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ARTICLE IN PRESS

Journal of Thermal Biology 32 (2007) 374–382
www.elsevier.com/locate/jtherbio

Does thermal-related plasticity in size and fat reserves influence
supercooling abilities and cold-tolerance in Aphidius colemani
(Hymenoptera: Aphidiinae) mummies?
H. Colineta, , P. Vernonb, T. Hancea
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, Belgique
b
Universite´ de Rennes 1,UMR 6553 CNRS, Equipe ICC, Station Biologique de Paimpont, 35380 Paimpont, France

a

Received 13 February 2007; accepted 24 March 2007

Abstract
(1) The parasitoid Aphidius colemani was reared at 15 or 25 1C to induce variation in size and fat reserves; SCP and cold-tolerance were
compared. Insects from both temperatures were also exposed to constant or fluctuating cold-exposure.
(2) The lower SCP in mummies reared at 25 1C may be partially explained by their smaller size, a negative relationship being observed
between SCP and size.
(3) A bimodality was observed in SCP distributions, with two modes around 26 and 22 1C, likely because of presence/absence of
gut content.
(4) The type of exposure had a striking impact, mortality being considerably lower under fluctuating regime.
(5) While energy storage is an important factor, vulnerability to chill-injury is supposed to be the primary factor regulating survival at
low temperature.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Parasitic wasp; Low temperature; Chill-injury; SCP; Fat reserves; Recovery

1. Introduction
Exposure to extreme temperatures, lethal or sub-lethal, is
a major factor shaping life history traits in insect
parasitoids (reviewed by Hance et al., 2007). Due to
seasonal cycles, many insect species are frequently exposed
to sub-optimal low temperatures in their natural environments. The underlying mechanisms allowing these organisms to increase their cold-hardiness have been extensively
reviewed (e.g. Sinclair et al., 2003). In insect parasitoids,
adaptations to low temperatures are similar to those of
most ectotherms (Hance et al., 2007). Exposures to
prolonged low temperature are known to have detrimental
Corresponding author. Tel.: +32 10 47 34 91; fax: +32 10 47 34 90.

E-mail addresses: hance@ecol.ucl.ac.be, colinet@ecol.ucl.ac.be
(H. Colinet).
0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtherbio.2007.03.005

effects on the survival of parasitoids (Langer and Hance,
2000; Lysyk, 2004; Tezze and Botto, 2004; Levie et al.,
2005; Colinet et al., 2006a, b). When the ‘‘dose’’ of coldexposure (a combination of time of exposure and
temperature) exceeds a specific threshold, chill-injuries
accumulate, become progressively irreversible and eventually lethal (Bale, 1996, 2002; Kostal et al., 2006). In many
insect species, the lethal cumulative injuries occur even at
temperatures above 0 1C, but the main causes of death are
still not well understood (Renault et al., 2002; Kostal et al.,
2004, 2006). In studies devoted to the consequences of coldexposure, the specific effects of starvation and cold are not
easy to identify as they are acting together. Hence,
consequences of cold-exposure may result either from cold
effects or starvation, or a combination of both phenomena.
Several studies emphasized that exposing insects to
fluctuating thermal regimes (FTR) (i.e. cold-exposure

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H. Colinet et al. / Journal of Thermal Biology 32 (2007) 374–382

