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ARTICLE IN PRESS
Insect
Biochemistry
and
Molecular
Biology
Insect Biochemistry and Molecular Biology 37 (2007) 1177–1188
www.elsevier.com/locate/ibmb

Proteomic profiling of a parasitic wasp exposed to constant and
fluctuating cold exposure
Herve´ Colineta, , Thi Thuy An Nguyenb, Conrad Cloutierb,
Dominique Michaudc, Thierry Hancea
a

Unite´ d’E´cologie et de Bioge´ographie, Centre de Recherche sur la Biodiversite´, Universite´ Catholique de Louvain, Croix du Sud 4-5,
B-1348 Louvain-la-Neuve, Belgium
b
De´partement de Biologie, Universite´ Laval, Cite´ Universitaire, Que´bec, Canada G1K 7P4
c
De´partement de Phytologie, Universite´ Laval, Cite´ Universitaire, Que´bec, Canada G1K 7P4
Received 3 May 2007; received in revised form 6 July 2007; accepted 7 July 2007

Abstract
When insects are exposed to fluctuating thermal regimes (FTRs) (i.e., cold exposure alternating with periodic short pulses to high
temperature), in contrast to constant low temperature (CLT), mortality due to accumulation of chill injuries is markedly reduced. To
investigate the physiological processes behind the positive impact of FTR, based on a holistic approach, two-dimensional electrophoresis
(2-DE) analysis were performed with the parasitic wasp Aphidius colemani. Parasitoid proteomes revealed 369 well-distinguishable
protein spots, where the overall response to cold exposure was clearly specific to treatments (CLT versus FTR). The reduced mortality
under FTR was associated with up-regulation of several proteins playing key roles in energy metabolism (glycolysis, TCA cycle, synthesis
and conversion of ATP), protein chaperoning (Hsp70/Hsp90), and protein degradation (proteasome). Our results also support the idea
that cytoskeleton components, particularly actin arrangement, could play a role in the higher survival rates of insects under FTR.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Proteomics; Parasitoid; Cold stress; Chill injuries; Recovery

1. Introduction
The development and survival of ectotherms is strongly
dependent on the thermal environment. Temperature
affects simultaneously numerous physiological processes
and biophysical structures. It influences metabolic activities, development rates and growth (e.g., Sinclair et al.,
2003). During seasonal cycles, many insect species are
frequently exposed to stressing low temperatures in their
natural environments (Hance et al., 2007). The cellular
stress response (CSR) can be defined as a reaction to the
threat of macromolecular damage. It comprises mechanisms that protect cells from sudden environmental change
or frequent fluctuations in environmental factors (Ku¨ltz,
2003). CSR involves a set of important cellular functions
Corresponding author. Tel.: +32 10 47 34 91; fax: +32 10 47 34 90.

E-mail addresses: colinet@ecol.ucl.ac.be (H. Colinet),
hance@ecol.ucl.ac.be (T. Hance).
0965-1748/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2007.07.004

including cell cycle control, protein chaperoning and
repair, DNA stabilization and repair, removal of damaged
proteins, and certain aspects of energetic metabolism
(Ku¨ltz, 2003). Proteins involved in key aspects of the
CSR are conserved in all organisms and can be identified
using proteomic approaches in combination with mass
spectrometry analysis (Ku¨ltz, 2005).
In many insect species, lethal cumulative chill injuries
(i.e., cold-induced damages without ice crystal formation)
occur at temperatures even above 0 1C. Though the
knowledge has substantially progressed in the field of
thermal biology, the main causes chill injuries are still not
fully understood (Renault et al., 2002; Kosˇ ta´l et al., 2004,
2006; Yocum et al., 2006). The complex nature of chill
injuries probably results from various physiological dysfunctions including a loss of membrane potential resulting
in the leakage of cytoplasmic solutes (Slachta et al., 2002),
neuromuscular injuries (Kelty et al., 1996), thermoelastic stress (Lee and Denlinger, 1991), ion homeostasis

