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Behavioural consequences of cold exposure on malesand females of Aphidius rhopalosiphi De Stephani Perez(Hymenoptera: Braconidae)
Delphine Bourdais • Philippe Vernon •
Liliane Krespi • Joan van Baaren
Received: 29 September 2010 / Accepted: 7 July 2011 / Published online: 11 August 2011
� International Organization for Biological Control (IOBC) 2011
Abstract Cold storage of insect parasitoids is
currently used before mass release in the field in
biological control programmes. The physiological
consequences of constant cold storage are known in
various species of parasitic wasps, but there are few
reports on the behaviour of survivors and even fewer
reports where both sexes were tested. In this study,
we observed the consequences of a long storage of
Aphidius rhopalosiphi De Stephani Perez (Hymenop-
tera: Braconidae), a parasitoid of the cereal aphid
Sitobion avenae Fabricius (Hemiptera: Aphididae), at
low temperature on some key behavioural decisions
that both sexes will make when released in the field.
Males are less tolerant than females and both sexes
suffer from a long exposure (28 days or more) at 4�C
during the pupal stage: alteration of olfactory
responses, decrease in mating capacity and decrease
in the efficiency of patch exploitation by females.
Keywords Thermal stress � Cold storage �Behaviour � Mating � Aphidius rhopalosiphi �Sitobion avenae
Introduction
Temperature is one of the most important environ-
mental factors, influencing nearly every aspect of
insect life, from direct effects on the kinetics of
enzyme reactions to behaviour and fitness (Lee and
Handling Editor: Stefano Colazza
D. Bourdais (&) � P. Vernon � J. van Baaren
UMR 6553 CNRS, EcoBio, Universite de Rennes I,
Equipe Impact des Changements Climatiques,
Station Biologique, 35380 Paimpont, France
e-mail: delphine.bourdais@uclouvain.be
P. Vernon
e-mail: philippe.vernon@univ-rennes1.fr
J. van Baaren
e-mail: joan.van-baaren@univ-rennes1.fr
D. Bourdais � P. Vernon � J. van Baaren
UMR 6553 CNRS, EcoBio, Universite de Rennes I,
Equipe Impact des Changements Climatiques, Campus
de Beaulieu, Avenue du General Leclerc,
35042 Rennes cedex, France
D. Bourdais
Biodiversity Research Center, Earth and Life Institute,
Universite Catholique de Louvain, Place Croix du Sud
4–5, 1348 Louvain-la-Neuve, Belgium
L. Krespi
Equipe d’Ecobiologie des Insectes Parasitoıdes,
Universite de Rennes I, Avenue du General Leclerc,
35042 Rennes cedex, France
e-mail: liliane.krespi@univ-rennes1.fr
123
BioControl (2012) 57:349–360
DOI 10.1007/s10526-011-9396-0
Denlinger 1991). Insect survival capacities and the
mechanisms allowing them to increase their cold-
hardiness have been a central theme in the field of
thermal biology and it is, thus, extensively docu-
mented (Bale 1987; Sinclair et al. 2003; Sømme
1982), especially in biocontrol agents like insect
parasitoids (Colinet et al. 2006; Colinet and Hance
2009; Hance et al. 2007; Langer and Hance 2000).
Indeed, cold storage is a valuable method for
prolonging insect development time and allows
suppliers to have a sufficient number of biocontrol
agents for biological control programmes.
Aphidius rhopalosiphi De Stephani Perez (Hyme-
noptera: Braconidae) is naturally present in cereal
fields in Europe (Langer et al. 1997). This species is
known to parasitise cereal aphid species such as
Sitobion avenae Fabricius and Metopolophium dirho-
dum Walker (Homoptera: Aphididae) (Farrell and
Stufkens 1990; Levie et al. 2000). Other species of
Aphidius, like Aphidius ervi Haliday or Aphidius
matricariae Haliday, are already commercialised for
biological control, particularly in glasshouses. A.
rhopalosiphi De Stephani Perez is presently studied
by firms for field releases, as its life cycle is close to
those of other Aphidiinae (Muratori et al. 2004),
allowing its commercial rearing. Because it is a
specialist of the habitat (cereal fields), this could
represent the most interesting species for field release
(Stilmant et al. 2008). The female parasitoid lays an
egg in the body of the aphid. When hatched, the
parasitoid larva feeds from the aphid body. Then, the
larvae stops feeding at the end of the third larval
instar and spins its cocoon inside the empty cuticle of
the aphid and pupates, forming a ‘‘mummy’’ (Mura-
tori et al. 2004). From that moment until emergence,
it does not feed, and all metabolic processes use
energy reserves accumulated during the larval instars.