interrupted by periodic short pulses at high temperature),
versus constant low temperatures (CLT), increased significantly the survival (Chen and Denlinger, 1992; Nedveˇd
et al., 1998; Renault et al., 2004; Colinet et al., 2006a). FTR
reduce the level of accumulated chill-injuries and thus the
mortality, either because less chill-injuries accumulate at
FTR, or because the effect of chilling is counteracted by the
high temperature intervals (Hancˇ and Nedveˇd, 1999;
Renault et al., 2004; Colinet et al., 2006a, 2007b).
During low temperature exposure, insects fast and basal
metabolism relies exclusively on body energy reserves
(Pullin, 1987; Lavy et al., 1997). Energy is needed not
only to survive to subzero temperatures, but also to survive
low, but above zero temperatures (Lavy et al., 1997).
Energy reserves, particularly from fat body, are consumed
during starvation at low temperature exposure (David and
Vannier, 1996; Renault et al., 2002, 2003). In a previous
study, it has been shown that parasitoid mass loss increased
with cold-exposure duration and was associated with a
marked decrease in dry mass, due to lipid reserves
depletion (Colinet et al., 2006b). Heavier insects with
larger fat reserves should presumably have a significant
advantage for survival at low temperature (Renault et al.,
2003; Colinet et al., 2006b), an assumption that will be
tested in this study.
There is strong evidence that an increase in lipid content
underlies increased resistance to starvation, as indicated by
numerous studies on Drosophila (reviewed by Hoffmann
and Harshman, 1999). An increase in body mass has also
been associated with increased starvation resistance (Chippindale et al., 1996; Harshman et al., 1999). The starvationresistance hypothesis (SRH) states that because energy
stores increase with size faster than metabolic rate,
resistance to starvation should increase with body size
(Cushman et al., 1993; Arnett and Gotelli, 2003).
The temperature-size rule states that ectotherms grow
larger at lower temperature, and this rule is supported by
numerous studies (reviewed by Atkinson, 1994; Atkinson
and Sibly, 1997; Angilletta and Dunham, 2003). As a
generally accepted guideline, decreased developmental
temperature results in slower growth rate, longer development time, and larger adult size in insects (Sibly and
Atkinson, 1994). Since, fat reserves are positively correlated with body mass (Ellers and van Alphen, 1997; Ellers
et al., 1998; Strohm, 2000; Rivero and West, 2002; Colinet
et al., 2007a), variations in developmental temperature is a
convenient way to obtain bigger and fatter individuals
(Colinet et al., 2007a).
Studies of insect cold-hardiness are generally achieved by
measuring their capacity to survive at low temperatures for
extended periods, and/or by measuring supercooling
abilities. The SCP should represent the lower lethal
temperature for freeze-intolerant insects, although death
sometimes occurs at temperatures well above SCP. The real
ecological value of the SCP thus remains ambiguous (Bale,
2002; Renault et al., 2002), but SCP can still be used as a
convenient comparative index, especially since it depends

375

on the organism’s native characteristics, such as body
composition and size (Renault et al., 2002).
In the present study we focused on the parasitoid
Aphidius colemani Viereck (Hymenoptera: Aphidiinae),
which is commercially produced and distributed as an
aphid biocontrol agent, targeting primarily Myzus persicae
Sulzer (Homoptera: Aphididae) in glasshouses in many
European countries. This parasitoid stops feeding at the
end of third larval stage (Muratori et al., 2004), the larva
then spins its cocoon inside the empty cuticle of the aphid,
forms a mummy and pupates (Hagvar and Hofsvang,
1991). From that moment until emergence, it does not feed
and all metabolic processes use energetic reserves accumulated during the larval stages.
Different developmental temperatures were used to
induce a phenotypic variation in insect size. We then tested
the hypothesis that body size may influence superccoling
abilities and/or cold-tolerance via SRH. We also tested the
crossed effect of the type of exposure (FTR versus CLT) on
insect survival during a medium-term cold-exposure.
2. Materials and methods
2.1. Insect rearing
The green peach aphid, M. persicae, was used as host for
the parasitoid rearing and laboratory cultures were
established from individuals collected in fields during
2000 at Louvain-la-Neuve, Belgium (50.31N Latitude).
Aphids were reared in 0.3 m3 cages on sweet pepper
(Capsicum annuum L.) under 1871 1C, 760% RH and
LD 16:8 h. A. colemani, originally obtained from Viridaxis
SA (Belgium), were subsequently reared in the laboratory
under the same conditions.
2.2. Parasitoid development
To obtain standard mummies, batches of 50 standardized 3-day old aphids were offered to a mated female
parasitoid for 4 h at 18 1C. Aphids were all synchronized at
the same age in order to avoid host–age effects on
parasitoid development (Colinet et al., 2005). The parasitoid females used were less than 48 h old, naı¨ ve, and
mated. After parasitism, the females were removed and the
aphids were immediately transferred to either 15 or 25 1C
to continue development until mummification. Developmental temperature is known to induce phenotypic
plasticity in body mass and fat reserves, wasps coming
from a rearing at 15 1C being larger (size), heavier (dry
mass) and fatter (fat content) than wasps developing at
25 1C (Colinet et al., 2007a).
2.3. Morphometric and gravimetric measures
For both developmental temperatures, one-day old
mummies were subjected to individual mass measurements
(Mettler-electrobalance Me22, sensitivity 1 mg) (n ¼ 84).

