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Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Interspecific variation in the response to low temperature
storage in different aphid parasitoids
H. Colinet & Th. Hance
´
´
Unite´ d’Ecologie
et de Biogeographie,
Biodiversity Research Centre, Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium

Keywords
Aphid parasitoid; cold storage; constant
temperature; fluctuating temperature.
Correspondence
´
Dr H. Colinet, Unite´ d’Ecologie
et de
´
Biogeographie,
Biodiversity Research Centre,
Universite´ catholique de Louvain, Croix du Sud
4-5, 1348 Louvain-la-Neuve, Belgium.
Email: herve.colinet@uclouvain.be
Received: 25 February 2009; revised version
accepted: 25 August 2009.
doi:10.1111/j.1744-7348.2009.00374.x

Abstract
Cold storage of natural enemies usually involves placing insects under constant
subambient temperatures. Even at non-freezing temperatures, a reduction
in survival is the norm. Using fluctuating thermal regimes (FTR) instead of
constant low temperature (CLT) has shown that mortality due to accumulation
of chilling injuries was significantly reduced in Aphidius colemani. Whether this
phenomenon can be generalised to other parasitoid species is not known. The
aim of this study was to analyse interspecific variation in the ability to tolerate
cold storage under CLT (continuous 2◦ C) versus FTR (daily cycle: 2◦ C for 22 h
and 20◦ C for 2 h) for various durations (0–20 days). Survival, sex ratio and
development of five different Aphidiine parasitoids were analysed: A. colemani,
Aphidius ervi, Aphidius matricariae, Ephedrus cerasicola and Praon volucre. A marked
interspecific variation in the ability to tolerate cold storage was observed:
A. matricariae and A. ervi were most chill tolerant, P. volucre and E. cerasicola
had an intermediate chill sensitivity and A. colemani was most chill sensitive.
In all species tested, FTR significantly reduced cold-induced mortality. This
phenomenon was manifested more in chill-sensitive species as they probably
accumulate chilling injuries more rapidly. The sex ratio remained unaffected
in all the species. Interspecific variation was also observed in developmental
responses to cold storage. Under CLT, time to adult emergence of A. matricariae,
A. colemani, A. ervi and P. volucre was temporarily stopped and in E. cerasicola
it increased. Under FTR, the short daily intervals at 20◦ C for 2 h allowed
parasitoids to continue development in all the species. Interspecific differences
are discussed. This study suggests that positive impact of FTR may apply to a
wide range of species.

Introduction
Major obstacles to the successful implementation of
augmentative biological control of insect pests are
difficulty, timing and cost of rearing beneficial insects in
very large numbers for mass release. Unlike pesticides,
most insects used in pest-control programs have a
relatively short shelf life and therefore they are produced
shortly before they are used. It is thus necessary to have
efficient storage methods in order to meet requirements.
The use of low temperature has proved to be a valuable
method for increasing shelf life of insects, allowing
flexibility and efficiency in mass production and providing
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

a steady and sufficient supply of insects for pest-control
programs. Cold storage also permits synchronised field
releases of natural enemies during the critical stages of
pest outbreaks (McDonald & Kok, 1990; Leopold, 1998;
Venkatesan et al., 2000). The application of cold storage
is not limited only to facilitating industrial rearing of
beneficial insects but it is also useful for maintaining
insect colonies under laboratory conditions for research
purposes. Moreover, there are other fields such as rearing
insects for pet food, fish bait, forensic indicators, as well
as rearing for rescue programs of endangered species
that may also profit from the advances in cold storage
technology (Leopold, 2007).
147