However, despite the critical stage of metamorphosis
that occurs during the ‘‘mummy stage’’, Levie et al.
(2005) have shown that the more favourable instar for
the cold storage of Aphidius species was one-day-old
mummies, and this instar is presently used for
commercial cold storage.
Physiological and biochemical consequences of
cold stress induced by cold storage during the pupal
stage of parasitoids have been well studied. Cold
storage increases development time (e.g. Campbell
et al. 1974; Sigsgaard 2000), and the rate of mortality
of pupae during the storage (Colinet et al. 2006;
Sømme 1982). It decreases longevity (e.g. Colinet
et al. 2006) and reproductive success acting, for
instance, on male sterility (Amice et al. 2008;
Carriere and Boivin 2001; Colinet and Hance
2009). Cold stress also alters morphogenesis (Sehnal
1991), acting on the morphology of wings (Colinet
and Hance 2009) and antennae (Amice et al. 2008;
Bourdais et al. 2006).
It is surprising that little is known of the future
behavioural effects of such stress in adult parasitoids.
Behavioural decisions that newly emerged males and
females make after cold storage are important for
controlling aphid populations in the field. Optimising
aphid population control depends on the efficacy of
parasitoid populations in the field. Indeed, rather than
simply measuring the emergence rates and sex ratios
of released males and females, it would be more
interesting to evaluate the contribution of these
individuals to maintaining the parasitoid population
in the field.
The female parasitoids contribution to the next
generation is determined by whether parasitism was
successful. This depends on a series of steps, such as
the efficiency of the host location process (attraction
to host odours) and their ability to lay eggs (Vinson
1981). The contribution of the males depends on the
number of progeny they father during their lifetime
(Arnqvist and Nilsson 2000). This is dependent on
their ability to acquire female mates (attraction to the
female’s odour) and to mate successfully. Altered
behaviour after a cold shock is known in both sexes
for some parasitoid species: for example, in Anaphes
victus Huber (Mymaridae), it was shown that learning
behaviour was altered (van Baaren et al. 2005), or in
Aphidius avenae Haliday, the mating rate was
reduced (Amice et al. 2008), but we are unaware of
any studies comparing the behaviours of both sexes.
The aim of our present research was to evaluate
behavioural consequences in surviving adults of both
sexes of A. rhopalosiphi De Stephani Perez exposed to a
classical cold stress during the pupal stage. Key
parameters related to their future efficiency when
eventually mass released in the field were, therefore,
analysed, such as odour recognition and mating success.
We used short storage durations, showing a strong
emergence rate, to determine if storage durations,
considered at this time as acceptable, do not induce
behavioural damages. The potential consequences for
successful biological control are discussed.
350 D. Bourdais et al.
123
Materials and methods
Biological material
Aphidius rhopalosiphi De Stephani Perez were col-
lected less than one year before the experiments in
cereal crops around Rennes (Lat. 48�0601000;Long. -01�4703900) (Brittany, France) and were
reared in the laboratory on a mixed-age culture of
S. avenae Fabricius originating from one partheno-
genetic female collected in 1990 in the same area
(SA1 clone, INRA-Zoology Collection). Aphids were
reared on winter wheat, Triticum aestivum, cv.
‘‘Boston’’. Colonies of hosts and parasitoids were
maintained in Plexiglas cages (50 9 50 9 50 cm)
placed in two different climate-controlled rooms at
20 ± 1�C, 70 ± 10% R.H. and a 16L: 8D photope-
riod. The rearing conditions of the parasitoids were
used as ‘‘control conditions’’ throughout our study.
Experimental design
Temperatures and exposure times were chosen in
order to obtain a high rate of emergence for each sex.
For aphid parasitoids, cold storage at a constant
temperature of 4�C is often used prior to mass release
in biological control programmes (Amice et al. 2008;
Colinet et al. 2006; Tezze and Botto 2004). To test
the effects of cold exposure at the mummy stage on
male and female adult parasitoids, one-day-old
mummies were exposed at a constant temperature
of 4�C for 14 and 28 days. Preliminary behavioural
tests after 28 days of cold exposure showed that
females seem to be more resistant to cold stress than
males. Therefore, a 42-day period of cold storage was
added in order to test the behavioural alterations on
females only.
