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PHYSIOLOGICAL ECOLOGY

Water Relations, Fat Reserves, Survival, and Longevity of a
Cold-exposed Parasitic Wasp Aphidius colemani
(Hymenoptera: Aphidiinae)
H. COLINET,1, 2 T. HANCE,1

AND

P. VERNON3

Environ. Entomol. 35(2): 228Ð236 (2006)

ABSTRACT Parasitoids exposed to low temperatures may suffer from extreme physiological conditions inducing direct or indirect chill injuries. Under long cold exposure (which also includes
starvation), individuals face a great challenge in maintaining both water balance and energy reserves.
Key parameters associated with individual Þtness and physiological parameters related to coldhardiness were analyzed. One-day-old mummies of the aphid parasitoid Aphidius colemani Viereck
(Hymenoptera: Aphidiinae) were exposed to cold (2 and 4⬚C) for various periods (1Ð3 wk) under a
high relative humidity (75 ⫾ 5% RH) and darkness. High mortality and shortened adult longevity
occurred with increasing duration of exposure at low temperatures. Individual parasitoid mass loss
increased with cold storage duration and was associated to a marked decrease in dry mass caused by
lipid reserve depletion. Water content (water mass/dry mass) slightly increased with cold exposure
duration because of starvation. Similar patterns were observed for both temperatures tested. This study
emphasizes (1) how energetic reserves may be critical to survive at low temperatures and (2) that
the survival and longevity are related, at least in part, to the depletion of energy reserves during
starvation.
KEY WORDS low temperature, cold hardiness, survival, water relations, fat reserves

OUR UNDERSTANDING OF THE responses of insects to low
temperatures has progressed substantially (Ramløv
2000, Sinclair et al. 2003). However, most research on
insect cold hardiness have been focused on the mechanisms by which insects survive in extreme environments, either by tolerating or by avoiding freezing
(Bale 1996). Many insects, however, die at temperatures higher than those at which they freeze (Bale
1993, 1996, Nedveˇ d and Windsor 1994, Mc Donald et
al. 1997, Rivers et al. 2000, Legrand et al. 2004a), and
the causes of death at low temperatures remain not
well understood (Denlinger and Lee 1998, Renault et
al. 2002a, Kosta´l et al. 2004). Until now, little attention
has been given to studies focused on the effect of
prolonged exposures to cold, both above and below
the freezing point (Sømme 1996).
Cold exposure may affect insects in many ways
(Sehnal et al. 2003), but mainly by inßuencing development time (Campbell et al. 1974, Sigsgaard 2000),
longevity (Stary´ 1970a), mortality (Sømme 1982,
Leather et al. 1993), and reproduction (Carrie` re and
Boivin 2001). Consequences of long cold exposures
1 Unite
´ dÕE´ cologie et de Bioge´ ographie, Biodiversity Research
Centre, Universite´ Catholique de Louvain, Croix du sud 4 Ð5, 1348
Louvain-la-Neuve, Belgium.
2 Corresponding author, e-mail: colinet@ecol.ucl.ac.be.
3 Universite
´ de Rennes 1, UMR 6553 CNRS ECOBIO, Equipe Impact des Changements Climatiques, Station Biologique, 35380 Paimpont, France.

may result either from starvation or temperature effects or a combination of both phenomena. Low temperature exposures may produce cumulative physiological damages and lead to a high level of mortality or
selection of individuals with poor reproductive success (Levie 2002). However, little is known about
physiological processes involved in cold exposure. In
dormant insects, many of the mechanisms used by
active insects to maintain their water balance are not
available, so water relationships can be critical (Danks
2000). Maintenance of body water homeostasis is a
great challenge particularly for dormant small-bodied
insects, which must prevent desiccation sometimes for
several months (Yoder et al. 1994, Tanaka 2000). Body
weight loss, associated not only with water loss but also
with the consumption of carbohydrate and lipid reserves, can be a signiÞcant factor of low temperature
survival (Leather et al. 1993). In cold temperatures
conditions, insect metabolism relies exclusively on
body energy reserves (Pullin 1987, Lavy et al. 1997).
Low temperatures can critically affect energy reserves
(David and Vannier 1996, Renault et al. 2002a). Lipids
are known to be used as energy source in insects
(Adedokun and Denlinger 1985, Pullin 1987, Van Handel 1993), but few studies describe energy consumption during cold exposure.
The parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiinae) is commercially produced and
distributed as an aphid biocontrol agent, targeting