Cold storage of aphid parasitoids

Cold storage of natural enemies (e.g. Hymenopteran
parasitoids) usually involves placing insects at some
constant subambient temperatures, generally above 0◦ C
(Leopold, 1998). Even at these non-freezing constant
temperatures, a reduction in survival is the norm (Hance
et al., 2007). In fact, in natural conditions (Legrand et al.,
2004), as well as under laboratory conditions (Langer
& Hance, 2000; Levie et al., 2005; Colinet et al., 2007c),
parasitoids die at conditions well above temperatures of
spontaneous ice crystallisation (i.e. supercooling point,
SCP). The consequences of a long exposure to low
but non-freezing temperatures are defined as ‘indirect
chilling injuries’ (Lee, 1991; Chown & Nicholson, 2004).
According to Koˇstal
´ et al. (2006), when the ‘dose of
cold exposure’ which corresponds to a combination
of exposure time and temperature exceeds a specific
threshold, indirect chilling injuries accumulate becoming
progressively irreversible and eventually lethal.
In a previous study (Colinet et al., 2006b), we have
emphasised that exposing a parasitic wasp Aphidius
colemani Viereck to fluctuating thermal regimes (FTR)
(i.e. cold exposure interrupted by periodic short pulses
of high temperature) versus constant low temperatures
(CLTs) significantly reduces mortality. Under FTR, lower
mortality is a result of periodic opportunities during
higher temperature intervals for physiological repair and
recovery from accumulated chilling injuries (Colinet et al.,
2007a,b). Whether this phenomenon can be generalised
to other parasitoid species is not known. As emphasised
by Leopold (1998), there is an immense variation in the
capacity of insect species to tolerate cold storage, making
any taxonomic generalisations across taxa unfeasible.
Because of this natural genetic variation between species,
individual studies are necessary to determine speciesspecific parameters for cold storage. Therefore, the aim
of this study is to analyse interspecific variation in
parasitoid ability to tolerate cold storage under CLT versus
FTR. We used five different parasitoid species specific
to aphids: A. colemani Viereck, Aphidius ervi Haliday,
Aphidius matricariae Haliday, Ephedrus cerasicola Stary´ and
Praon volucre Haliday, all belonging to the subfamily of
Aphidiines (Hymenoptera: Braconidae: Aphidiinae).

Materials and methods
Rearing aphids and parasitoids
The green peach aphid Myzus persicae Sulzer (Homoptera:
Aphididae) was used as host for rearing the parasitoid
species. Laboratory cultures of M. persicae were established
from individuals collected in the field during 2000 at
Louvain-la-Neuve (Belgium). Aphids were reared in
0.3 m3 cages on sweet pepper (Capsicum annuum L.)
148

H. Colinet & Th. Hance

under laboratory conditions of 18 ± 1◦ C, ± 60%
relative humidity (RH) and light : dark (LD) 16:8 h.
The parasitoids A. colemani and A. ervi were originally
provided by Viridaxis SA (Belgium) in 2008. A. matricariae
was collected in the field in 2005 at Louvain-la-Neuve
(Belgium). E. cerasicola was collected in the field in 2006
at Corroy-le-Grand (Belgium) and P. volucre was collected
in 2008 at Fleurus (Belgium). All the parasitoid species
were reared on M. persicae under laboratory conditions for
several generations before the experiment.
To obtain standard aphid mummies containing parasitoid pupae for the experiments, batches of 50 standardised 3-day-old aphids were offered to a female
parasitoid wasp for 4 h to allow parasitism. Parasitoid
wasps were less than 48-h old and mated. The resulting parasitised aphids were then reared under laboratory
standard conditions (18 ± 1◦ C, ± 60% RH and LD 16:8 h)
until mummification. Newly formed mummies were left
to develop for 1 day for Aphidius sp. or 2 days for E.
ceriscola and P. volucre under the same rearing conditions
to allow complete mummy/cocoon formation. Mummies
were then exposed to low temperature treatments.
Thermal treatments
In order to extend shelf life, natural enemies are exposed
to cold for various periods of time at species-specific
temperatures, generally ranging between 0◦ C and 15◦ C
(van Lenteren & Tommasini, 1999). The temperature
choice for cold storage has to be low enough to decrease,
or even stop completely parasitoid development. This
is particularly true for Aphidiines, which are shipped
at the mummy stage. Undesirable emergence during
refrigeration occurs at 7◦ C in A. colemani (Hofsvang &
Hagvar, 1977) and at 8◦ C in A. matricariae (Shalaby &
Rabasse, 1979). Therefore in the present experiment, we
choose 2◦ C for cold storage. Mummies were exposed
to low temperature inside thermo-regulated cooled
incubators (Model 305, LMS Ltd, Sevenoaks, Kent,
UK) with saturated RH and complete darkness. Groups
of mummies were randomly assigned to either CLT
(continuous exposure at 2◦ C) or FTR (the 2◦ C exposure
was interrupted by daily transfer to 20◦ C for 2 h) (see
Colinet et al., 2006b). Control groups of mummies were
allowed to continue their development until emergence
at 20◦ C.
Survival assays, sex ratio and development
To test whether thermal treatments (FTR versus
CLT) had a differential impact on parasitoid survival, for each species and each experimental condition (treatment × duration), we used 4 replicates of
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