To obtain cold-stored parasitoids, only mummies
less than 24 h old (the more resistant instar––Levie
et al. 2005) were collected each day from the mass
rearing, placed individually in gelatine capsules to
avoid contact between future emerged individuals
and then randomly exposed to cold. After cold
storage, mummies were returned to control condi-
tions (i.e. the standard rearing conditions). The size of
these mummies was not standardised, as we wanted
to have an estimate of the effects of cold storage on a
population of stored individuals. Newly emerged
individuals were checked for daily and maintained
individually for 24 h in small cages with honeydew
and water before behavioural tests. Control individ-
uals consisted of mummies non-exposed to any cold
stress, i.e. isolated in gelatine capsule the day of the
mummy formation, and maintained at 20�C for all of
their development.
Measured traits
Developmental aspects
To test whether thermal treatments had a differential
impact on parasitoid survival, we calculated the
emergence rate. After each cold exposure, mummies
were placed at 20�C in standard rearing conditions
and survival was assessed as the number of adults that
had successfully emerged. Mummies from which no
adult had emerged at least ten days after reintroduc-
tion to control conditions, or ten days after the
normal date of emergence for control mummies,
were dissected to determine the rate of mortality
(unemerged adults) and the rate of arrest in the
development (i.e. when third-stage larvae were still
present alive). A total of 4,412 mummies were
exposed as controls. For cold exposures, 213
mummies were exposed for 14 days, 450 for 28 days
and 176 for 42 days.
Male recognition of females
The ability of males to detect virgin females (\24 h
old) with aphids and wheat at long range and direct
themselves toward them was tested using a Y-tube
olfactometer, which is a common method used to test
the attraction of parasitoids to odours (Ardeh et al.
2004; Perez-Maluf and Kaiser 1998; van Baaren and
Nenon 1996). Reconstituted compressed air (80%
nitrogen and 20% oxygen) was circulated through a
water bottle and entered into the Y-tube through the
two arms (the length of each branch was 15 cm,
diameter was 3 cm). Air left the tube through the
central branch, which was covered with a fine mesh
to prevent insects from escaping from the device.
This experiment was carried out with light (circular
neon light source that provided a homogenous
illumination of 400 lux) and the air velocity was
chosen in order to obtain a normal walk (airflow
adjusted to 200 ml min-1, as in a Petri dish without
air) from the males. The olfactometer setup was
Behavioural consequences of Aphidius rhopalosiphi 351
123
placed in a temperature-controlled room (20 ± 2�C).
Five virgin control females (\24 h old) with a small
mixed colony of ten aphids on a small wheat leaf
(3 cm in length) were used as the odour source and
were placed at the end of one branch of the
olfactometer in a glass tube (4 cm in length, 3 cm
in diameter), covered at each extremity by a fine
mesh to prevent females escaping and allowing the
male to have access to the females. Between every
observation, the position of the odorous branch was
changed and the Y-tube was washed with 95� alcohol
and rinsed with water. Parasitoids were released
individually into the stem of the Y-tube and allowed
10 min to choose one of the arms. We considered a
choice to be made when the male reached a line
(placed 7 cm after the intersection between the two
branches) placed on the Y-tube branch containing the
odour source. Males choosing the control branch
were considered to be unattracted by the odour and
males staying in the central branch were considered
as non-responding and were excluded from the
statistical analyses on choice. Parasitoids were
directly observed. Ninety-four control males were
used in the experiment but because of the percentage
of emergence of the exposed individuals, we were
able to use 40 males exposed for 14 days and 18
males exposed for 28 days at 4�C in our behavioural
experiments.