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H. Colinet et al. / Insect Biochemistry and Molecular Biology 37 (2007) 1177–1188

perturbation (Kosˇ ta´l et al., 2004, 2006) and inhibition of
critical gene(s) expression (Yocum et al., 2006). Moreover,
low temperatures can induce disruption of the fine balance
between substrates and products because of the thermal
dependence of different metabolic pathways (Knight et al.,
1986). The non-proportional decrease (imbalance, decoupling) in enzymatic reactions and transport may either
result in the accumulation of potentially toxic metabolic
substances, such as lactic acid (Nedve˘d et al., 1998) and
free radicals (Rojas and Leopold, 1996), or induce the lack
of essential metabolites, such as ATP (Hochachka, 1986;
Churchill and Storey, 1989a).
Several studies have shown that exposing insects to
fluctuating thermal regimes (FTRs) (i.e., cold exposure
interrupted by periodic short pulses to high temperature),
versus constant low temperatures (CLT), significantly
reduces mortality in most species tested to date (Chen
and Denlinger, 1992; Leopold et al., 1998; Nedve˘d et al.,
1998; Renault et al., 2004; Colinet et al., 2006a; Kosˇ ta´l et
al., 2007). It was frequently suggested that chill injuries,
which accumulate during the cold periods, are ‘repaired’
during the brief high-temperature intervals. The interest in
understanding the functional processes behind this phenomenon has only recently developed. Kosˇ ta´l et al. (2007)
suggested that under low temperatures, unfavourable for
gene transcription, mRNA splicing and protein translation
and folding, aberrant proteins may increase; and therefore,
stress proteins may be up-regulated during the recovery
periods. An RNA-based study recently found that expression of heat shock proteins (Hsps) was increased under
FTR, however, there was a discrepancy between maximum
survival and Hsps accumulations (Wang et al., 2006). The
role of cryoprotectants has also been investigated, and it
was shown that some of them may contribute to the
increased survival under FTR (Wang et al., 2006; Lalouette
et al., 2007). Kosˇ ta´l et al. (2007) also showed that ion
pumping systems could re-establish electrochemical gradients during high-temperature pulses. Finally, we recently
reported very different metabolic responses in free amino
acids levels (FAA) under CLT versus FTR, where FAA
significantly accumulated under CLT, probably because of
protein breakdown, disruption of metabolic pathways, and
reduction of protein synthesis (Colinet et al., 2007). Since
temperature simultaneously affects many physiological
processes, the nature of chill injuries and the physiological
mechanisms involved during the recovery periods, are
likely very complex.
To broadly investigate the physiological processes underlying the positive impact of FTR, proteomics offer a great
tool, since it allows the examination of the entire complement
of proteins in an organism, tissue, or cell type (Shi and
Paskewitz, 2006). Proteomics goes one step further in
functionality than RNA-based studies, which cannot ensure
that transcripts are actually translated (Li et al., 2007; Gygi
et al., 1999). By working at the protein level, it may be
possible to discover post-translational modifications involved
in key processes of the recovery from chill-injuries. Along

this same line, using proteomics, Li et al. (2007) discovered
that many more Hsps were present in diapausing flesh fly
than it was previously anticipated.
To investigate changes in protein profiles during
recovery from chilling stress, we carried out two-dimensional electrophoresis (2-DE) analysis using the chillsusceptible parasitic wasp Aphidius colemani Viereck
(Hymenoptera: Aphidiinae) as study model. This small
parasitoid is commercially produced and distributed as an
aphid biocontrol agent, targeting primarily Myzus persicae
Sulzer (Homoptera: Aphididae) in glasshouses of several
European and North American countries.
2. Materials and methods
2.1. Rearing aphids and parasitoids
The green peach aphid, M. persicae, was used as a host
in parasitoid rearing and laboratory cultures. They were
established from individuals collected in agricultural fields
around Louvain-la-Neuve, Belgium (50.31N latitude) in
2000. Aphids were reared in 0.3 m3 cages on sweet pepper
(Capsicum annuum L.) under laboratory conditions of
18 1C, 760% RH and long-day photoregime (L:D, 16:8 h).
The parasitoid A. colemani, originally provided by
Viridaxis SA (Belgium), was reared in the laboratory on
M. persicae under the same conditions.
To obtain standard aphid mummies containing parasitoid pupae, batches of 50 standardized 3-day-old aphids
were offered to a female parasitoid wasp for 4 h. Parasitoid
wasps were less than 48 h old, naı¨ ve, and mated. The
resulting parasitized aphids were then reared under
laboratory conditions (18 1C, 760% RH and L:D 16:8 h)
until mummification. Newly formed mummies were left to
develop for 1 day, under the same rearing conditions,
before cold exposure.
2.2. Thermal treatments and chill-susceptibility assays
The mummies were cold exposed to 4 1C, a temperature
known to affect A. colemani survival (Colinet et al.,
2006a, b). One-day-old mummies were placed in small
plastic Petri dishes. Mummies were then exposed to low
temperature inside thermo-regulated LMSs incubators,
with saturated relative humidity and complete darkness.
Mummies were randomly assigned to either constant or
fluctuating temperature regimes:




CLT: 4 1C for the entire duration of the experiment.
FTR: the 4 1C exposure was interrupted daily (every
22 h) by a transfer to 20 1C for 2 h.