50 mummies (n = 4 × 50 individuals) to calculate the
mean emergence rate. After 0, 5, 10, 15 and 20 days
of cold exposure, mummies were placed at 20◦ C, and
survival was measured as the number of adults that
successfully emerge (emergence rate).
The sex ratio at emergence was determined and
expressed as the proportion of males. It was assumed
that any significant difference in sex ratio between test
sample and control would indicate a differential mortality
between sexes.
Finally, for each species and each experimental
condition, the time to adult emergence after mummies
were placed at 20◦ C following cold exposure was
individually recorded. Data were used to determine the
cumulative emergence function in relation to time after
cold storage and the corresponding time required for 50%
of emergence (EM50 ) (median emergence peak).

Statistics
Arcsin square root transformation was required to normalise distribution of proportional data (i.e. emergence
rate and sex ratio) (Hardy, 2002). For each species, emergence rate and sex ratio were analysed using two-way
analysis of variance with thermal treatment (FTR versus
CLT) and duration of cold exposure (from 0 to 20) as
fixed factors. Subsequently for each duration, Bonferroni
post-test was used to compare thermal treatments. Data
presented in figures are untransformed. A significance
level of α = 0.05 was used for all tests.
Daily emergence after cold exposure was cumulated
which gave a sigmoid-shaped function for each species
and each experimental condition. Non-linear regression
method was used to fit the relationship between the
dependent variable Y (the cumulative emergence) and
the independent variable X (the time after cold exposure).
EquationY = 100/[1 + 10(A – X)∗B ] was used to fit the data
and to estimate the parameters A and B, which represent
EM50 and the slope, respectively. For each species,
treatment-specific changes in EM50 values with cold
storage duration were analysed using linear regressions.
Assuming that if development occurs during storage,
EM50 values should decrease with time and therefore
slopes should be different from 0 (i.e. Ho : slope = 0).
Finally, for each species, slope homogeneity tests were
performed to determine if changes in EM50 values with
cold storage duration were differentially affected by
treatments (FTR versus CLT).
All tests were performed using SPSS V16.0.1 (SPSS
Inc., Chicago, IL, USA) or Prism V5 (GraphPad Software
Inc., San Diego, CA, USA).
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

Results
Survival and sex ratio during cold storage
When mummies were stored under CLT conditions,
reduction of parasitoid emergence was observed in all
species and the ability to tolerate these prolonged cold
exposures showed strong interspecific variation (Fig. 1).
Aphidius matricariae and A. ervi showed a relatively good
tolerance to exposure under CLT (Fig. 1). After 10 days of
cold storage, survival was still similar to the control value
(more than 85%) while reduction in emergence began to
start after 15 days. At the end of the experiment, there
was still approximately 65% of surviving adults under
CLT in both species. Survival of A. matricariae and A. ervi
mummies exposed to FTR was only slightly affected by
low temperature with more than 85% of individuals
being able to emerge after 20 days of cold storage
(similar to control) (Fig. 1). Statistical analysis showed
that the emergence rate was significantly affected by
treatment (FTR versus CLT) and by cold storage duration
in both species (Table 1). The significant interactions
indicate that temporal variations in emergence rates were
treatment specific (Table 1). Bonferroni tests revealed
that in A. matricariae, emergence was greater under FTR
than under CLT at 20 days whilst in A. ervi emergence
under FTR was greater at 15 and 20 days. The sex ratio
was female biased in A. matricariae (approximately 40% of
males) and male biased in A. ervi (approximately 65–70%
of males) (Fig. 2). The sex ratios were not affected by
treatment or duration (Table 1).
Ephedrus cerasicola and P. volucre displayed an intermediate chill sensitivity (Fig. 1). Under CLT, for E. cerasicola
reduction of adult emergence already occurred after
5 days and after 10 days emergence had reduced to only
40%, whereas for P. volucre, emergence remained similar
to the control value after 10 days reducing to less than
40% after 20 days. Under FTR, emergence after 20 days
was still 74.5% for E. cerasicola and 84.5% for P. volucre,
clearly higher than under CLT (Fig. 1). Statistical analysis
showed that emergence rates were significantly affected
by treatment and duration in both species and that there
were significant interactions between the two factors
(Table 1). Bonferroni tests revealed that emergence was
greater under FTR than under CLT from 5 days duration in E. cerasicola and for 15 and 20 days durations in
P. volucre. The sex ratio was male-biased in both species
(approximately 70% of males) (Fig. 2) and was unaffected
by treatment and duration (Table 1).
Aphidius colemani appeared to be the most chillsensitive species among those tested (Fig. 1). In this
species, strong temporal reduction of adult emergence
was observed under CLT. After 10 days, emergence
reached less than 50% and only 3.5% of individuals
149