Female recognition of aphids on a piece of wheat
The ability of 24 h old virgin females to detect hosts
was tested using a circular chamber (Ø = 9 cm,
1 cm high) divided into three equal parts (3 cm width
each at their central part), covered by a fine mesh
surmounted by a lid (Ø = 9 cm, 1 cm high), making
the device inaccessible to the female. One of the two
end parts contained the odour source, consisting of a
mixed colony (different larval instars) of ten aphids
on a small wheat leaf (3 cm in length). The aphids
had been on this leaf since birth, and the leaf presents
some traces of honeydew. Between every observa-
tion, the position of the odorous part of the chamber
was changed and the chamber with the fine mesh was
washed with 95� alcohol and rinsed with water. The
female was introduced into the upper central part and
its behaviour was directly observed for 10 min. The
number of females entering into each peripheral
zone (the zones on the right and on the left of the
introduction zone) was checked. We considered that
a female had recognised the odour composed by
aphids and wheat and was attracted by it when the
relative proportion of those entering this zone was
significantly higher than in the one that did not
contain aphids (binomial test with 1/2:1/2 propor-
tions) and a new index (1 for succeeded and 0 for
failed) was attributed to these females. Females that
did not move from the central introduction zone were
considered to be unattracted and not used in the
statistical analyses. Thirty-eight control females were
used in the experiment but because of the percentage
of emergence of the exposed individuals, we were
able to use 34 females exposed for 28 days and ten
females exposed for 42 days at 4�C in our behav-
ioural experiments.
Mating behaviour of males
Male courtship and mating ability were assessed by
placing them individually with one control virgin
female in a small tube (1 9 0.5 cm2) for 30 min.
Males were given a maximum of two opportunities to
mate with two different control females. Behavioural
observations of 22 mating sequences from 22 differ-
ent control males give us the mating ethogram of the
species (Table 1). The recorded behaviour of the
males during the mating behavioural sequence was
then analysed using the method described by Pierre
and Kasper (1990) and was used in several different
ethological analyses (Roux et al. 2005; van Baaren
et al. 1993, 2003). This method provided a descrip-
tion of the sequential structure of behavioural
patterns placed in a factorial space, their distance
being inversely related to the frequency of their
temporal succession. This analysis yielded a flow
chart on factorial maps in which two patterns
occurring frequently in succession will appear close
and be linked by thick arrows. Conversely, two
patterns occurring rarely in succession will be
represented as far apart and linked by thin arrows.
By comparing the occurrences of the different
behaviours of control and treated males, any influ-
ence of thermal stress on the mating sequence could
be determined (Table 1). In the mating experiments,
the individuals were those previously tested in the
olfactometry trials (all of the previous males were
used, whether they succeeded or not in the odour
recognition test).
352 D. Bourdais et al.
123
Table 1 Ethogram and number of occurrences of the different behavioural patterns (mean ± SE) of the mating behaviour sequence
of Aphidius rhopalosiphi De Stephani Perez males
Behavioural patterns observed during the mating
sequence and definitions
Code
used
in
Fig. 2
Control 14 days of
cold exposure
28 days of
cold
exposure
Mating No mating Mating No mating No mating
Phase 1: detection of the female
The male walks continuously with its wings down 1a 8.9 ± 3.4 51.7 ± 7.5 6 ± 2.8 27.3 ± 4.2 37.3 ± 5.5
The male walks continuously with its wings up
(wing fanning)
1b 10.3 ± 4.0 1.7 ± 0.8 8.8 ± 2.8 0.4 ± 0.2 1.9 ± 1.1
Antennal contact from the male to the female 1c 7.2 ± 2.6 24.9 ± 4.2 5.6 ± 2.1 19.4 ± 4.0 28.2 ± 3.1
The male stops and fans its wings 1d 1.3 ± 0.4 4 ± 0.8 1.8 ± 0.8 7.4 ± 2.4 6.4 ± 1.7
The male stops to put down its wings 1e 0.5 ± 0.2 0 0.6 ± 0.3 0 0
Phase 2: courtship
The male mounts the female 2a 1.6 ± 0.3 0 1.2 ± 0.2 0 0
The male makes alternative antennal movements
when it is on the back of the female
2b 2.2 ± 0.3 0 1.3 ± 0.4 0 0
The female moves when the male is on its back 2c 1.2 ± 0.2 0 1.5 ± 0.4 0 0
The male lowers itself on the female before the
genitalia contact
2d 0.6 ± 0.3 0 1.2 ± 1.1 0 0
Phase 3: mate acceptance
The male lowers its abdomen 3a 1.3 ± 0.1 0 1.3 ± 0.3 0 0
The female moves its abdomen up and down but no
contact occurred
3b 0.1 ± 0.1 0 0 0 0
Phase 4: copulation