0046-225X/06/0228Ð0236$04.00/0 䉷 2006 Entomological Society of America

April 2006

COLINET ET AL.: PHYSIOLOGICAL AND FITNESS COSTS OF COLD EXPOSURE IN A. colemani

primarily Myzus persicae Sulzer (Homoptera: Aphididae) in glasshouses in many European countries. In
Europe, its distribution in nature is restricted to the
Mediterranean area (Stary´ 1975). In the life cycle of
most Aphidiinae, the parasitoid stops feeding at the
end of third larval stage (OÕDonnell 1987, Muratori et
al. 2004), and the larva spins its cocoon inside the
empty cuticle of the aphid and pupates (Hagvar and
Hofsvang 1991), forming a “mummy.” From that moment until emergence, it does not feed, and all metabolic processes, including metamorphosis, use energetic reserves accumulated during the larval stages.
In cold natural conditions, most Aphidiinae arrest
their development and enter hibernal quiescence
(dormancy) inside the cocoon in a mummiÞed aphid
(Stary´ 1970a, Jarry and Tremblay 1989). Quiescent
mummies are usually used for cold storage in insectaries before mass releases, because they are more
tolerant to cold exposure than adults (Stary´ 1970b).
For aphid parasitoids, low temperatures used for
long cold storage depend on the species and generally
range between 0 and 7⬚C (Archer et al. 1973, Scopes
et al. 1973, Polga´r 1986, Singh and Srivastava 1988,
Whitaker-Deerberg et al. 1994, Rigaux et al. 2000).
Survival of insects exposed to low temperatures depends on their cold-hardiness (Zachariassen 1985).
Biochemical changes associated with cold-hardiness
are often substantial, and maintenance of cold-hardiness is expected to be energy-dependent (Leather et
al. 1993).
The aim of this research was to study the consequences of a low temperature exposure and its duration on A. colemani; key parameters related to Þtness
(immature survival and adult longevity) and physiological parameters (water and lipids relations) associated to the cold-hardiness process were analyzed.
Materials and Methods
Rearing Aphids and Parasitoids. Laboratory cultures of M. persicae were established from individuals
collected in Þelds during 2000 at Louvain-la-Neuve,
Belgium (50.3⬚ N latitude). Aphids were reared in
0.3-m3 cages on oilseed rape (Brassica napus L., variety
Mesural) under 20 ⫾ 1⬚C, 60 ⫾ 10% RH, and a L 16:D
8 regimen. A. colemani originating from commercial
stocks of Biobest Co. were reared in the laboratory
under 20 ⫾ 1⬚C, 60 ⫾ 10% RH, and a L 16:D 8 regimen.
To obtain mummies for the experiments, batches
of 50 2-d-old aphids were offered to female parasitoids
for 4 h. Aphids were synchronized at the same age to
avoid host-age effects on parasitoid development
(Colinet et al. 2005). The parasitoid females used were
⬍48 h old, naõ¨ve, and mated. To ensure mating, each
female was placed with two males for 24 h and fed on
water and honey. Resulting parasitized aphids were
reared under 20 ⫾ 1⬚C, 60 ⫾ 10% RH, and a L 16:D 8
regimen to mummiÞcation. Newly formed mummies
were left to develop for 1 d before cold exposure
(under the same rearing conditions). One-day-old
mummies were used in the experiments because
young mummies are generally more tolerant to cold