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

Figure 1 Survival, expressed as the proportion of emerged adults (mean percent ± SE), as function of cold exposure duration for each species and each
thermal treatment. Black bar for control, white bars for constant low temperature (CLT) and grey bars for fluctuating thermal regimes (FTR). Symbols
Am for Aphidius matricariae, Ae for Aphidius ervi, Ac for Aphidius colemani, Pv for Praon volucre and Ec for Ephedrus cerasicola. Symbol (*) indicates
significant difference between thermal treatments (CLT versus FTR) for each duration (Bonferroni post-test).

could survive 20 days exposure. The beneficial impact
of FTR was again very clear, but it seems that some
individuals were not able to recover completely as
mortality was around 50% after 20 days under FTR.
Emergence rate was significantly affected by treatment
and duration in A. colemani and there was significant
interaction between treatment and duration (Table 1).
Bonferroni tests revealed that emergence was greater
under FTR than under CLT from 5 days duration. Sex
ratio was female biased (approximately 45% of males)
(Fig. 2) and was not affected by treatment and duration
(Table 1).
Time to emergence
For each species and condition, a cumulative emergence
function was determined in relation to the time after
cold exposure. The adjusted parameters of these sigmoid
functions [i.e. time required for 50% of emergence
(EM50 ) and slopes] are summarised in Table 2. The EM50
gives an accurate estimation of the median emergence
peak. The three Aphidius species required approximately
4–5 days to complete development until adult emergence
under control conditions, whereas E. cerasicola and
P. volucre required longer time to complete development
(EM50 = approximately 7–8 days for control) (Table 2).
In P. volucre, we occasionally noticed few unusual
150

individuals that emerged long time after emergence
peak. For each species, treatment-specific changes
in EM50 values were regressed against cold storage
duration (Table 3; Fig. 3). It was assumed that if some
development occurs during storage, EM50 values should
decrease linearly with time and therefore slopes should
be different from 0. In A. matricariae, A. colemani,
A. ervi and P. volucre, changes in EM50 values did
not differ significantly from 0 under CLT, suggesting
that development was momentarily stopped or very
reduced at constant 2◦ C. On the contrary, significant
linear decreases in EM50 values with cold storage
duration were observed under FTR, indicating that
development proceeded under fluctuating temperatures
(Table 3; Fig. 3). After 20 days of cold storage under
FTR, individuals emerged 1.8, 2.2, 2.4, 2.0 days before
control individuals in A. matricariae, A. colemani, A. ervi
and P. volucre, respectively (Table 2; Fig. 3). E. cerasicola
exhibited a different pattern: significant increase in EM50
values was observed under CLT whilst EM50 values
decreased significantly with cold storage duration under
FTR (Table 3; Fig. 3). After 20 days of cold storage,
E. cerasicola individuals emerged 1.1 days after and
1.6 days before control individuals under CLT and FTR,
respectively (Table 2; Fig. 3). Based on regression model
equations, the X-intercepts (i.e. when EM50 = 0) were
calculated (Table 3). These values predict time required
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