The male flicks its antennae in parallel and keeps
them in contact with those of the female
4a 2.2 ± 0.2 0 1.2 ± 0.2 0 0
Contact between the two genitalia 4b 1 0 1 0 0
Phase 5: end of copulation
The male moves when it is on the female 5a 0.5 ± 0.2 0 0.3 ± 0.3 0 0
The male is redressed on the back of the female but
does not lose antennal nor genitilia contact
5b 1 0 0.8 ± 0.2 0 0
Loss of contact between antennae but still contact
between genitilia
5c 1 0 1 0 0
Phase 6: separation of the partners
Loss of contact between the two genitilia 6a 1 0 1 0 0
The male escapes 6b 1 0 1 0 0
The female escapes 6c 1 0 1 0 0
Other behavioural patterns
Grooming: the wasp rubs its antennae and/or abdomen
with its legs
7a 0.3 ± 0.2 2.1 ± 0.6 0 1.2 ± 0.4 0.2 ± 0.2
Immobility of more than 1 s 7b 0 32.7 ± 8.7 0.3 ± 0.3 4.5 ± 0.9 11.6 ± 7.9
The wasp tries to fly away 7c 0 8.9 ± 6.5 0 1.7 ± 0.4 3.8 ± 1.1
n = 10 observations were made in each cold-stored period and in controls
Behavioural consequences of Aphidius rhopalosiphi 353
123
Female patch exploitation and mate acceptance
All females previously used in the olfactometry test
were placed individually in a Petri dish (Ø = 9 cm)
with ten 2nd instar aphids on wheat (3 cm in length)
for 30 min. The total number of eggs laid by the
female was estimated to be the same as the number of
larvae observed in aphid dissections after four days.
A previous study on this species has shown that more
than 96% of the parasitised aphids contain a larva
after four days. In the case of superparasitism, the
dead defeated larvae are still visible at the time of
dissection (van Baaren et al. 2009). Superparasitism
was calculated as the percentage of aphids containing
more than one parasitoid larva. The individuals were
the same females as used previously in the olfactory
tests whether successful or not in the first test. Mate
acceptance was also assessed by presenting to
females a maximum of two control males and noting
if mating occurred or not within 30 min.
Effect on population
The parasitoids for which a cold exposure as
mummies had no behavioural effect (i.e. individuals
that were able to recognise odour and mate success-
fully) were considered to be the proportion poten-
tially of use in a mass release programme. It was
compared to the proportion of control individuals
successful in both tests. To do this, we created a new
index based on the success of each individual to each
of the behavioural tests (1 for succeed and 0 for
failed).
Statistical analysis
Emergence rates (i.e. proportions of adults that
successfully emerge), death rates (i.e. proportions of
adults that died before emergence) and arrest of
development on third instar rates were compared
between treatments using v2 tests (PROC FREQ, SAS
Institute, Cary, NC, USA, 1990). Rates of success of
mating for both sexes were also compared between
treatments using v2 tests. In odour recognitions trials,
v2 goodness-of-fit tests were used to test the hypoth-
esis that the distribution of side-arm choices deviated
from a null model when odour sources were chosen
with equal frequency. One-way analysis of variance
(ANOVA) (Proc GLM, SAS Institute, Cary, NC,
USA, 1990) was used to analyse the effect of cold
storage on the number of eggs laid by females and
superparasitism. Multiple comparisons were then
performed using Tukey’s honestly significant differ-
ence (HSD) test to describe differences between
treatments.
Results
Developmental aspects
The proportion of emerged individuals decreased with
cold exposure (v2 = 49.71, df = 3, P \ 0.0001). The
non-emerged individuals were those for which cold
storage either stopped their development or killed
them. This decreased emergence in the 14 days of
cold storage treatment could be explained by more
individuals ceasing development, whereas for the
longer exposures to cold (28 days), it was due rather
to an increased mortality (Fig. 1). For the individuals
stored for 42 days, the decreased emergence was also
due to an increased mortality (Fig. 1).
Male recognition of females and female
recognition of aphids and wheat
The cold exposure did not have any effect on the
proportion of males that responded in the olfactom-
eter trial (v2 = 0.18, df = 2, P = 0.91), meaning that
the same proportion of males did not move in our trial
independently of the cold exposure duration. We
found an effect of cold duration on the proportion of
males that succeeded in the test (v2 = 12.37, df = 2,
P = 0.0021). About 95% of control males that had
moved succeeded in choosing the branch containing
the odour composed of females and aphids on a piece
of wheat. This proportion decreased significantly
with exposure to cold stress (control males vs.
