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Research Article
Received: 15 March 2013 Revised: 15 July 2013 Accepted article published: 26 July 2013 Published online in Wiley Online Library: 22 August 2013
(wileyonlinelibrary.com) DOI 10.1002/ps.3619
Factors inuencing supercooling capacity
of the koinobiont endoparasitoid Venturia
canescens (Hymenoptera: Ichneumonidae)
Stefanos S. Andreadis,
a,b
Christos G. Spanoudis,
a
Christos G. Athanassiou
c
and Matilda Savopoulou-Soultani
a
Abstract
BACKGROUND: Venturia canescens is aparthenogenetic koinobiont endoparasitoidof several pyralidmothlarvaethat aremajor
pests of stored products. Low temperatures have been extensively used to control stored-product insects as an alternative
to the application of traditional pesticides. However, most studies have focused on the cold hardiness prole of the major
stored-product pests. The objective of this study was to investigate how factors such as age, food, host availability and
acclimation affect the cold tolerance of V. canescens by determining its supercooling capacity.
RESULTS: Youngadults displayedsignicantlylower supercoolingpoints (SCPs) thanolder adults, irrespectiveof theavailability
of a host. Host availability had a moderate effect on supercooling, whereas food consumption resulted in a signicant
enhancement of SCP. Acclimation to low temperatures increased the supercooling capacity considerably. Furthermore, an
increase in the duration of exposure to acclimation temperature resulted in lower SCPs.
CONCLUSION: Adults of V. canescens displayed an enhanced ability to supercool, however, they appear to be less cold tolerant
than their respective hosts. This information would be useful in determining the potential of using V. canescens as a biological
agent in Integrated Pest Management (IPM) programs, taking into consideration the adverse effects of lowtemperatures on its
survival.
c 2013 Society of Chemical Industry
Keywords: Ichneumonidae; Ephestia kuehniella; supercooling point; age; food; acclimation
1 INTRODUCTION
Venturia canescens Gravenhorst (Hymenoptera: Ichneumonidae)
is a parthenogenetic solitary, koinobiont endoparasitoid that
develops in the larvae of several pyralid moths, which are major
pests in stored products, such as Ephestia kuehniella Zeller,
Ephestia elutella (H ubner), Plodia interpunctella (H ubner) and
Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae).
1
Several
laboratory studies have been conducted using this parasitoid as
a model organism because of its rapid mass rearing and its large
body size, which allows several observations that are not always
possible with other parasitoid species.
25
It is widely known that temperature has a major impact on
insects. Nearly every aspect of an insects life is inuenced by
temperature, including its biology, behavior, physiology and
evolution. The exposure of insects to lowor subzero temperatures
is a major factor that should be considered as having a great
impact ontheir life table characteristics andbehavior. The capacity
of an organismto survive exposure to lowtemperatures is referred
toas coldhardiness or coldtolerance. Avariety of factors affect this
capacity, including development stage, nutritional status, genetic
potential and thermal history (acclimation).
6
To estimate the cold
hardiness of an insect species various parameters should be taken
into consideration, such as the seasonal uctuation of cryoprotec-
tants, supercoolingabilityandlethal temperatures after prolonged
exposure to subzero temperatures above the supercooling point
(SCP). The SCP is dened as the temperature at which body
water spontaneously freezes and latent heat is released upon
crystallization.
7
SCP itself is inuenced by several factors. In
the European corn borer, Ostrinia nubilalis Hubner (Lepidoptera:
Crambidae), age appeared to affect the supercooling ability of
non-diapausing larvae. Late fth instars were more supercooled
than early ones, probably due to the cessation of feeding prior
to pupation.
8
Moreover, food consumption is generally known
to have a negative effect on SCP, mainly due to the presence
of ice-nucleation bacteria.
9
Acclimation for a few days at low
temperatures considerably improved the supercooling ability of
Lobesia botrana (Denis &Schiffermuller) (Lepidoptera: Tortricidae),