Table 1 Results of two-way analysis of variance for emergence rate and
sex ratio, in the five Aphidiine species testeda

Species

Parameter

Emergence
Aphidius
matricariae
Sex ratio

Emergence
Aphidius ervi
Sex ratio

Emergence
Aphidius colemani
Sex ratio

Emergence
Praon volucre
Sex ratio

Emergence
Ephedrus
cerasicola
Sex ratio

Source of
Variation

F

P

Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction
Treatment
Duration
Interaction

15.99
15.20
4.841
0.002
0.187
0.369
12.49
7.222
3.192
3.643
0.931
2.137
205.3
131.1
21.83
0.024
2.366
2.624
78.80
36.12
22.64
0.755
2.523
0.936
105.50
26.84
9.542
0.298
1.067
1.434

<0.001
<0.001
0.004
0.995
0.943
0.828
0.001
0.003
0.026
0.065
0.459
0.100
<0.001
<0.001
<0.001
0.876
0.096
0.073
<0.001
<0.001
<0.001
0.392
0.063
0.457
<0.001
<0.001
<0.001
0.589
0.390
0.246

a Fixed factors are thermal treatment (fluctuating thermal regimes versus
constant low temperature) and duration of cold exposure (from 0 to
20 days).

to emergence of adults during storage period. Finally,
heterogeneity tests were significant for all the species
indicating that changes in EM50 values with cold storage
duration were treatment specific (Table 3).

Discussion
Exposure to low temperatures, lethal or sublethal, is
a major factor shaping life history traits in insect
parasitoids (Hance et al., 2007). As observed in other
studies, exposure to prolonged CLTs has a detrimental
effect on the survival of parasitic wasps (Hofsvang &
Hagvar, 1977; Jarry & Tremblay, 1989; Langer & Hance,
2000; Levie et al., 2005; Colinet et al., 2006a; Marwan &
Tawfiq, 2006). Generally when the ‘dose’ of cold exposure
exceeds specific thresholds, chilling injuries accumulate,
become progressively irreversible and eventually lethal
(Bale, 1996, 2002; Koˇstal
´ et al., 2006). As expected,
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

detrimental effects of low temperature increased with
duration of cold exposure and this was particularly
pronounced in chill-sensitive species such as A. colemani.
The tolerance to cold storage of the five Aphidiine
species analysed in this study showed interspecific
variations. A. matricariae and A. ervi were found to be most
chill tolerant, with approximately 65% of emergence after
20 days under CLT. This observation is in accordance
with literature, where A. matricariae was found to display
a survival higher than 70% after 23 days of cold storage
at 2◦ C (Polgar, 1986) as well as after 3 weeks at 3.5◦ C
(Marwan & Tawfiq, 2006). Concerning A. ervi, there are
no published data on the chill tolerance for this species,
except for data of Langer & Hance (2000) who analysed
the cold-hardiness of diapausing versus non-diapausing
wasps. Results of their study corroborate ours, as nondiapausing A. ervi were found to tolerate cold exposure
relatively well, with 80% survival after 20 days at 0◦ C.
P. volucre and E. cerasicola displayed an intermediate chill
sensitivity, with less than 50% of individuals surviving
after 20 days at CLT. There are no data published on
cold-tolerance of P. volucre, therefore this study provides
a first look at response to cold exposure in this species.
Concerning E. cerasicola, the only report of cold storage
comes from Hofsvang & Hagvar (1977) who found that
E. cerasicola was able to tolerate cold storage at around
0◦ C with survival ranging from 80 to 30% after 4 weeks,
depending on the age of the mummies. Hofsvang &
Hagvar (1977) also found that A. colemani was not suited
for prolonged cold storage. In the present study, only
3.5% of individuals could survive after 20 days of cold
exposure under CLT confirming the high chill sensitivity
of this species, as was previously observed by Colinet
et al. (2006a,b). A. colemani seems to accumulate chilling
injuries to such an extent that FTR may not be sufficient
to allow a complete recovery, an increasing mortality
being also observed under FTR.
The interspecific variation in the ability to tolerate
prolonged cold exposure may result, at least in part,
from specific thermal adaptations which may be linked
to geographical origins and distribution. The distribution
area of A. ervi covers North America, Europe and part of
Asia. A. matricariae originated in northern India but is now
found in both North and South America, Australia and
various parts of Europe (Stary,
´ 1970, 1999). E. cerasicola is
widely distributed in Europe including Northern countries
(Stary,
´ 1970; Hofsvang & Hagvar, 1977). P. volucre is
widely distributed in Europe and Asia (Stary,
´ 1970,
1999), while A. colemani manifests a subtropical–tropical
distribution and is restricted to warm climate areas of
Mediterranean Europe, parts of Asia, Africa, Australia
and South America (Stary,
´ 1975, 1999). The ability of
A. ervi and A. matricariae to withstand long periods of
151