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376

The mummies were dried at 60 1C for 3 days in an air oven
and then weighed to measure dry mass (DM). The lean dry
mass (LDM) was measured after an extraction in chloroform
methanol (2:1) (see Terblanche et al., 2004; Colinet et al.,
2006b). Each mummy was left for two weeks in an
Eppendorfr tube containing 1 ml of the extracting solution
agitated daily. The mummies were then placed for 12 h in an
air oven at 60 1C to eliminate residues of the extracting
solution before measurement of the LDM. The fat mass
(FM) (FM ¼ DM-LDM), and the fat content (FC)
(FC ¼ FM/LDM), were calculated (see Colinet et al., 2006b).
For both developmental temperatures, mummy size was
characterized by measuring maximum length (L) and
maximum width (W). The volume (V) was estimated by
the formula V ¼ p.L.W2/6 (Atkinson, 1979), assuming
each mummy was a regular ellipsoid (Legrand et al., 2004)
(n ¼ 84). A video module connected to a monitor was used
to capture digital pictures, which were subsequently
analysed using Image Pro Plus software (V4.0, Media
Cybernetics, USA).
2.4. Determination of the supercooling point
The supercooling point (SCP), i.e. the temperature of
crystallization, of at least 25 mummies aged up to 48 h was
measured for individuals coming from a rearing at 15 and
25 1C (treatments MF15 and MF25, respectively). Measurement were performed on isolated mummies placed
inside a 1 ml Eppendorfr tube, fixed to the tip thermocouple (K-type) with petroleum jelly, the tube being closed
with parafilmr. The thermocouple was connected to high
resolution thermometers (model HI 93531R, HANNA
instruments, Belgium) (resolution 0.1 1C) coupled to a
computer for recording the temperature every second. The
tube was placed inside a refrigerated Cryostat circulator
(Model FP50-ME, Julabo, Germany) programmed to cool
at a rate of 1 1C min 1. The SCP was taken as the start of
the exotherm produced by the latent heat of freezing. To
investigate the potential relationship between SCP and
mummy size, the mummy volume was determined (as
described before) in another set of 30 individuals reared
under standard conditions (i.e., 1871 1C, 760% RH and
LD 16:8 h) before measuring the corresponding SCP.
2.5. Medium-term cold-exposure
To determine the susceptibility to long chilling periods,
young mummies (one-day old), from both rearing temperature (15 and 25 1C), were exposed to two different
types of cold-exposure, either CTL or FTR:





Treatment 15F: rearing at 15 1C and cold-exposure at
fluctuating thermal regime.
Treatment 25F: rearing at 25 1C and cold-exposure at
fluctuating thermal regime.
Treatment 15 1C: rearing at 15 1C and cold-exposure at
constant low temperature.



Treatment 25 1C: rearing at 25 1C and cold-exposure at
constant low temperature.