Correspondence to: S.S. Andreadis, Swedish University of Agricultural Sciences,

Department of Plant Protection Biology, Unit of Chemical Ecology, Box 102,
23053 Alnarp, Sweden. E-mail: stefanos.andreadis@slu.se
a Laboratory of Applied Zoology and Parasitology, Aristotle University of
Thessaloniki, Thessaloniki, Greece
b Department of Plant Protection Biology, Swedish University of Agricultural
Sciences, Alnarp, Sweden
c Department of Agriculture, Plant ProductionandRural Environment, University
of Thessaly, N. Ionia, Volos, Greece
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Cold tolerance of V. canescens www.soci.org
which might be related to the accumulation of cryoprotective
substances, such as glycerol and many other sugars.
10,11
The exposure of insects to low or freezing temperatures is a
major measure that must be considered as a potential alternative
to chemical control for protecting stored commodities frominsect
infestation.
12,13
Despite the great potential that cooling has for
the control of stored-product pests, negative effects of cold tem-
peratures on non-target natural enemies are also likely to occur.
14
To date, the low-temperature tolerance of many parasitoids has
received little attention.
15
The objective of this study was to inves-
tigate factors that may affect the cold hardiness of V. canescens. In
a series of experiments we examined the effect of food, age, hsot
availability, acclimation temperature and duration of acclimation
on the supercooling capacity of the endoparasitoid V. canescens.
2 MATERIALS ANDMETHODS
2.1 Insect cultures
Ephestia kuehniella larvae were maintained in plastic boxes
(17 11 5 cm) containing 150200 moth eggs and 200250 g
of semolina, which provided the larvae with sufcient food
throughout their development. Emerging adults remained in
the same boxes. A colony of V. canescens was maintained on
E. kuehniella as a host. Adults of V. canescens were reared in clear
plastic boxes (20 20 20 cm) and provided with honey solution
(10%) as food. Ten 5th instar larvae from the host colony were
placed in each box together with two adult parasitoids. The
following day the parasitized larvae were transferred individually
to small plastic cups (3 cm high and 4 cm in diameter) until
adult emergence. The procedure of parasitism in the colony was
repeated every 2 days until the death of the adult parasitoids.
Both hosts and parasitoids were collected in autumn 2007 from
the Kavala region (41

01

N, 24

22

E) of northern Greece, from

dried nuts and were reared in incubators set at 25 1

C, 60 5%
relative humidity (RH), with a 16:8h light : dark photoperiod.
2.2 Determination of SCPs
Each parasitoid was placed individually into a transparent plastic
capsule (16 mm high and 7 mm in diameter) and immobilized
with cotton. A copper constant thermocouple (Digitron 2000T;
Torquay, UK) was attached to the surface of each individual,
securely positionedwitha sensor tomonitor its body temperature.
The capsule bearing the parasitoid with the sensor was placed
separately in a testtube (17.5 cm high and 1.7 cm in diameter),
which was then immersed in a circulating bath (Model 9505;
PolyScience, Niles, IL. USA) with a solution of ethylene glycol and
water (1:1). The cooling rate was set at 1

Cmin
1
with a starting
temperature of 20

C. The lowest temperature reached before an

exothermic event due to the release of latent heat was taken as
the SCP of the individual.
2.3 Effect of age, food and host availability
In separate experiments, we compared the SCPs of young vs. old
and fed vs. unfed adults of V. canescens. The effect of age on SCP
was determined by separating young fed adults (15 days; n =40)
fromolder ones (1020 days; n =40) andplacingthemin different
clear plastic boxes (20 20 20 cm). Groups were further divided
(n =20) according to the availability or non-availability of their
host. Those that were treated in the presence of the host, in
addition to the honey solution (10%), were also provided with
10 fth instar larvae of E. kuehniella from the host colony. This
procedure was repeated every 2 days until the SCP was recorded.
Respectively, those that were treated in the absence of the host
were provided with honey solution only (10%). To determine the
effect of food, newly eclosed adults (<24 h) from the same cohort
were placed in different clear plastic boxes (20 20 20 cm), with
(n =10) or without (n =10) honey solution (10%) and their SCPs
were recorded. All adults derived from the same cohort and were
held under the same laboratory conditions as above prior to
determination of the SCP.
2.4 Effect of acclimation
To test whether acclimation temperature and time might affect
supercooling capacity and the release of latent heat fromadults of
V. canescens, groups of fed adults (n =10) were placed in growing
chambers (Precision Scientic, General Electric, Louisville, KY, USA)
at 5 and 10

C, and with a 16:8 h light : dark photoperiod for a

period of 10 or 20 days. Groups were further divided according
to the availability or non-availability of their host, as previously
described. As soon as they reached the desired age (10 or 20 days)
ineachchamber, they were removedandusedas describedabove
for the determination of SCP.
2.5 Data analysis
The independent two-tailed Students t-test was used to test
the effect of age, feeding status and host availability. Analysis
of variance (ANOVA) was carried out to test the effect of
acclimationtimeandacclimationtemperatureonthesupercooling
capacity of V. canescens. Means were compared using the Tukeys
b-test for multiple comparisons. Treatment differences were
considered signicant at P <0.05.
16
Prior to analysis, SCPs were
subjected to the AndersonDarling (AD) goodness-of-t test
for the normalization of the variables.
17
Statistical analyses were
performed using Minitab 16 Statistical Software.
18
3 RESULTS
3.1 Effect of feeding status on SCPs
Food consumption (10% honey solution) signicantly reduced
the ability of V. canescens adults to supercool. Mean SCP values
for 1-day-old V. canescens adults that were not fed was 1.5-fold
lower thanfor those that were fed(21.7 0.6 and15.2 0.2