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

Figure 2 Sex ratio, expressed as male proportion (mean proportion ± SE), as function of cold exposure duration for each species and each thermal
treatment. Black bar for control, white bars for constant low temperature (CLT) and grey bars for fluctuating thermal regimes (FTR). Symbols Am for
Aphidius matricariae, Ae for Aphidius ervi, Ac for Aphidius colemani, Pv for Praon volucre and Ec for Ephedrus cerasicola.

storage is perhaps linked to their temperate latitude
origins (Scopes et al., 1973). A. colemani is probably less
adapted to low temperature conditions. Because of the
natural variation between species, individual studies are
necessary to determine species-specific parameters for
cold storage. However, even if genetic variation between
species can significantly influence variability in cold
storage tolerance, phenotypic plasticity is also expected to
account for a part of the variability observed.
Survival at low temperature is known to be related to
the depletion of energy reserves during starvation which
may progressively become critical (Colinet et al., 2006a).
In addition, CLT induces the accumulation of chilling
injuries, as a result of various physiological dysfunctions
such as (a) loss of membrane potentials (Slachta et al.,
2002), (b) neuromuscular injuries (Kelty et al., 1996),
(c) thermoelastic stress (Lee & Denlinger, 1991), (d) ion
homeostasis perturbations (Koˇstal
´ et al., 2006) and (e)
inhibition of critical gene(s) expression (Yocum et al.,
2006). Several studies have shown that under FTR,
short warm intervals give opportunities to physiologically
recover from accumulated chilling injuries (Colinet et al.,
2007a, b; Koˇstal
´ et al., 2007; Lalouette et al., 2007). One
objective of the present study was to test whether the
positive impact of FTR could be generalised to parasitoid
species other than A. colemani. According to our results,
it seems that FTR significantly reduces cold-induced
mortality in all the species tested. This phenomenon
152

seems to be more pronounced in chill-sensitive species as
they accumulate chilling injuries more rapidly.
The sex ratio at emergence was measured and it was
assumed that any significant change would indicate a
differential mortality between sexes. We did not observe
any consistent trend in sex ratio. Other studies on
Aphidiine parasitoids also concluded that cold storage
does not affect sexes differentially (Hofsvang & Hagvar,
1977; Jarry & Tremblay, 1989; Rodrigues et al., 2003;
Levie et al., 2005). Although contrasting conclusions
were reached in other studies where increase in male
population has been observed (Okine et al., 1996; Foerster
& Nakama, 2002; Chen et al., 2008).
We observed different developmental patterns in the
five parasitoid species tested. In the three Aphidius species,
time necessary to compete mummy-adult development at
20◦ C was short (4–5 days), which corresponds to values
from the literature (Hofsvang & Hagvar, 1975a; Scopes &
Biggerstaff, 1977; Sigsgaard, 2000; Sampaio et al., 2007).
P. volucre and E. cerasicola required more time to complete
mummy-adult development (7–8 days). This observation
also corroborates values found in the literature for
these two species (Hofsvang & Hagvar, 1975b; Sigsgaard,
2000). Moreover, interspecific variation was observed
in developmental responses to cold storage. Under CLT
development of A. matricariae, A. colemani, A. ervi and
P. volucre was temporarily arrested or very reduced.
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

Table 2 Parameter estimates (with their 95% confidence intervals, CI) and adjusted coefficient of determination (r2 ) of the sigmoid models describing the
cumulative emergence (Y) in relation to time after cold storage (X), for each species and each experimental conditiona
Treatment
Control
CLT
Aphidius matricariae
FTR

Control
CLT
Aphidius ervi
FTR

Control
CLT
Aphidius colemani
FTR

Control
CLT
Praon volucre
FTR

Control
CLT
Ephedrus cerasicola
FTR

Duration (days)
0
5
10
15
20
5
10
15
20
0
5
10
15
20
5
10
15
20
0
5
10
15
20
5
10
15
20
0
5
10
15
20
5
10
15
20
0
5
10
15
20
5
10
15
20

EM50 (days)

EM50 − 95% C.I.