For CLT, the low temperature was 4 1C for the entire
duration of the experiment, a temperature known to affect
A. colemani survival (Colinet et al., 2006a, b). For the
FTR, the 4 1C exposure was interrupted by daily pulses at
20 1C for 2 h (see Colinet et al., 2006a, 2007b). Mummies
were placed inside thermo-regulated LMSs incubators,
with saturated relative humidity and in complete darkness.
For each treatment, three batches of 50 mummies were
removed at weekly intervals and kept at 20 1C to measure
the survival after one week, two weeks and three weeks of
cold-exposure. The survival was assessed as the number of
adult parasitoids that successfully emerged from the
mummies (i.e. percentage emergence). A non cold-exposed
control (i.e. three batches of 50 mummies) was maintained
at 20 1C.
2.6. Statistical analysis
The normality of all data sets were first checked using the
Shapiro–Wilk statistic (a ¼ 0.05). The SCP data sets failed
to fit normality (Po0.05) and tended to show a form of
bimodal distribution. Hence, analyses based on comparison of the distributional patterns were used, since
statistical tests based on mean values are not relevant in
this case (Worland et al., 2006). We use Kolmogorov–
Smirnov two-sample tests (KS-stat) to compare SCP
distributions (Worland et al., 2006). Morphometric data
were compared using one-way ANOVA (Proc GLM,
SAS Institute, 1990). Mummy FC was analyzed using
ANCOVA (PROC GLM, SAS Institute, 1990) with
developmental temperature (15 or 25 1C) as main effect
and DM as covariate. The potential relationship between
mummy size and SCP was analyzed using linear regression
model (Proc REG, SAS Institute, 1990). Arcsin square root
transformation was required to normalize the distributions
of percentage emergence, which were analyzed using twoway ANOVA (Proc GLM, SAS Institute, 1990) with
treatment (25 1C, 15 1C, 25F, 15F) and duration of coldexposure as fixed factors. Multiple comparisons were then
performed on each factor using Tukey’s HSD test to
describe differences between groups. Data presented in
figures are untransformed.
3. Results
3.1. Mass and size
Insects grown at the lower temperature (15 1C) were
significantly larger than those reared at 25 1C (Table 1),
significant increases in all morphometric measures were
observed: mummy length (ANOVA1: F ¼ 88.74, Po0.001),
mummy width (ANOVA1: F ¼ 216.32, Po0.001), and
mummy volume (ANOVA1: F ¼ 182.88, Po0.001).

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As predicted, the dry mass and the fat content were also
variable in response to rearing temperature (Fig. 1). The
mummy fat content was positively related to the dry mass
(ANCOVA: F ¼ 16.03, Fo0.001), indicating that larger
individuals contained a higher proportion of fat. The
treatment effect revealed that mummies coming from a
rearing at 15 1C were fatter (FC) than the ones coming
from a rearing at 25 1C (ANCOVA: F ¼ 58.92, Fo0.001)
(Fig. 1).
3.2. Supercooling points
The SCP frequency distributions are shown in Fig. 2.
For both developmental temperatures (MF25 and MF15),
a bimodal tendency was observed with two modes around
26 and 22 1C. Bimodal distribution has already been
reported in insects, caused by a sexual dimorphism (Salin
et al., 2000). Since individuals inside the mummies could be
from both sexes, we analysed an additional set of mummies
to verify whether bimodality was related to the sex. Since
unmated females only give males in the progeny, we
measured SCP in a set of mummies derived from virgin
females and grown at 25 1C (treatment M25). The resulting
SCP distribution also showed a bimodality for male

Table 1
Morphometric measurements of mummies reared at 15 or 25 1C: mean
(7SEM) width, length and volume
Parameters

Rearing
temperatures (1C)

n

Mean

Width (mm)

15
25
15
25
15
25

84
84
84
84
84
84

1.1270.02
1.0070.01
1.5270.01
1.3870.01
1.0070.01
0.7370.01

Length (mm)
Volume (mm3)

377

mummies (Fig. 2). Kolmogorov–Smirnov tests showed a
significant effect of developmental temperature on the SCP
frequency distributions (MF15 versus MF25, KS-stat ¼
0.345, P ¼ 0.038), whereas the absence of females in a set
did not (MF25 versus M25, KS-stat ¼ 0.284, P ¼ 0.162).
The median values of SCP distributions were 23, 25 and
25 1C for treatment MF15, MF25 and M25, respectively. A
broad range of SCP values were observed, and mummies
coming from the lower developmental temperature (15 1C)
tended to show lower supercooling abilities. The Fig. 3
illustrates the negative linear relation observed between
SCP and mummy volume (F ¼ 6.15, P ¼ 0.019, r2 ¼ 0.18).
3.3. Cold-exposure
The survival, expressed as percentage emergence (Fig. 4),
decreased significantly with duration of cold-exposure
(ANOVA2: F ¼ 197.42, Po0.001), and it was particularly
manifest under CLT. Tukey’s test revealed that among all
treatments, survival was the highest in control mummies
(no cold-exposure, duration ¼ 0), then it decreased with
increasing durations (rank order: 0414243 weeks).
The percentage emergence was also significantly affected
by the treatments (ANOVA2: F ¼ 169.54, Po0.001)
(Fig. 4). The type of exposure (FTR versus CLT) had a
dramatic impact on emergence, mortality being clearly
lower under FTR than CLT. After three weeks of coldexposure, the survival was still close or higher than 70%
under FTR (i.e. 15F and 25F), whereas it was lower than
25% under CLT (i.e. 15 and 25 1C). The effect of
developmental temperature (25 1C versus 15 1C) was noticeable under FTR but less manifest under CLT. Tukey’s
HSD tests indicated that among all treatments, survival
was the highest for mummies under FTR followed by
mummies exposed to CLT (rank order: 15F425F4
15 1C425 1C). The significant interaction between treatment and duration revealed that temporal reductions in

