 2005) indicating that a large
part of phenotypic variation of cold tolerance can be due to
environmental effects.
We found no evidence of DA affecting recovery time,
while thermoperiodic acclimation of only few days (GA)
was beneficial for chronic cold tolerance. Similarly, Terblanche & Chown (2006) reported a large effect of GA on
critical thermal minimum (CTmin), while DA had little
effect on this trait. The absence of a DA effect suggests that
acclimation has different effects on chill coma (noninjurious) and chilling injury (that cause defects that cannot be
recovered). These traits are likely caused by different mechanisms (Macmillan & Sinclair 2011). Our data suggest that
thermoperiodic acclimation experienced during development is not important for chronic cold tolerance, while the
proximal thermal environment experienced in adults just
before chronic cold stress is influential. This supports observations of Fischer et al. (2010) who recently reported that
thermal environment experienced during the last day prior
to testing strongly impacted recovery time, while the effects
of previous thermal experience were more subtle. The
absence of a DA effect on recovery time may at first seem
surprising because significant effects of developmental temperature had previously been reported (Ayrinhac et al.
2004). However, in that study, flies were kept at their
growth temperature for several days before being subjected
to chronic cold stress. It resulted that flies were acclimated
during both development and as adults. Our data highlight
how acclimation-related plasticity in cold tolerance is a
remarkable example of adaptive phenotypic plasticity. We
corroborate the predictions that (i) combinations of acclimatory treatments show cumulative effects, (ii) the benefits
of each type of acclimation differ in magnitude and (iii)
effects differ in response to acute vs. chronic cold stress.
Our molecular work provides some of the first evidence
for an acclimation-related plasticity of stress gene expression during recovery. Thermoperiodic acclimation of adults
(GA) was responsible for changes in expression, while no
such modulation resulted from DA or RA. Expression patterns involved the same set of genes in response to acute
and chronic cold stress, except for Hsp68 which only varied
in flies exposed to acute cold stress. Hsp70, Hsp40, Stv and

2011 The Authors. Functional Ecology 2011 British Ecological Society, Functional Ecology, 26, 84–93

Cold acclimation and stress response 91
Fst displayed similar patterns of expression: their levels
were lower in the cold-tolerant phenotypes. Given the
relationship of Hsps (particularly Hsp70) to thermotolerance (Sørensen, Kristensen & Loeschcke 2003), low Hsp
expression would at first seem maladaptive. However, if
expression of these genes is an indicator of damage because
of stress, then lower expression in cold-tolerant phenotypes
might be expected as these should accumulate less chilling
injuries. The concomitant expression of Hsp70, Hsp40 and
Stv is not surprising as Hsp40 and Stv are essential co-chaperones of Hsp70 involved in its regulation (Cobreros et al.
2008; Colinet & Hoffmann 2010). Previous results have
shown that upregulation of responsive Hsps and Fst during
recovery is related to some undefined repairing functions
(Colinet, Lee & Hoffmann 2010a,c), and this was further
supported by tests involving RNAi experiments (Kosta´l &
Tollarova´-Borovanska´ 2009; Colinet, Lee & Hoffmann
2010b,c). In copper butterflies, high-altitude populations
show increased cold resistance compared with low-altitude
ones; this natural phenotypic variation is matched by differences in Hsp70 expression, which is substantially lower in
high-altitude populations (Fischer & Karl 2010). Along the
same line, in Drosophila buzzatii Hsp70 expression decreases
in response to heat stress when selected for heat resistance,
and this is attributable to the cost of Hsp70 expression
(Sørensen et al. 1999). We suggest that corresponding
patterns between cold tolerance and expression of stress
responsive genes may partially underlie the mechanistic
differences between GA-related phenotypes. The absence of
DA- and RA-dependent modulations in molecular stress
response of Hsps indicates that other physiological adjustments underlie phenotypic differences following these types
of acclimation. Previous studies suggested that induction of
Hsp70 and other stress genes regulated by heat shock factor
are not directly involved in RA response (Kelty & Lee 2001;
Nielsen et al. 2005; Wang et al. 2011). Other systems of protection such as increasing glucose and trehalose (Overgaard
et al. 2007) and lipid remodelling in membranes (Overgaard
et al. 2005, 2008) may underlie phenotypic effects resulting
from DA and RA. In this study, we looked at mRNA levels
of stress genes during their peak of expression; however,
there is generally a delay in temporal dynamics of mRNA
and the onset of Hsp production (Bahrndorff et al. 2009;
Kosta´l & Tollarova´-Borovanska´ 2009). It would therefore
be interesting to test whether the acclimation-related plasticity of stress gene expression is also manifested at the
protein level.
Based on the assumption that cold-responsive genes
are involved in repair functions, our expectation was
that flies expressing high levels should accumulate more
chilling injuries and therefore be the least cold tolerant.
This pattern is confirmed for five genes (Hsp70, Hsp40,
Hsp68, Stv and Fst); however, surprisingly an opposing
pattern was found for Hsp23. In all treatment combinations, Hsp23 and Hsp22 were highly up-regulated during
the recovery, confirming their function during the recovery (Colinet, Lee & Hoffmann 2010a,b). However,

Hsp22 showed no acclimation-induced plasticity of
expression whereas Hsp23 did. This corroborates the
notion that both genes may contribute to cold tolerance
in different ways (Colinet, Lee & Hoffmann 2010b). It
is unclear, however, whether high expression of Hsp23
in cold-tolerant phenotypes reflects protective rather
than repairing function. High expression of Hsp70 (and
its co-chaperone machinery) induces significant resource
cost because of the shut-down of normal cell functions
(Sørensen, Kristensen & Loeschcke 2003). This might
affect plastic changes of other gene ⁄ protein synthesis.
Fst was the only gene whose expression varied according to both GA and RA. In all the cases, expression
levels were lower in the more cold-tolerant phenotypes.
This pattern is consistent with the notion that Fst is
primarily involved in repairing functions during the
recovery (Colinet, Lee & Hoffmann 2010c). The changes
in Fst expression following RA suggest that this gene is
good candidate for rapid cold hardening response (Qin
et al. 2005).
In this study, cold tolerance was assessed at the end of
a thermoperiod cycle (i.e. when temperature was at the
minimum). As cold tolerance varies with the thermoperiod
(Kelty 2007), it will be interesting to test whether the
responses observed are conserved when flies are tested at
different time points in the thermoperiod cycle. Moreover,
full-genome transcriptomics or proteonomics studies may
reveal information on new candidate genes. Overall this
study has shed light on both phenotypic and molecular
aspects of thermal acclimation. Different types of acclimation have varying benefits for different types of cold
stress. Cumulative effects suggest the mechanistic basis of
the different types of acclimation vary, which was also
supported by the molecular patterns of response to stress.
These results provide a connection between ecological
conditions and molecular responses and a basis for understanding the evolution of plastic responses under different
thermal conditions.

Acknowledgements
We are grateful to Nancy Margaret Endersby and Jason Axford for assistance
and access to incubators. This study was supported by Fonds de la Recherche
Scientifique – FNRS, and the Australian Research Council via their Discovery
and Fellowship schemes. This paper is number BRC206 of the Biodiversity
Research Centre.

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