C,
respectively; t =9.68; P <0.001).
3.2 Effect of age and host availability on SCP
In the absence of host, mean SCP for adults of V. canescens was
signicantly affectedby age. MeanSCPfor youngadults (15 days)
was signicantly lower than that of older adults (1020 days)
(t =3.18; P <0.01; Fig. 1). When adults were provided with host,
mean SCP decreased further for both young and older adults.
Hence, mean SCP for young adults in the presence of the host
was signicantly lower than that of older adults (t =2.22; P <0.05;
Fig. 1). Host availability hada signicant effect onthe supercooling
capacity of old adults (t =2.12; P <0.05), but not young adults
(t =1.33; P >0.05).
3.3 Effect of acclimation temperature on SCP
The results for the SCP of V. canescens adults that were acclimated
for 10 and 20 days at different temperatures are presented in
Fig. 2. In the absence of host, signicant differences in mean SCPs
were obtained among adults that were maintained for 10 days
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t s o h h t i w t s o h t u o h t i w
Age Mean SE Minimum Maximum Age SE Minimum Maximum
young -15.9 0.2 -17.9 -14.2 un yo g
Mean
-16.5 0.4 -17.3 -13.6
old -14.2 0.5 -16.6 -9.5 old -15.5 0.3 -15.5 -12.1
old young
-5
-10
-15
-20
old young
S
u
p
e
r
c
o
o
l
i
n
g

p
o
i
n
t

(

C
)
Figure 1. Boxplot and results of descriptive analysis of SCP of young (15 days) and old (1020 days) adults of V. canescens in the presence or absence of
E. kuehniella larvae (n =20). The line between a box marks the median, the boundaries of a box indicate the 25th and 75th percentiles, error bars indicate
the 10th and 90th percentiles, and the points represent data beyond these limits.
at room temperature, 5 and 10

C (for 10 days, F(2, 29) =12.13,

P <0.001). The lowest SCP was obtained for adults that were
acclimated at 5

C, and the highest for those that were not

acclimated (Fig. 2). Similarly, signicant differences in mean SCPs
were recorded among V. canescens adults that were maintained
for 20 days at room temperature, 5 and 10

C (for 20 days, F(2,

29) =3.40, P <0.05). A signicantly lower SCP was obtained for
adults that were acclimated at 5

C compared with those that were

not acclimated (Fig. 2).
In the presence of host, signicantly lower mean SCPs were
obtained for V. canescens adults that were acclimated for 10 and
20 days at 5

Ccompared with adults obtained at 10

C(for 10 days,
F(2, 29) =5.50, P <0.05, Fig. 2; for 20 days, F(2, 29) =4.55, P <0.05;
Fig. 2). However, no signicant differences were recorded in SCPs
among treatments that were acclimated at 10

C and those that

were kept at room temperature for either 10 or 20 days.
4 DISCUSSION
Physical control methods, such as use of extreme temperatures,
particularly low temperatures, have been extensively used
to control stored-product insects as a viable and effective
non-chemical alternative to the application of traditional
pesticides.
13,1921
To date, most studies have focused on the
cold hardiness prole of the major stored-product pests,
8,13,22
whereas the tolerance to low temperatures of benecial species
that may occur in stored-product facilities is poorly understood.
Recently, Foray et al
23
demonstrated that development at low
temperatures improves the coldtolerance of adults of V. canescens
by means of measuring the chill coma recovery time of 1-day-old
adults of V. canescens after exposure to7

Cfor 7 h. This is the rst

study on the cold-tolerance prole of V. canescens in which the
supercooling capacity is estimated by taking into consideration
the feeding status, age and acclimation temperature. Overall, our
results stand in accordance with the above observations, given
that the previous exposure of V. canescens adults signicantly
reduced their SCP.
Inlight of the current ndings, foodaccess (10%honey solution)
has a marked inuence on SCP of V. canescens. One-day-old adults
of V. canescens that had no access to food presented a 1.5-fold
lower SCP compared with those that were provided with food.
The presence of food particles in the gut is known to serve as
potential ice nucleators, which promote ice formation at relatively
high temperatures.
6,14,2426
This hypothesis is consistent with the
decrease in supercooling ability detected in this study. A similar
relationshipbetweenpresenceof foodinthegut andsupercooling
capacity has been documented in unfed females of the ectopar-
asitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae)
which displayeda signicantly lower (8