To investigate the effects of the thermal treatments on
parasitoid survival, three batches of 50 mummies were
removed from each experimental condition and kept at
20 1C. The survival after 5, 10, and 15 days of cold
exposure, expressed as the emergence rate, was assessed as

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the number of adult A. colemani wasps that successfully
emerged from the mummies when replaced at 20 1C. A
non-cold-exposed control (i.e., three batches of 50 mummies) was maintained at 20 1C, and wasp emergence was
similarly monitored.
2.3. Conditions for proteomics
To investigate the impact of thermal treatments on
protein expression, proteomics was performed on:





individuals coming from the control condition (Co),
individuals exposed to CLT for 5 and 10 days (C5 and
C10, respectively), and
individuals exposed to FTR for 5 and 10 days (F5 and
F10, respectively).

For each experimental condition, several groups of 50
individuals were removed from incubators, immediately
frozen in liquid nitrogen and kept at 80 1C until protein
extraction. For conditions F5 and F10, the individuals
were frozen at the end of the 2-h-warm interval.
2.4. Protein extraction
Groups of 50 mummies were ground to fine powder in
liquid nitrogen. The proteins were directly precipitated for
2 h at 201C in a solution containing 10% trichloracetic
acid (TCA) and 0.07% (v/v) 2-mercaptoethanol in acetone.
After centrifugation at 20,000g for 30 min at 4 1C, the pellet
was washed two times with 10% (v/v) distilled water in
acetone with a centrifugation between each wash. The
pellet was then solubilized in an electrophoretic sample
buffer consisting of 8 M urea (Sigma-Aldrich) containing
2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3–10, and
60 mM dithiothreitol (DTT) (Sigma-Aldrich). Protein
concentrations in the samples were determined using the
method of Bradford (1976), with ovalbumin as standard
protein according to the supplier recommendation (Biorad). Final sample volumes of each extract were adjusted to
standardize the amount of protein per sample (i.e., 300 mg/
250 ml) for electrophoresis.
2.5. Two-dimensional electrophoresis
For the first, isoelectric focusing (IEF) step, 250 ml
samples were loaded on immobiline IPG strips (13 cm) with
non-linear pH 3–10 gradients, and resolved using the
IPGphor apparatus (Amersham Biosciences, Baie d’Urfe´,
Que´bec, Canada). After active rehydration for 12 h, IEF
was performed following a voltage step-gradient (100 V for
1 h, 500 V for 1 h, 1000 V for 1 h, 5000 V for 1 h, and 8000 V
to reach 19,000 V-h). Before the second step (SDS–PAGE),
the IPG strips were first equilibrated for 15 min in a
solution containing 6 M urea, 50 mM Tris–HCl pH 8.8, 2%
(w/v) SDS, 30% (v/v) glycerol, and 60 mM DTT, and then
for 15 min in the same solution, substituting DTT with 5%

1179

iodoacetamide (Sigma-Aldrich). The second dimension was
carried out on a 1 mm thick 12% (w/v) polyacrylamide gel.
Gels were run at constant 30 mA until the bromphenol blue
dye front migrated 2 cm from the bottom. The proteins
were fixed overnight in water containing 10% (v/v) acetic
acid, 50% (v/v) methanol, and stained with GelCode blue
reagent (Pierce, Rockford, IL, USA) following the
manufacturer’s instructions. Three different replicate gels
from individuals exposed to the same experimental
conditions were performed to allow subsequent statistical
assessments.
2.6. Image analysis and protein identification
The 2-DE gels were digitized and analyzed using the
Phoretix 2D Expression software, v. 2005 (NonLinear
USA Inc., Durham, NC, USA). Protein spots intensities
were normalized using method of ‘total spot volume’ (i.e.,
each spot being expressed as a percentage of the total spot
volume on that gel) to account for variations in emission
levels between images and the background subtraction was
performed following the supplier’s recommendations.
Detection by gel to gel matching was performed to identify
differences among them. The spots selected for identification were excised from gels manually and sent to Genome
Quebec Innovation Centre (McGill University, Montreal,
Canada) for mass spectrometric analysis (tandem MS–MS:
ESI-QUAD-TOF). Protein identification was performed
using the MASCOT algorithm (http://www.matrixscience.
com) with the following parameters: Drosophila or ‘other
metazoa’ of NCBI database, fixed modification were
carbamidomethyl of cysteine residues and methionine in
oxidized form, a peptide mass tolerance of 0.5 Da, 2+ and
3+ as peptide charge, and monoisotopic experimental
mass values.
2.7. Statistical analyses
Arcsin square root transformation was required to
normalise the distribution of the emergence rate, which
was analysed using a two-way ANOVA (Proc GLM, SAS
Institute, Cary, NC, USA, 1990) with thermal treatment
and duration of cold exposure as factors. Multiple
comparisons were then performed using Tukey’s HSD
tests to describe differences between treatments. Data
presented in the figures are untransformed.
For proteomic analysis, normalized volumes of each spot,
reflecting protein expression levels, were analysed using
ANOVAs (Proc GLM, SAS Institute, Cary, NC, USA,
1990) with two crossed factors ‘thermal treatment’ and
‘duration of cold exposure’ (i.e., comparison among C5,
C10, F5, F10). Dunnett’s pairwise multiple comparison
t-tests were also used to compare the each treatment value
with control mean (Co). Because spot by spot ANOVAs
does not provide a comprehensive way to separate treatment
effects (Nguyen et al., 2007), we also used a multivariate
approach, whereby treatment-specific variation factors from