Slope

Slope − 95% C.I.

r2

4.312
4.261
4.03
3.768
3.957
4.024
3.376
2.813
2.53
5.016
5.088
4.744
4.630
4.698
4.637
3.721
3.068
2.688
4.813
5.157
4.5
4.843
4.448
4.482
4.092
3.385
2.64
7.55
8.347
7.394
7.636
8.359
7.377
6.048
5.789
5.539
8.083
8.46
8.545
8.961
9.132
8.046
6.891
6.776
6.472

4.281–4.356
4.182–4.340
3.898–4.161
3.646–3.891
3.862–4.053
3.951–4.098
3.311–3.440
2.766–2.859
2.466–2.594
4.988–5.055
5.056–5.120
4.691–4.796
4.577–4.684
4.641–4.756
4.589–4.684
3.686–3.756
2.710–3.164
2.620–2.756
4.768–4.840
5.121–5.192
4.491–4.509
4.796–4.890
4.343–4.552
4.382–4.582
3.968–4.215
3.320–3.450
2.617–2.663
7.491–7.610
8.302–8.392
7.332–7.455
7.572–7.700
8.262–8.456
7.254–7.500
5.950–6.146
5.716–5.862
5.506–5.571
7.945–8.259
8.401–8.519
8.465–8.625
8.898–9.024
9.069–9.196
8.015–8.077
6.775–7.008
6.646–6.907
6.404–6.541

1.017
0.928
0.941
1.271
1.141
1.202
1.020
1.116
0.843
1.318
1.100
1.453
1.246
1.489
0.841
0.943
0.969
1.490
0.987
1.354
1.897
1.200
0.772
0.777
1.060
0.836
1.446
0.859
0.738
0.899
1.058
0.647
0.747
0.990
1.489
1.145
0.757
1.156
0.697
1.037
1.022
1.061
0.836
0.853
0.841

0.919–1.113
0.791–1.064
0.683–1.199
0.855–1.686
0.823–1.460
0.912–1.493
0.895–1.145
0.988–1.242
0.754–0.932
1.151–1.484
1.007–1.194
1.235–1.672
1.107–1.386
1.266–1.712
0.771–0.906
0.880–1.005
0.766–1.172
1.234–1.746
0.894–1.080
1.190–1.517
1.863–1.931
1.039–1.361
0.648–0.895
0.657–0.896
0.735–1.385
0.746–0.925
1.371–1.521
0.772–0.944
0.688–0.786
0.802–0.995
0.926–1.189
0.565–0.729
0.609–0.884
0.771–1.208
1.125–1.853
1.075–1.215
0.569–0.944
1.025–1.286
0.618–0.775
0.877–1.197
0.875–1.169
0.978–1.144
0.665–1.006
0.659–1.047
0.746–0.935

0.991
0.977
0.993
0.925
0.995
0.970
0.981
0.987
0.980
0.995
0.996
0.989
0.989
0.997
0.993
0.995
0.954
0.974
0.993
0.999
1.000
0.990
0.966
0.970
0.928
0.984
0.997
0.990
0.995
0.989
0.986
0.978
0.962
0.997
0.978
1.000
0.934
0.988
0.984
0.985
0.986
0.996
0.961
0.951
0.987

∗B

The following equation was used to fit the data, Y = 100/[1 + 10(A – X) ] and to estimate the parameters A which represents the time required for 50%
of emergence (EM50 ) and B which represents the slope.
a