229

exposure (Hofsvang and Hagvar 1977, WhitakerDeerberg et al. 1994, Levie et al. 2005).
Immature Survival and Adult Longevity After
Cold Exposure. Cold storage of A. colemani at 0⬚C
results in low emergence, and storage at 7⬚C seems to
be difÞcult because the wasps develop and emerge at
this temperature (Hofsvang and Hagvar 1977). Therefore, we chose two temperatures between 0 and 7⬚C:
2 ⫾ 0.3 and 4 ⫾ 0.3⬚C, situated below and above the
estimated thermal threshold (2.8⬚C) of A. colemani
(Elliott et al. 1995). Before the experiment, the relative humidity inside the incubators was progressively
increased by water evaporation until it reached a stable value of 75 ⫾ 5% RH for all the cold storage
duration. Temperature and relative humidity were
checked inside each thermo-regulated LMS incubator
using Onset Hobo data loggers (model: “H8 RH/
Temp/Light/external”). Batches of 1-d-old mummies
were exposed to cold conditions, and to avoid coldshock, an acclimation was performed by progressively
lowering the temperature by 5⬚C every 2 h, as described by Levie et al. (2005). During the cold exposure, mummies inside the incubators were maintained
in complete darkness in small unsealed plastic petri
dishes (4.5 cm diameter). For each temperature, the
cold-exposed mummies were removed at weekly intervals and kept at 20⬚C, so that we were able to
measure the survival after 1, 2, and 3 wk of cold
exposure, plus a control batch that had not been exposed to cold. Because an increasing mortality was
expected with duration of cold storage, a batch of 175,
200, and 250 randomly chosen mummies was exposed
for 1, 2 and 3 wk, respectively, at both temperatures.
A batch of 150 mummies was left under 20 ⫾ 1⬚C as a
control. After exposure, a deacclimation back to 20 ⫾
1⬚C was conducted according to the same pattern used
for the acclimation process for each batch of mummies. A parasitoid was considered as surviving when
complete emergence of the adult occurred within 7 d
after cold exposure. Adults that had broken partially
through the mummy were not considered as surviving.
Nonemerged mummies were collected and dissected
to identify the stage reached before death (i.e., teneral
or melanized adult).
For each temperature and duration, 16 emerged
adults (8 males and 8 females) were maintained at
20 ⫾ 1⬚C with food supply (solution of honey), and
survival was checked daily, until the last parasitoid
died, to determine adult longevity.
Physiological Parameters. For each treatment
(combination of temperature and duration of cold
exposure), 25 mummies were subjected to individual
mass measurement (micro-electrobalance; sensitivity,
0.1 ␮g). To determine mass loss (ML) occurring
during cold exposure, fresh mass was measured just
before (FMb) and after (FMa) cold exposure; ML
corresponds to the difference and was expressed as a
percentage of initial mass (ML ⫽ FMb ÐFMa/FMb ⫻
100). Mummies were immediately dried at 60⬚C for 3 d
in an air oven and reweighed to measure dry mass
(DM) and to deduce water mass (WM) determined
as the difference between FMa and DM. The water