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Fig. 1. Percentage of emerging adults (mean7S.E., n ¼ 3) as a function of duration of cold exposure for each treatment: constant low temperature (filled
squares) and fluctuating thermal regime (open circles).

control, were considered as data in the analysis for the whole
proteome and thermal treatments as variables. For each
protein, the mean variation ratios (i.e., fold factor) based on
the three replicate gels of each experimental condition were
considered in the analysis. This analysis was carried using
principal component analysis (PCA) (Proc PRINCOMP,
SAS Institute, Cary, NC, USA, 1990) allowing to the
detection of the structure in the relationships between
variables.

3. Results
3.1. Survival during cold exposure
As expected, the survival expressed as emergence rate
(Fig. 1), decreased significantly with duration of cold
exposure (ANOVA2: F ¼ 57.05, Po0.001), and this was
particularly manifest under CLT. Tukey’s tests revealed that
among both conditions, survival to emergence was the highest
in control mummies (no cold exposure, duration ¼ 0), then it
decreased in mummies cold exposed for 5, 10, and finally 15
days. The emergence rate varied significantly according to
treatments (ANOVA2: F ¼ 51.83, Po0.001). The mortality
of individuals under FTR was clearly lower than under CLT
over periods of time superior to 5 days.
3.2. Proteomic analysis
The 2-DE pattern revealed approximately 369 individual
spots corresponding to A. colemani proteins with molecular
masses ranging from 6.5 to 200 kDa, and isoelectric point
between 3 and 10 (Fig. 2). The cold exposure had a clear
impact on expression of several proteins. Spot by spot twoway ANOVA revealed only 55 spots showing a duration
effect (Po0.05). Variation in spot intensities was much
more manifest between thermal treatments, with 123 spots
out of 369 showing a significant treatment effect (Po0.05).

Fig. 2. 2D-PAGE reference gel separation of proteins from A. colemani.
Positions of molecular size markers (kDa) located on the left side of the gel
and PI range for isoelectric focusing is indicated on the top. Spot numbers
are indicated on proteins successfully identified by mass spectrometry
analysis.

Among all proteins detected, only 22 showed a significant
interaction between treatment and duration (Po0.05). To
get an overview of the general treatment-specific reactions
to the cold-exposure, PCA was performed using variation
ratio based on control (i.e. fold factor) as data set and
thermal treatments as variables. Fig. 3 illustrates the
distribution of scores in the space formed by the first two
principal components (PC) extracted (with eigenvalues
41). The PC1 explained 59.37% of the variance, and PC2

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identifications revealed eight proteins (spots 15, 29, 36,
54, 65, 95, 280, 345) playing crucial roles in energy
metabolism (including glycolysis, TCA cycle, and ATP
production), two Hsps (spots 7, 217), a proteolytic enzyme
(spot 145), a protein involved in signal transduction (spot
84), a protein involved in actin regulation (spot 186), and
finally an hypothetical protein (spot 79) (see Table 2 and
Fig. 4). Among successful identifications, four spots were
up-regulated under CLT (specifically or not) and corresponded to a chitin-binding protein (spot 123), a cuticular
protein (spot 321), a protein involved in energy metabolism
(spot 111), and a protein with unknown function (spot 210)
(see Table 2 and Fig. 4).
4. Discussion
Fig. 3. Results of the principal component analysis based on specific
expression level (fold factors) of all proteins detected in thermal
treatments. The first two principal components cumulate 88% of the
variability: 59% and 29% for factor 1 and 2, respectively. Each cross
refers to a single protein and red squares indicate spots selected for
identifications. Superimposed arrows represent direction of eigenvectors
on the first two axes. Spots strongly decentred show high modulation
response from control.