The estimated thermal threshold (T0) for mummyadult development is 7.9◦ C for A. matricariae, 5.9◦ C
for A. colemani, 6.6◦ C for A. ervi and 5.5◦ C for P. volucre
(Scopes & Biggerstaff, 1877; Sigsgaard, 2000; Sampaio
et al., 2007). Therefore, based on estimated T0 found
in the literature, development should be momentously
Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

stopped or very reduced at constant 2◦ C. Our observations
thus support the data found in the literature. In
E. cerasicola, time to emergence significantly increased
with duration of cold storage indicating a developmental
delay. A delay in pre-emergence period is a phenomenon
that has been observed in other parasitoids. This seems
153

Cold storage of aphid parasitoids

H. Colinet & Th. Hance

Table 3 Results of linear regression models describing treatment-specific changes in the time required for 50% of emergence (EM50 ) according to cold
exposure durationa
Species
Aphidius matricariae
Aphidius ervi
Aphidius colemani
Praon volucre
Ephedrus cerasicola

Treatment

F

P

r2

CLT
FTR
CLT
FTR
CLT
FTR
CLT
FTR
CLT
FTR

7.781
161.0
7.778
146.4
1.467
90.87
0.321
26.98
95.47
22.55

0.069
0.001
0.068
0.001
0.312
0.002
0.610
0.013
0.002
0.017

0.722
0.982
0.721
0.979
0.328
0.968
0.096
0.90
0.969
0.882

Y-intercept (X = 0) X-intercept (Y = 0)
4.306
4.366
5.054
5.071
4.961
4.971
7.676
7.583
8.116
8.152

179.0
45.72
231.0
40.73
237.6
45.66
NA
67.58
NA
90.74

Slope

Slope heterogeneity

F

P

−0.024
−0.095
−0.021
−0.124
−0.020
−0.108
0.0181
−0.112
0.051
−0.089

yes

38.94 <0.001

yes

62.90 <0.001

yes

18.09

0.005

yes

11.40

0.014

yes

52.06 <0.001

a Based on linear models, Y- and X-intercepts are calculated, the latter predicting when adults should emerge during storage. For each species, slope
homogeneity tests are used to determine if changes in EM50 values with cold storage duration are differentially affected by treatments (fluctuating
thermal regimes versus constant low temperature).

to be a sublethal effect of low temperature (Ballal
et al., 1989; Kivan & Kilic, 2005; Chen & Leopold,
2007; Luczynski et al., 2007). Under FTR, short daily
intervals at 20◦ C for 2 h allowed parasitoids to continue
development. This may limit the use of FTR technique
because Aphidiines are commercially distributed not as
adults but at mummy stage. Based on regression model
equations and X-intercepts (Table 3), it was possible
to predict that adults under FTR should emerge after
40–45 days for Aphidius species, after 67 days for P. volucre
and after 90 days for E. cerasicola. Therefore, undesirable

development under FTR should not impact negatively
insects stored for relatively short periods.
Exposure to constant suboptimal temperature usually
negatively affects reproductive potential of parasitoids
(Hance et al., 2007). Apart from beneficial impact on
survival, FTR allows preservation of male reproductive
ability and conservation of mobility performances in
A. colemani (Colinet and Hance, 2009). This is particularly
important for successful biological control. From an
applied point of view, benefit of using FTR for cold storage
of all tested Aphidiine species is clear. Our study thus

Figure 3 Least-squares linear regressions modelling the changes in the time required for 50% of emergence (EM50 ) according to cold exposure duration.
Dashed lines and circles for constant low temperature (CLT), plain lines and squares for fluctuating thermal regimes (FTR). Symbols Am for Aphidius
matricariae, Ae for Aphidius ervi, Ac for Aphidius colemani, Pv for Praon volucre and Ec for Ephedrus cerasicola.

154

Ann Appl Biol 156 (2010) 147–156 © 2010 The Authors
Journal compilation © 2010 Association of Applied Biologists

H. Colinet & Th. Hance

suggests that the positive impact of FTR may apply to a
wide range of species.

Acknowledgements
We thank J. Shirriffs and L. Rako from Centre
for Environmental Stress and Adaptation Research in
Melbourne (AU) for very constructive comments on the
manuscript. This study was supported by Fonds de la
Recherche Scientifique (FNRS), Belgium. This paper is
number BRC141 of the Biodiversity Research Centre.

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