C) mean SCP in compar-

ison with honey-fed females.
14
Furthermore, the presence of solid
food in the midgut signicantly raised the SCP of the boll weevil,
Anthonomus grandis Boheman (Coleoptera: Curculionidae).
27
Likewise, the mean SCP of younger adults of V. canescens was
signicantly lower than that of older adults regardless of the
presence or absence of the host. This indicates that supercooling
capacity is affected by physiological changes induced by aging.
Indicators of aging are observed at different levels of cellular
metabolism and are manifested as changes in metabolome,
transcriptome and proteome.
28
Similar results are observed by
Slosser et al
29
who demonstrated that younger diapausing boll
weevils, A. grandis, could tolerate freezing conditions better than
older weevils. Generally, regarding several major stored-product
moth species, induction to diapause is highly moderated by the
exposure to low temperatures.
30
By contrast, late fth instar non-
diapausing larvae of the European corn borer, Ostrinia nubilalis
(H ubner) (Lepidoptera: Crambidae), were more cold tolerant than
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Cold tolerance of V. canescens www.soci.org
Figure 2. Boxplot and results of descriptive analysis of SCP of adults of V. canescens maintained for 10 and 20 days at room temperature, 5

C and 10

C in
the presence or absence of E. kuehniella larvae (n =10). The line between a box marks the median, the boundaries of a box indicate the 25th and 75th
percentiles, error bars indicate the 10th and 90th percentiles, and the points represent data beyond these limits.
younger ones, probably due to cessation of feeding prior to
pupation and evacuation of the gut.
8
Another remarkable feature
that addresses the reduced supercooling capacity of the older
adults of V. canescens is the enhanced tness cost due to the
continuous search for a host for oviposition. Energy loss through
host-seeking behavior is denitely positively correlated with age
of V. canescens adults, and this trend is expressed more vigorously
when the host is present.
4,31
Acclimation for a few days at low temperatures considerably
improves cold hardiness, although not always.
10,32,33
Similar
results have been reported for several major stored-product
insect species, with moths being generally more tolerant to
low temperatures than beetles.
13
Apart from cold hardiness,
acclimation below optimum developmental temperature is likely
to increase the survival of major stored-product insects 210
times following exposure to low temperatures, provided that the
temperature decline is gradual.
13
Allowing adults of V. canescens
to adapt to low temperatures (i.e. 5 and 10

C) resulted in a
noticeable increase in supercooling ability. Considering the
overall data, adults of V. canescens displayed an enhanced ability
to supercool, given that the mean SCP of non-acclimated adults
ranged from16.5 to 13.7

C, depending on the treatment (with

the exception of 1-day-old unfed adults). However, this value was
much higher than the respective SCPs of its host, E. kuehniella,
and also of other pyralid moths that are hosts of V. canescens.
Mean SCP values for late instars, pupae and adults of E. kuehniella
were 19.5, -23.3 and 21.6

C, respectively.
8
The same trend was
observed for P. interpunctella for which SCP values for eggs, rst
instars, pupae and adults were 24.4, 23.5, 22.2 and 22.4

C,
respectively.
34
In both cases, the host appears to be more cold
tolerant than the parasitoid itself. This might have an adverse
effect on the survival of V. canescens regarding the application
of low temperatures in stored-product facilities. Furthermore,
after the low temperature interval, the increase in temperature
may allow moths to build up high rebound population densities,
without the presence of parasitoids. Additional experimental work
is needed to indicate cold tolerance of V. canescens immatures.
In summary, our results clearly demonstrate that food, age
and acclimation to low temperatures inuence the supercooling
capacity of V. canescens adults, and these factors may be
considered further in order to obtain parasitoid populations that
have the capacity to survive at temperatures close to E. kuehniella
SCP. By contrast, host availability had a moderate effect on super-
cooling capacity. Continued investigations into how well this
parasitoid tolerates low temperatures will be useful in evaluating
its potential as a biological control agent of stored-product moths
in temperate regions, but also in better understanding parasitoid
life table characteristics at low temperatures, for which very few
data are available to date.
ACKNOWLEDGEMENTS
The authors thank Styliani Kamperidou for laboratory assistance
and two anonymous reviewers for useful comments. They also
express their gratitude to Christina Soultani for her language
editing services.
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