29.03%, thus cumulatively accounting for 88.4% of the
variability. The PC1 separated proteins responding by a
down- or up-regulation (on the negative and positive part
of the axis, respectively), while PC2 well-separated thermal
treatments (CLT and FTR on the negative and positive
part of the second axis, respectively). The PC3, which
opposed exposure durations (5 days versus 10 days)
(eigenvalue ¼ 0.27) explained only 6.95% of the variance
and was thus not informative.
To better understand which physiological processes may
be involved during the high temperature intervals, we were
particularly interested in spots showing opposite reaction
under FTR and CLT, and particularly those that were upregulated under FTR. Twenty-five proteins were selected
for identification based on the specificity and magnitude of
the response, abundance, and reproducibility among
replicates. Spots that were too faint, fused or placed at
the border of the gel were rejected. On 25 selected proteins,
18 were successfully identified by mass spectrometry
analysis followed by database matching. Identified proteins
related to spots numbers are presented in Table 1 and the
differential expressions of these proteins are illustrated in
Figs. 4 and 5.
Apart from spots 111 and 321, all identified proteins
showed a significant treatment effect (CLT versus FTR)
(Table 2). When comparing to control, the majority were
significantly up-regulated under FTR, except spots 111,
123, and 210 which were up-regulated under CLT. The
spot 321 was chosen because it was significantly accumulated under CLT and FTR, and because it was the most
abundant protein observed in the proteome (see Fig. 2).
Among the 18 identified proteins, 14 spots showed a
specific up-regulation under FTR. Within this class,

As shown in our previous studies (Colinet et al., 2006a,
2007), the mortality of A. colemani mummies was markedly
reduced when the cold exposure was interrupted by daily
short pulses to 20 1C. The clear difference in survival
between CLT and FTR indicates that the experimental
conditions had a strong impact on physiological and
metabolic responses. In fact, different metabolic reactions
in A. colemani FAA pools, induced by the same thermal
treatments (CLT versus FTR), were previously reported
(Colinet et al., 2007), confirming that these experimental
conditions actually induce different metabolic responses to
stress. Since metabolites, such as FAA, are downstream of
both gene transcripts and proteins, and are involved in
many metabolic pathways (Malmendal et al., 2006),
differential protein expressions, according to thermal
treatments were also expected.
To investigate changes in protein profiles during
recovery from chilling stress, we carried out 2-DE analysis
of the total insect proteins. Simple analyses of variance on
protein expressions revealed that 1/3 of proteins were
differentially expressed according to thermal treatments.
The PCA analysis clearly separated the two thermal
treatments (CLT versus FTR), confirming that at the level
of proteins, the overall response to cold exposure was also
specific to treatments. Some proteins showed a duration
effect, and in very few cases, interactions were also
observed between duration and treatment factors. Duration factor includes time-dependent effect of low temperature and a potential developmental effect (differential or
not). Development proceeds under CLT, and probably a
bit faster under FTR, but previous study (Colinet et al.,
2006a) has shown that difference in time-to-adult emergence between both conditions (CLT versus FTR) was less
than 1 day after 21 days of cold exposure. Thus, in the
present study, after 5 and 10 days, the potential distortion
in development was clearly a minor determinant.
Though the Honeybee genome has recently been
sequenced (The Honeybee Genome Sequencing Consortium, 2006), the protein database for hymenopteran insects
is still poor and many protein functions still remain
unknown. The task of identifying proteins and their

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Table 1
List of significantly modulated proteins identified in A. colemani by tandem MS/MS (ESI-QUAD-TOF)
Spot
no.

7

15
29
36
54
65
79
84
95
111
123
145
186

210
217
280
321
345

Source species

Accesion no.b

Function

4

Apis mellifera

110756123

Heat shock chaperonin

43

1

Apis mellifera

48100966

Energy production and conversion

72

3

91084043

Energy production—TCA cycle

283

10

Tribolium
castaneum
Apis mellifera

110763826

Energy production-glycolytic enzyme

267

13

Apis mellifera

110748949

306

7

86160922

40
644

5
19

Blattella
germanica
Aedes aegypti
Apis mellifera

686

43

67043759

Energy production—glycolytic
enzyme
Energy production and conversion—
phosphorylation—phosphagen
Function unknown
Regulation of signal transduction—
regulation of Phosphorylation
Energy production—TCA cycle

267

5

24658560

Energy production and conversion

339
126

7
4

114052326
91087659

571

9

110751158

Chitin metabolic process
Multicatalytic endopeptidase
complex, proteolytic complex
Actin regulatory protein