Fig. 1. Relationship between mummy fat content (FC) and dry mass (DM); FC is expressed as the ratio between fat mass (FM) and lean dry mass (LDM).
The straight lines are positive linear relationships between DM and FC in mummies reared at 15 1C (’) and 25 1C (J). Every square represents an
individual value, n ¼ 84 for each rearing temperature.

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H. Colinet et al. / Journal of Thermal Biology 32 (2007) 374–382

378

MF15

Relative frequency (%)

40%

MF25

M25

30%

20%

10%

-27 -26 -25 -24 -23 -22 -21 -20 -19

-27 -26 -25 -24 -23 -22 -21 -20 -19

-27 -26 -25 -24 -23 -22 -21 -20 -19

SCP (°C)

SCP (°C)

SCP (°C)

Fig. 2. Supercooling point frequency distributions in mummies of Aphidius colemani: males or females reared at 15 1C (MF15, n ¼ 30), males or females
reared at 25 1C (MF25, n ¼ 31), only males reared at 25 1C (M25, n ¼ 26).

3
Mummysize (volume mm )

2.5

2.0

1.5

1.0

-27

-26

-25

-24

-23

-22

SCP (°C)
Fig. 3. Relationship between mummy size (expressed as volume) and SCP.
The straight line is the least-squares linear regression modelling the
negative relationship between size and supercooling ability. Every square
represents an individual value (n ¼ 30).

percentage emergence were treatment-specific (ANOVA2:
F ¼ 27.33, Po0.001).
4. Discussion
During exposure at temperature close to developmental
threshold, insect development is severely reduced and, to
stay alive, individuals require energetic resources to
maintain basal metabolism. In a previous study, parasitoid
mass loss was shown to increase with cold-exposure
duration as a result of lipid reserves depletion during
starvation (Colinet et al., 2006b). We hypothesized that

insects with larger fat reserves should presumably have a
significant advantage for survival at low temperature. We
used the knowledge of temperature-size rule (Atkinson,
1994) to induce a phenotypic variation in parasitoid body
size and we compared cold-tolerance and supercooling
abilities in A. colemani.
As previously observed (Colinet et al., 2007a), A. colemani
follows the typical pattern of ectotherms, development time
and body size being negatively correlated with temperature
during development. Developmental temperature induced
a plastic response in the size of mummies: individuals from
the lower temperature were significantly larger than
parasitoid reared at 25 1C. As previously observed in
insects, fat reserves were positively correlated with body
mass (Pullin, 1987; Ellers et al., 1998; Strohm, 2000; Rivero
and West, 2002; Colinet et al., 2007a), and individuals
coming from the lower temperature were also fatter than
the ones coming from the higher temperature.
A.colemani exhibited a rather high supercooling capacity, and SCP values were in the range of values generally
observed in closely related parasitoid species (Hofsvang
and Hagvar, 1977; Langer and Hance, 2000; Rigaux et al.,
2000; Tullett et al., 2004). However, in the aphid parasitoid
Diaeretiella rapae, SCP values reported were lower than
ours (around 30 1C), but individuals were suspected to be
in diapause (Nowierski and Fitzgerald, 2002). As observed
in the pupae of the parasitoid Habrobracon hebetor
(Carrillo et al., 2005), SCP values showed a quite wide
range (from –19 to 27 1C) and SCP distributions departed
significantly from normality. A bimodal tendency was
observed here and, to our knowledge, it is the first report of
such a distribution pattern in a parasitic wasp.
The SCP has long been known to show substantial
variation within a population and its frequency distribution sometimes showed evidence of bimodality, as observed
in Collembola (Sømme and Block, 1982; Coulson et al.,
1995; Worland et al., 2006), in Coleoptera (Salin et al.,
2000, 2003), in Homoptera (Powell, 1976), and in Diptera,