230

ENVIRONMENTAL ENTOMOLOGY

content (WC) was calculated as a proportion of DM
(WC ⫽ WM/DM) (Kosta´l et al. 2004). Lean dry mass
(LDM) was measured by extracting lipids in a chloroform/methanol solution (2:1) (Vernon and Vannier
1996, Terblanche et al. 2004). Each pierced mummy
was left for 2 wk in an Eppendorf tube containing 1 ml
of the extracting solution agitated daily. The mummies
were placed for 12 h in an air oven at 60⬚C to eliminate
residues of the extracting solution before measurement of LDM. Body fat mass (FM) corresponds to
DM ⫺ LDM, and fat content (FC) was calculated as
a proportion of LDM (FC ⫽ FM/LDM). The deacclimation process was also executed for each group of
25 weighed mummies. As a control, all measurements
were performed on a batch of 25 mummies that were
not exposed to cold, except for ML being null, as FMb
⫽ FMa.
Statistical Analyses. Emergence rates (i.e., proportions of adults that successfully emerge) were compared between treatments using ␹2 tests (PROC
FREQ; SAS Institute 1990). Changes in gravimetric
measures were analyzed using analysis of covariance
(ANCOVA; PROC GLM; SAS Institute 1990) with
temperature (2 or 4⬚C) as the main effect (independent variable) and duration of cold exposure as the
covariate. Changes in log-transformed adult longevity
were analyzed using ANCOVA (PROC GLM; SAS
Institute 1990) with temperature (2 or 4⬚C) and sex as
main effects (independent variables) and duration of
cold exposure as the covariate. The equivalent slopes
assumption was veriÞed before each ANCOVA by
checking interaction in the full model.
Results
Immature and Adult Survival. Parasitoid emergences after cold exposure were clearly reduced at
both temperatures with increasing duration of exposure (Fig. 1). After 3 wk of continuous cold exposure,
only 14 and 9% of immature parasitoids emerged from
mummies at 2 and 4⬚C, respectively, as opposed to 93%
in the control set. After 1 wk of exposure, emergence
rates did not differ from control (4⬚C: ␹2 ⫽ 0.27; df ⫽
1; P ⫽ 0.60; 2⬚C: ␹2 ⫽ 2.0; df ⫽ 1; P ⫽ 0.15). Emergence
rates were signiÞcantly lower than in untreated mummies after 2 wk of cold exposure, (4⬚C: ␹2 ⫽ 59.28; df ⫽
1; P ⬍ 0.001; 2⬚C: ␹2 ⫽ 22.36; df ⫽ 1; P ⬍ 0.001) and after
3 wk (4⬚C: ␹2 ⫽ 141.17; df ⫽ 1; P ⬍ 0.001; 2⬚C: ␹2 ⫽
125.43; df ⫽ 1; P ⬍ 0.001). In contrast, the emergence
rates were similar for both temperatures (1 wk: ␹ 2 ⫽
0.81; df ⫽ 1; P ⫽ 0.36; 3 wk: ␹ 2 ⫽ 1.22; df ⫽ 1; P ⫽ 0.26).
After 2 wk of exposure, the emergence rate was signiÞcantly higher at 2 than at 4⬚C (␹2 ⫽ 11.59 df ⫽ 1;
P ⫽ 0.01). Dissections of nonemerged cold-stored
mummies revealed that they contained a majority
(⬎80%) of well-formed adults (Table 1). The proportion of fully formed adults in dead mummies was the
same among all treatments (␹ 2 ⫽ 8.65; df ⫽ 5; P ⫽
0.124).
Adult longevity (Fig. 2) decreased signiÞcantly
with duration of cold exposure (F ⫽ 53.31; df ⫽ 1,123;
P ⬍ 0.001); this reduction was similar for both sexes

Vol. 35, no. 2

Fig. 1. Emergence rate showing a signiÞcant decrease
with cold exposure duration.

(F ⫽ 0.01; df ⫽ 1,123; P ⫽ 0.93) and both temperatures
(F ⫽ 2.42; df ⫽ 1,123; P ⫽ 0.12).
Gravimetric Measures. Mummy ML (Fig. 3) occurring during low temperature storage increased significantly with duration of cold exposure (F ⫽ 46.10; df ⫽
1,197; P ⬍ 0.001) and similarly at both temperatures
(F ⫽ 4.06; df ⫽ 1,197; P ⫽ 0.054). Mummies lost 14.9
and 11.6% of their initial FM after 3 wk of cold storage
for the mummies stored at 2 and 4⬚C, respectively.
DM and water mass WM (Fig. 4) decreased significantly with duration of cold exposure (DM: F ⫽ 27.69;
df ⫽ 1,197; P ⬍ 0.001; WM: F ⫽ 16.81; df ⫽ 1,197; P ⬍
0.001) and similarly at both temperatures (DM: F ⫽
0.54; df ⫽ 1,197; P ⫽ 0.46; WM: F ⫽ 0.88; df ⫽ 1,197;
P ⫽ 0.35). WC (Fig. 4) increased slightly but signiÞcantly with duration of cold exposure (F ⫽ 12.76; df ⫽
1,197; P ⬍ 0.001) and similarly at both temperatures
(F ⫽ 0.02; df ⫽ 1,197; P ⫽ 0.90).
LDM (Fig. 5) remained constant and was not dependent on duration of cold exposure (F ⫽ 0.16; df ⫽
1,197; P ⫽ 0.68), with mean values ranging from 97.4 ⫾
17.1 to 100.9 ⫾ 15.7 ␮g. FM and FC (Fig. 5) decreased
Table 1.

Results of the dissections

No
Storage
No
Temperature
dissected Percent Percent
duration cold-stored
(⬚C)
dead
adults immatures
(weeks) mummies
mummies
4
4
4
2
2
2

1
2
3
1
2
3

175
200
250
175
200
250

16
116
228
22
68
216

87.50
89.66
93.86
86.36
82.35
91.20

12.50
10.34
6.14
13.64
17.65
8.80

For each condition, no. of mummies initially used for cold storage,
no. of dead mummies (i.e. the parasitoid did not successfully emerge)
used for dissection, percent of dead mummies containing an adult, and
percent of dead mummies containing an immature parasitoid.