40

1

118782571

Function unknown

248

4

66501507

Heat shock chaperonin

895

39

67043757

90
426

2
8

Energy production—glycolytic
enzyme
Component of the rigid cuticle
Energy production—TCA cycle

Protein name

Mowse
scorea

Similar to Hsp70/Hsp90
organizing protein homolog
CG2720-PA isoform 1
Similar to bellwether (F0F1-type
ATP synthase, alpha subunit)
Similar to fumarate hydratase
(fumarase)
Similar to phosphoglycerate
kinase isoform 1
Similar to aldolase CG6058-PF,
isoform F
Arginine kinase

196

Hypothetical protein
Similar to receptor for activated
protein kinase C (RACK 1)
Mitochondrial malate
dehydrogenase
Bellwether CG3612-PA (F0F1type ATP synthase, alpha subunit)
Gasp precursor
Similar to proteasome subunit
beta type 6 precursor
Similar to cofilin/actindepolymerizing factor (protein
twinstar)
Leucine-rich repeat (LRR) protein
ENSANGP00000012086
Similar to heat shock protein
cognate 5 (Hsp70)
Glyceraldehyde-3-phosphate
dehydrogenase
Similar to pupal cuticular protein
Similar to aconitase CG9244-PB

No. of
matched
peptides

Lysiphlebus
testaceipes
Drosophila
melanogaster
Bombyx mori
Tribolium
castaneum
Apis mellifera

Anopheles
gambiae
Apis mellifera
Lysiphlebus
testaceipes
Lonomia obliqua
Apis mellifera

108876622
48104663

58370540
48098039

c

a

Mowse score derived from Mascot algorithm, indicating identity or extensive homology (po0.05).
Protein accession numbers from the NCBI Genbank database.
c
Protein identified using EST database from the NCBI Genbank.
b

functions thus remains a challenge. To our knowledge, this
is the first proteomic study on a parasitic wasp. Among the
25 proteins selected for identification, 18 could be identified
by MS/MS.
The most abundant spot in the gels (number 321) was a
structural protein corresponding to a pupal cuticular
protein. Low temperature could have induced modifications in cuticular characteristics. Temperature-related
cuticular modifications have already been reported, particularly on cuticular hydrocarbons (Yoder et al., 1992,
1995). The up-regulation of cuticular proteins may be a
consequence of the cold-exposure, but this proteins is
probably not responsible for the higher survival under
FTR, as it was also up-regulated (relative to control) under
CLT. Alternatively, even if the development is known to be
strongly reduced at 4 1C, individuals coming from both
conditions continue to develop very slowly (Colinet et al.,

2006a), the onset of pre-pupal stage could possibly explain
why pupal cuticular proteins accumulated.
A gasp precursor (spot 123) was up-regulated under
CLT. The gasp precursor is a peritrophin-like protein
found in chitin-binding proteins associated with the
peritrophic matrix (PM) of insects (Coudron et al., 2006).
The PM is a structure composed primarily of chitin,
protein, and glycoproteins (Wang and Granados, 1997;
Barry et al., 1999). The PM is secreted by the midgut of
insects and acts as a barrier against mechanical damages,
pathogens, toxins, and is also involved in ultrafiltration in
the gut lumen (Lehane, 1997). The reason why this protein
was up-regulated after continuous cold exposure is unclear.
But it seems that peritrophins may play more roles than
previously thought, since they have been reported in other
tissues than the PM including tracheae and ovaries (Barry
et al., 1999). They are clearly expressed at important

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Spot No

Protein name

7

Hsp70/Hsp90 homolog

15

ATP synthase

29

Fumarase

36

Phosphoglycerate kinase

54

Aldolase

65

Arginine kinase

79

Conserved hypothetical protein

84

Guanine nucleotide-binding protein
(activated protein kinase Receptor)

95

Mitochondrial malate dehydrogenase

111

Bellwether ATP synthase

123

Gasp precursor

145

Proteasome

186

Cofilin/actin-depolymerizing factor
(Proteint winstar)

210

Leucine-rich repeat (LRR) protein

217

Heat shock protein cognate 5
(Hsp 70)

280

Gyceraldehyde-3-phosphate
dehydrogenase

321

Homolog to pupalcuticular protein

345

Aconitase

Control

CLT

1183

FTR

Fig. 4. Selected 2D-PAGE gel areas related to A. colemani proteins differentially expressed according to treatments (control, and constant or fluctuating
cold exposure). Protein identifications related to spots numbers are presented in Table 1.