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Percentage of emergence (%)

Percentage of emergence (%)

H. Colinet et al. / Journal of Thermal Biology 32 (2007) 374–382

15F

25F

15C

25C

379

100

75

50

25

100

75

50

25

0

1

2

3

Cold exposure duration (weeks)

0

1

2

3

Cold exposure duration (weeks)

Fig. 4. Percentage of adults emerging (mean+SEM) as a function of the duration of cold-exposure for each treatment : (15F) mummies reared at 15 1C
and FTR, (25F) mummies reared at 25 1C and FTR, (15 1C) mummies reared at 15 1C and CLT, (25 1C) mummies reared at 25 1C and CLT.

parasitized or not (Block et al., 1987). Since mummies
could be from both sexes, we first suspected that bimodal
tendency might be related to a sexual dimorphic response,
as has already been reported in Alphitobius diaperinus
(Salin et al., 2000). In this study, the bimodal distribution
was likely not to be related to sex, since the absence of
females in a set (i.e. M25) gave a similar distribution than
when both sexes were present (i.e. MF25). Other factors
influencing SCP may be involved.
The temperature at which a supercooled arthropod
freezes is influenced by a wide range of factors including
cooling rate, hydration, cryoprotectant levels, stage of
development, ecdysis, feeding status, quality and quantity
of nucleating agents, body size, and sex (e.g. Sømme, 1982;
Vernon and Vannier, 1996; David and Vannier, 1996; Salin
et al., 2000, 2003; Carrillo et al., 2005; Worland et al.,
2006). The influence of feeding status on SCP distribution
has been well studied, and presence/absence of gut content
has been revealed to be a reliable explanatory factor
for supercooling bimodality in insects (Powell, 1976;
Sømme & Block, 1982; Salin et al., 2000). The gut,
containing foreign elements (bacteria, food, particles, dust,
products of digestion), is a major site of non-specific
nucleation in insects (Zachariassen, 1985; Storey and

Storey, 1989; Duman, 2001). In Aphidiinae, the mummy
development is characterized by the following sequence:
cocoon spinning, pre-pupal and pupal stage characterized
by meconium ejection and finally gradual formation of
adults, all these processes lasting around 4–5 days at 20 1C
(Pennacchio and Digilio, 1990). Individuals used in this
experiment were all aphids mummified for less than 48 h, so
that some individuals may have excreted meconium and
others not. Krunic´ and Radovic´ (1974) reported differences
in SCP values between stages (larvae, pre-pupae and pupa)
in Megachile rotundata and even in its parasitoids, and
suggested that it may be related to evacuation of gut
content in pupal stage (i.e. meconium). Similarly, Block
et al. (1987) suggest that increased variability in SCP
bimodal distributions observed in Delia radicum, parasitized by Trybliographa rapae, may be related to meconium evacuation at puparial stage. Meconium excretion in
some individuals, and not in others, might explain the
bimodal tendency observed in this study.
Another factor that may have influenced the SCP is the
body size of individuals. The capacity to supercool
decreases as body mass increases, because the probability
that an ice embryo spontaneously forms increases with
size (Lee and Costanzo, 1998). There is evidence that