14 days of cold exposure males: v2 = 4.52, df = 1,
P = 0.033, with 82.75% of cold-exposed males that
succeeded and control vs. 28 days of cold exposure:
v2 = 13.15, df = 1, P = 0.0003, with 69.23% of
cold-exposed males that succeeded).
There was no effect of cold exposure duration on
the proportion of females that prefer the part of the
chamber that contained aphids (v2 = 1.63, df = 2,
P = 0.44; comparison between control vs. 28 days of
354 D. Bourdais et al.
123
cold exposure: v2 = 0.05, df = 1, P = 0.83, with
47% of cold-exposed females that succeeded and
comparison between control vs. 42 days of cold
exposure: v2 = 0.84, df = 1, P = 0.35, with 30% of
cold-exposed females that succeeded).
Mating behaviour of males
The mating behaviour sequence of the control males
used here could be divided into six parts appearing on
the factorial map (Fig. 2). Usually, a male became
excited just after its introduction into the glass vial
containing the virgin female, and almost immediately
started wing fanning (phase 1). When the male
encountered a female (e.g. when an antennal contact
occurs), he tried to mount her (phase 2). The female’s
reluctance stopped when the male began to flick its
antennae alternatively against those of the female and
then the male could finally lower its abdomen on the
female (phase 3). The fourth phase corresponded to
contact between the two genitalia whilst the male
flicked its antennae against those of the female. Then,
antennal contact terminates (phase 5) before, finally,
the genitalia and partners separate (phase 6).
Cold exposure affected the mating capability of
males (v2 = 19.55, df = 2, P = 0.001). There was
no statistical difference following 14 days of cold
exposure (27.5% effective mating, v2 = 0.012,
df = 1, P = 0.91), but no mating was observed for
the males exposed at 4�C for 28 days (v2 = 6.163,
df = 1, P = 0.013). For control and cold-stressed
males that did not mate, the typical sequence of
mating was stopped in the first phase and only a few
males sometimes tried to mount the female (Table 1).
We also observed a higher proportion of selfish
behaviour (i.e. behaviours not directed towards the
female), such as antennae or leg grooming, phases of
immobility or phases of jumps when no mating
occurred.
Female patch exploitation and mate acceptance
Females from cold-exposed mummies had a tendency
to lay fewer eggs (represented as the number of
larvae found in the aphid, F = 6.38, df = 2, 74,
P \ 0.0001) and to parasitise fewer aphids
(F = 8.39, df = 2, 74, P = 0.0005) (Fig. 3). Control
and cold-exposed females had a mean rate of self-
superparasitism of around 30%. We observed an
effect of cold stress duration on the rate of superpar-
asitism (F = 4.94, df = 2, 74, P = 0.009) but the
differences between treatments were not significant
(Tukey’s test, P [ 0.05).
Cold stress decreased the mating acceptance rate
of the females (v2 = 10.24, df = 2, P = 0.017).
Fig. 1 Effects of the duration of cold exposure on the
emergence rate (a), mortality (b) and arrest of development
in third instar (c) of Aphidius rhopalosiphi De Stephani Perez.
The mean % are shown ± SE. Means indicated by the same
letters were not statistically different at the 5% level (v2 test
with Yates’ continuity correction)
Behavioural consequences of Aphidius rhopalosiphi 355
123
Mating involved 75% of control females, but only
42% of females exposed to cold as mummies for
28 days (v2 = 7.56, df = 1, P = 0.006) and 28.57%
of females exposed for 42 days (v2 = 5.75, df = 1,
P = 0.016).
Effect on population
The percentage of cold-stored individuals that suc-
ceeded in our two behavioural tests (i.e. odour
recognition and mating and/or patch exploitation)
decreased with cold exposure duration (males:
v2 = 14.9, df = 2, P = 0.005; females: v2 = 19.9,
df = 2, P \ 0.001). A 14-day cold storage treatment
had no influence on the percentage of males that
succeeded in the two behavioural tests (v2 = 3.39,
df = 1, P = 0.065). Since none of the 18 tested
males succeeded in mating when exposed for 28 days
at 4�C, we concluded that none of them are adequate
for biological control in the population after such a
cold storage. When we observed the other sex, an
increasing proportion of females were also ineffective
following cold storage. For the longer exposure to
cold stress (42 days), none of the females succeeded
in the two behavioural tests that were proposed to
them. It is of interest to notice that a 28-day cold
Fig. 2 Flow chart on the
factorial map obtained with
mating sequences of 22
different A. rhopalosiphi De
Stephani Perez control
males showing the different
phases explained in
Table 1. Axes I and II are
the two axes of the factorial
correspondence analysis.