interfaces of insects with their environment, particularly
where nutrient or gas exchange is important (Barry et al.,
1999).
According to Ku¨ltz (2005), a key aspect of the response
to stress is the modulation of major pathways of energy
metabolism. Among identified spots, eight up-regulated

proteins under FTR were enzymes involved in energy
metabolism. Respiration can be divided into three main
parts: glycolysis, TCA cycle, and electron transport in
mitochondria. Aldolase (spot 54), glyceraldehyde-3-phosphate dehydrogenase (spot 280) and phosphoglycerate
kinase (spot 36) are enzymes playing successive important

Author's personal copy
ARTICLE IN PRESS
H. Colinet et al. / Insect Biochemistry and Molecular Biology 37 (2007) 1177–1188

1184
0.20

spot 7

*

0.3

*

spot 15

*
0.10

0.15

spot 29

0.06
0.10

0.04

0.1

0.05

0.02
0.00

0.00

0.0
Co

C5

C10

F5

F10

Co

*

spot 54
0.25

C5

C10

F5

F10

Co

*

0.10

C5

C10

F5

spot 79

*

spot 65
*

*

0.15

0.2

0.10
0.05

*

0.20

*

0.08
*

spot 36

*

F10
*

0.10

Co
0.4

0.20

0.08

0.08

0.3

0.15

0.06

0.06

0.2

0.10

0.04

0.04

0.05

0.02

0.02

C5

C10

F5

F10

*

spot 84

0.1
0.0

Co
0.6

C5

C10

F5

spot 95

F10
*

*
0.4

0.2

Co
0.25

C5

C10

F5

*

spot 111

F10

Co
0.5

C5
*

C10

F5

F10

Co

spot 123

C5

C10

spot 145

0.20
0.15

0.3

0.10

0.2

0.05

0.1

F10

*

0.15
0.4

F5

*

*
0.10

0.05

0.00

0.0
Co

C5

C10

F5

spot 186
*

0.12

F10
*

Co
0.60

C5
*

C10
*

F5

F10

spot 210

Co
0.08

C5

C10

F5

spot 217

F10
*

*
0.06

Co
0.30

C5

C10

F5

F10

*

spot 280

*

0.24

0.40
0.08
0.20

0.04

0.00

0.18

0.03

0.12

0.02

0.06

0.00
Co

6.0

0.05

C5

spot 321

C10

F5

Co

*

*

*

F10

C5

C10

F5

spot 345

4.5

0.05

3.0

0.03

1.5

0.02

0.0

Co

C5

C10

F5

F10

Co

C5

C10

F5

F10

*

0.06

*

F10

0.00
Co

C5

C10

F5

F10

Co

C5

C10

F5

F10

Fig. 5. Illustrations of differential expression of identified proteins according to each experimental condition (Co: control, C5 and C10: CLT for 5 and 10
days, F5 and F10: FTR for 5 and 10 days). Each bar represents the mean value of three replicate gels and error bars are standard errors. Symbol
indicates a significant difference from control, based on Dunnett’s t-test. Spot number is indicated on top of each graph.

Author's personal copy
ARTICLE IN PRESS
H. Colinet et al. / Insect Biochemistry and Molecular Biology 37 (2007) 1177–1188
Table 2
Analysis of variance on normalized volumes of identified proteins
(reflecting expression levels) with thermal treatment (CLT and FTR)
and duration of cold-exposure (5 and 10 days) as crossed factors
Spot no.

7
15
29
36
54
65
79
84
95
111
123
145
186
210
217
280
321
345

Duration

Treatment

Duration treatment

F

P

F

P

F

P

4
0.04
1.2
0.03
0.07
0.1
7.94
10.29
3.17
0.13
1.96
0.99
0.03
0.02
0.67
1.78
1.63
18.25

0.081
0.851
0.306
0.867
0.804
0.757
0.023
0.013
0.113
0.726
0.199
0.350
0.875
0.898
0.436
0.219
0.238
0.003

24.42
16.12
29.36
33.8
18.77
33.48
29.31
23.54
110.21
3.32
8.18
21.53
10.48
28.66
15.84
57.08
0.1
17.22

0.001
0.003
o0.001
o0.001
0.002
o0.001
o0.001
0.001
o0.001
0.105
0.021
0.002
0.012
o0.001
0.004
o0.001
0.946
0.003

1.46
0.12
1.03
0.21
0.29
0.04
10.92
4.44
3.17
2.61
0.49
1.16
0.01
0.03
0.16
1.06
0.12
15.25