April 2006

COLINET ET AL.: PHYSIOLOGICAL AND FITNESS COSTS OF COLD EXPOSURE IN A. colemani

Fig. 2. Changes in adult longevity in relation to duration
of cold exposure at 2 and 4⬚C, for males (f) or females (E).
Each point represents the mean ⫾ SE. Lines represent the
linear relationships between longevity and cold storage duration.

signiÞcantly with duration of cold exposure (FM: F ⫽
133.71; df ⫽ 1,197; P ⬍ 0.001; FC: F ⫽ 154.21; df ⫽ 1,197;
P ⬍ 0.001) and similarly at both temperatures (FM:
F ⫽ 0.68; df ⫽ 1,197; P ⫽ 0.41; FC: F ⫽ 0.31; df ⫽ 1,197;
P ⫽ 0.58).
Discussion
As observed in many studies, an exposure to prolonged low temperatures has a detrimental effect on
the survival of parasitic wasps (Polga´r 1986, Jarry and
Tremblay 1989, Whitaker-Deerberg et al. 1994, Langer
and Hance 2000, Lysyk 2004, Tezze and Botto 2004).
In this experiment, parasitoid survival was strongly
reduced after 2 wk of cold exposure at both temperatures tested (2 and 4⬚C). A previous study indicated
that A. matricariae was easier to maintain at a low
temperature (4⬚C); after 20 d, the survival was still
⬎90%. The ability of A. matricariae to withstand long
periods of cold storage is perhaps linked to its temperate latitude origins (Scopes et al. 1973). However,
A. colemani manifests a subtropical worldwide distribution (Stary´ 1975) and is therefore probably less
adapted to low temperature conditions.
Low temperatures are known to induce parasitoid
mortality caused by chill injury at temperatures well
above the supercooling point, and cold injuries are
expected to be more severe as temperature is lowered
or as the length of exposure is prolonged (Nedveˇ d
et al. 1998, Renault et al. 1999). Low temperature

Fig. 3. Changes of fresh mass loss (ML) at 2 and 4⬚C
according to cold exposure duration. Lines represent the
linear relationships between ML and cold storage duration.

231

injuries are probably caused by disruption of metabolic regulation (e.g., enzyme activity), particularly
cellular energetics that, when too severe or prolonged,
can lead to irreversible lethal metabolic imbalance
(Storey and Storey 1988, Kosta´l et al. 2004). According
to De Bach (1943), there are three main causes of
death during low temperature exposure: freezing
damage, desiccation, and starvation. In the case of
A. colemani, the Þrst cause can be excluded because
mummies were kept under nonfreezing temperatures.
The individuals inside the mummies could have suffered from desiccation even if water loss of cold-stored
individuals often occurs very slowly (De Bach 1943).
For insects at rest, water loss by respiration is negligible in comparison with cuticular losses through
evaporation (Edney 1977). In fact, a slight but significant decrease in water mass was observed during
cold storage. Evaporation depends on the activity gradient between atmospheric and body water. Water
represents 95Ð99% of body molecules (Danks 2000):
body water activity is between 0.95 and 0.99, and the
atmospheric water activity is the relative humidity
divided by 100 (Wharton 1985). When the body water
activity is higher than the atmospheric water activity,
which was the case, water evaporates from the insect.
Water loss could then have occurred even if aphid
parasitoids are well protected against desiccation inside mummies: one of the main roles of the cocoon is
to protect the immature from desiccation (Yoder et al.
1994, Tagawa 1996, Danks 2004, Legrand et al. 2004b).
It could be argued that part of the dehydration resulted from individuals that may have died during the
course of the cold storage. However, the dissections of
nonemerged mummies revealed that mortality occurred principally after metamorphosis, indicating
that they were actually still alive at the end of cold
storage period.
The decreases in DM and WM observed were associated to an increase in WC. This is because DM
decreased at a higher rate than WM, resulting in an
increase in the individual hydration, even if body
water was decreasing. Because of constraints linked to
the exoskeleton, arthropods have to maintain internal
turgidity and body volume. Therefore, during starvation, lost tissues mass must be replaced, in some species, by water which can result in an increase in hydration level if an easy access to water is possible
(Hervant et al. 1999). Similar increase in WC under
food deprivation associated with low temperatures has
previously been observed (Lavy et al. 1997, Renault et
al. 2002b, Levie 2002). Moreover, like dormant insects,
quiescent mummies maintain a low metabolism,
which requires and consumes energy. Metabolized
lipids produce water that may partially account for
limited water loss (Danks 2000). The slight increase of
tissue hydration indicates that water accumulation,
probably of metabolic origin, may compensate for the
loss of mass caused by fat use (Nedve˘ d and Windsor
1994). WC variations are complex and can result from
both cold adaptation and starvation (Yoder et al. 1994,
Renault et al. 2003). The mummies used in this study
were of similar mass (LDM), meaning that they were