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H. Colinet et al. / Journal of Thermal Biology 32 (2007) 374–382

supercooling ability is negatively correlated with body size
(David et al., 1996), body mass (Powell, 1976; Block et al.,
1987), and even fat content (Lombardero et al., 2000;
David and Vannier, 1996) in several species. Our results
confirm this negative relationship, as supercooling ability
decreases with size in A. colemani mummies (Fig. 3). Since
mummies reared at 15 1C were larger, heavier and fatter
than mummies reared at 25 1C, it may partially explain the
relative greater proportion of individuals showing lower
supercooling abilities in this group.
In order to determine cold-tolerance of parasitoids at
temperatures above the SCP, mummies reared at 15 and
25 1C were exposed to either constant low temperature or
fluctuating temperature. As previously observed (Colinet
et al., 2006a), the type of exposure (CLT versus FTR) had a
striking impact on emergence, mortality being considerably
lower under FTR than CLT. It has been suggested that the
normal physiological state of the cold-exposed insects may
be re-activated during high temperature pulses, reducing
the speed and the amount of accumulated injuries
(Laloulette et al., 2007; Kostal et al., 2007). Additionally,
high temperatures may counteract the negative effects of
the cold-exposure by allowing a repair of the cumulative
damages due to cold (Nedveˇd et al., 1998; Renault et al.,
2004; Colinet et al., 2006a, 2007b).
Even if consumption of energy reserves must be reduced
under low temperatures, since it depends on metabolic rate
which increases with temperature (Karan and David,
2000), insect metabolism relies on body energy reserves to
survive during cold-exposure (Pullin, 1987; Lavy et al.,
1997). The costs of fasting may thus be crucial especially in
terms of lipids. It has been demonstrated in A. colemani
that fat reserves were critically depleted in mummies
exposed to cold (Colinet et al., 2006b). Consequences of
cold exposures may thus result from a combination of both
chill-injuries and exhaustion of energy reserves.
An increase in body mass has been associated with
increased starvation resistance in some insect species
(Chippindale et al., 1996; Harshman et al., 1999). There
is also strong evidence that an increase in lipid content
underlies increased resistance to starvation (reviewed by
Hoffmann and Harshman, 1999). In Alphitobius diaperinus,
survival at cold was positively correlated to fresh mass
(Renault et al., 2003). In the parasitoid Trichogramma
carverae, a reduction in wasp size was observed with time
of cold-exposure, because resources are used to survive at
the expense of size at emergence (Rundle et al., 2004). In
this study, mortality observed in mummies of the treatment
15F was nearly cancelled, after three weeks of coldexposure survival was still 90%, suggesting that chillinjuries were reversed completely and that lipid depletion
has no consequence on survival. In the other treatments,
the survival decreased with cold-exposure duration, but
with much more intensity under CLT. Since survival in
treatment 15F (i.e., low level of chill-injury+high lipid
reserves) was higher than in treatment 25F (i.e., low level of
chill-injury+low lipid reserves), we suggest that when chill-

injuries are inconsequential, secondary factors, such as
starvation effect, may affect cold-survival. According to
starvation-resistance hypothesis (SRH), large-bodied individuals should be able to ride out longer periods of
unfavourable conditions before succumbing to starvation
(Cushman et al., 1993; Arnett and Gotelli, 2003). It results
that larger and fatter mummies reared at 15 1C may have
survived longer. Survival in treatment 15 1C (i.e., high level
of chill-injury+high lipid reserves) was only slightly
superior to treatment 25 1C (i.e., high level of chillinjury+low lipid reserves), suggesting that when insects
accumulate chill-injuries (i.e., in treatments 15 1C and
25 1C), the starvation effects are minor, accumulation of
chill-injuries being the limiting factor for survival. We
argue, therefore, that while energy storage is an important
factor for cold-survival, the vulnerability to chill-injuries is
the primary factor regulating survival at low temperature
in A. colemani. The combination of fluctuating temperature
for chill-injuries recovery, and high energy reserves to
increase starvation resistance, reduce significantly the
detrimental impact of cold-exposure on parasitoid survival.
Acknowledgements
This study was supported by ‘‘Ministe`re de la Re´gion
wallonne – DGTRE Division de la Recherche et de la
Coope´ration scientifique’’. FIRST EUROPE Objectif 3.
We are grateful to Christian Michel for useful help and to
Veterinary Sciences Unit (UCL) for equipment access. This
paper is BRC 115 of the Biodiversity Research Centre.
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