0.262
0.739
0.341
0.660
0.606
0.844
0.011
0.068
0.113
0.145
0.505
0.312
0.929
0.870
0.696
0.334
0.740
0.005

roles in the glycolytic pathway (Lehninger et al., 1993).
Aconitase (spot 345), fumarase (spot 29) and malate
dehydrogenase (spot 95) are involved in the TCA cycle
(Lehninger et al., 1993). During low temperature exposure,
insects are fasting and basal metabolism must rely
exclusively on body energy reserves to survive (Pullin,
1987; Lavy et al., 1997). It has been shown that lipid
reserves were consumed during cold exposure in A.
colemani (Colinet et al., 2006b). The energy supplies,
depleted during cold exposure, may be regenerated during
the pulses of warm temperature, as suggested by Chen and
Denlinger (1992). The up-regulation of energy production
pathways, during the recovery phase, may be important to
provide energy equivalents to various physiological processes. First, since cold-induced metabolic disorders cause
the lack of necessary products, such as ATP (Hochachka,
1986; Churchill and Storey, 1989a), up-regulation of the
energy production pathways during the intervals at 20 1C
may allow the restoration of the normal cellular energy
status. Second, the induction of energy metabolism may be
necessary to provide the reducing and energy equivalents to
diverse stress-related processes such as: (1) chaperoning
using Hsps, observed during the pulses to high temperature
(see below), as Hsps are known to be ATP-dependent
(Pratt and Toft, 2003); (2) synthesis of cryoprotectants, an
elevation in glycerol level was observed under FTR
(Lalouette et al., 2007); its synthesis being known to be
ATP-consuming (Churchill and Storey, 1989b; Storey and
Storey, 1990); (3) the redistribution of ions through
membranes (particularly [K+]), observed during the hightemperature pulses (Kosˇ ta´l et al., 2007), and which involves
an ATP-dependent pumping system (Kosˇ ta´l et al., 2004,

1185

2006); (4) proteolytic degradation, observed during the
warm intervals (see below), which is also ATP-dependent
(Hersko and Ciechanover, 1998; Mykles, 1999). The
increased expression of proteins involved in energetic
pathways during the warm intervals may come along with
the increase in ATP demand, and thus represents a
protective mechanism against supply–demand energy imbalance.
Apart from enzymes involved in glycolysis and TCA
cycle, a high level of expression of ATP-synthase subunit
(spot 15), was observed under FTR, corroborating that
energy metabolism was particularly galvanized during
warm intervals. The multisubunit ATP-synthase is present
in the membranes of chloroplasts, eubacteria, and mitochondria. In animals, the mitochondrial ATP-synthase is
responsible for the synthesis of the majority of cellular
ATP, thereby playing a crucial role in energy metabolism
(Hochachka and Lutz, 2001; Talamillo et al., 2004). An
ATP-synthase subunit was also up-regulated under CLT
(spot 111), especially after 5 days. Proteomic studies on
chilling stress responses in plants reported similar observations, where ATP-synthase subunits appeared to increase
in abundance during prolonged chilling-stress (Taylor
et al., 2005; Yan et al., 2006). These proteins were
considered as breakdown products rather than intact ones,
because Mr values were much smaller than theoretical ones
(Taylor et al., 2005; Yan et al., 2006). The spot position in
our gels indicates that experimental Mr correspond to
theoretical one (59.58 kDa) for spot 15 but not for spot
111, indicating that the proteins were probably breakdown
products rather than intact ones under CLT.
A protein corresponding to arginine kinase (AK) (spot
65) was up-regulated under FTR. AK is the sole phosphagen kinase found in several major invertebrate groups,
including arthropods and molluscs (Tanaka et al., 2007). It
plays an important role in the maintenance of ATP levels
by the phosphorylation of phosphagen, which then serve as
a high-energy source from which ATP can rapidly be
regenerated (Newsholme et al., 1978). Members of this
enzyme family thus play key roles as ATP-buffering
systems in animal cells that display high and variable rates
of ATP turnover (Ellington, 2001; Tanaka et al., 2007).
Churchill and Storey (1989a) observed in cold-adapted
Epiblema scudderiana (Lepidoptera), that multiple cycles of
temperature influenced cellular energetics; a drop in energy
charge (including ATP) under cold was followed by a
recovery during re-warming, where an increase in arginine
phosphate (i.e., phosphagen) was observed. Since energetic
metabolism seems to be galvanized during the warm
intervals in A. colemani, up-regulation of AK should be
important for buffering ATP levels.
According to Ku¨ltz (2005), molecular chaperones are part
of the minimal stress proteome. Two spots were identified as
heat shock chaperonins, one similar to Hsp70/Hsp90 (spot 7)
and the other to Hsp70 cognate (spot 217). Molecular
chaperones are involved in diverse functions including
transport, folding, unfolding, assembly, disassembly, and


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