232

ENVIRONMENTAL ENTOMOLOGY

Vol. 35, no. 2

Fig. 4. Changes of dry mass (DM), water mass (WM), and water content (WC) at 2 and 4⬚C according to cold exposure
duration. Lines represent the linear relationships between independent variables (DM, WM, and WC) and cold storage duration.

likely to be of equal size (Wharton 1985), so differences in water relations may not be attributed to
differential exposed surface area.

The increase in ML and decrease in DM observed
with cold exposure duration indicate that quiescent
parasitoids were actually consuming biomass. The DM

April 2006

COLINET ET AL.: PHYSIOLOGICAL AND FITNESS COSTS OF COLD EXPOSURE IN A. colemani

233

Fig. 5. Changes of lean dry mass (LDM), fat mass (FM), and fat content (FC) at 2 and 4⬚C according to cold exposure
duration. Lines represent the linear relationships between independent variables (FM and FC) and cold storage duration.

decrease was a result of depletion of fat reserves, as
observed in other starved parasitoids (Ellers 1996,
Olson et al. 2000). FC also declines regularly during

cold exposure. Body energy reserves, particularly
from fat body, may be lost during low temperature
exposure (David and Vannier 1996, Renault et al.

234

ENVIRONMENTAL ENTOMOLOGY

2002b). Prolonged exposure at low temperatures results in depletion of nutriments necessary for the parasitoid to complete development to emergence. This
may account for the increasing preemergence mortality observed with increased duration of cold exposure. Indeed, dissections of nonemerged dead mummies revealed that mortality occurred principally after
metamorphosis to adult. The inability of fully formed
adults to emerge from mummies probably result, at
least in part, from insufÞcient available energy to
break through the cocoon as noticed in the parasitoid
Diadegma insulare (Okine et al. 1996). Moreover, cold
exposure decreased adult longevity, as observed in
other parasitoids (Uc¸ kan and Gu¨ lel 2001, Rundle et al.
2003). In A. colemani, the amount of fat available for
potential emerging adults declined with duration of
cold exposure, and a corresponding decrease of adult
longevity was observed. Emerged parasitoids thus had
an energy stock proportional to cold exposure duration before emergence; this could have inßuenced
adult longevity. It has been shown that the longevity
of parasitoid is sharply linked to the amount of fat
reserve (Ellers 1996). Feeding of adult parasitoids on
honey solution did not compensate the depleted FC,
suggesting that wasps were unable to synthesize lipids
de novo as observed in other parasitoids (Ellers et al.
1998, Olson et al. 2000, Rivero and West 2002, Giron
and Casas 2003). The consequence of the lack of
lipognenesis makes the fat reserve a nonreplaceable
resource potentially limiting for parasitoids cold
stored during relatively long periods. As the fat reserves increase with size (Ellers et al. 1998, Rivero and
West 2002), heavier mummies with larger fat reserves
should presumably have a signiÞcant advantage for
survival at low temperature.
Acknowledgments
We thank D. B. Rivers for the review of an early version
of this manuscript and D. Giron for very constructive comments. Many thanks to G. Van Impe for useful help on the
manuscript. This study was supported by Ministe` re de la
Re´ gion WallonneÐDGTRE Division de la Recherche et de la
Coope´ ration ScientiÞque, FIRST EUROPE Objectif 3. We
are also grateful to the Station Biologique de Paimpont,
University of Rennes 1, for facilities. This paper is BRC 085
of the Biodiversity Research Centre.

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Received for publication 4 October 2005; accepted 3 January
2006.


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