Each behavioural pattern in
the circles corresponds to
one abbreviation given in
Table 1. The circles
represent, from the smallest
to the largest, respectively,
less than 20, 21–50,
51–100, 101–200 and more
than 200 behavioural
patterns. The arrows
represent the successions
between two patterns. Small
dashed lines represent less
than ten successions, while
solid lines are directly
proportional to the number
of successions (10–200)
356 D. Bourdais et al.
123
stored treatment did not influence the percentage of
females that had succeeded in the two behavioural
tests (v2 = 0.45, df = 1, P = 0.51), as also observed
for males.
Discussion
Cold exposure had lethal effects on our population of
A. rhopalosiphi De Stephani Perez, increasing the
total mortality during metamorphosis. This phenom-
enon was also reported in other insect groups, such as
Coleoptera (Ali et al. 1997) and Diptera (Brevault
and Quilici 2000). Death after cold exposure may be
due to a progressive blockage of metabolic activity
(Hochachka and Somero 2002). According to the
intensity and length of chilling exposure, organisms
suffer from various chill injuries that induce diverse
detrimental consequences, from minor or major
weakness of the central nervous system, to death
(Lee and Denlinger 1991, 2010). More generally,
thermal stress may impair brain functioning, but
consequences for behaviour are not yet well docu-
mented, even if a recent study has convincingly
emphasised that thermal stress in Drosophila
melanogaster Melgen (Diptera: Drosophilidae) larvae
significantly affects subsequent learning in adults
(Wang et al. 2007).
Cold stress is known to affect reproductive
behaviour and mating success (Shreve et al. 2004),
and, more specifically, male physiology (David et al.
2005; Rinehart et al. 2000) or male mating capacity
(Amice et al. 2008; Colinet and Hance 2009). For the
first time, we have shown that the male’s capacity to
detect odours (here, a mix of virgin females, aphids
and wheat) is also affected by cold stress and this
could be partly responsible for the unsuccessful
mating. We propose here that the decreased mating
rate of both sexes could be due to an inability to
recognise each other, resulting in an unaccepted
copulation. Indeed, when mating did not occur, the
courtship behaviour was stopped at the beginning of
the second phase, i.e. just after the antennal contact
between partners, which was shown to be a crucial
phase of mating behaviour in other species (Guerrieri
et al. 2001; Isidoro et al. 1996). Moreover, in
A. rhopalosiphi De Stephani Perez, Bourdais et al.
(2006) have already shown that the chemoreceptors
of the antennae can be modified by thermal stresses.
These behavioural alterations could also be accom-
panied by physiological problems.
In the host habitat location process, chemical cues
also elicit a series of directed responses by the female
that serve to reduce and restrict the area of habitats
searched and the number of species of host thus
located. Antennal sensillae are important sensory
receptors implicated in these behaviours, as demon-
strated by various tests involving partial and total
antennal excisions (Hay and Vinson 1971; Weselow
1972), as well as antennal electrophysiological
experiments reported by Ochieng et al. (2000). In
this study, we found that cold exposure altered the
female capacity to recognise potentially attractive
odours and to exploit an aphid patch. Other studies on
Fig. 3 Mean number of A. rhopalosiphi De Stephani Perez
larvae found in Sitobion avenae Fabricius (±SE) resulting from
egg laying by each female during the 30 min of the experiment
(a) and number of parasitised aphids per female (mean ± SE)
(b). Means indicated by the same letters were not statisti-
cally different at the 10% level (comparisons with Tukey’s
HSD test)
Behavioural consequences of Aphidius rhopalosiphi 357
123
parasitoids also reported that cold has a negative
effect on female patch exploitation and olfaction
(Hanna 1935; Herard et al. 1988; Rinehart et al. 2000;
Scott et al. 1997; van Baaren et al. 2005). We found
that cold-stressed females parasitised fewer aphids
than control ones. This was maybe due to a reduced
capacity to recognise hosts’ suitability and would
seriously affect their patch exploitation ability.
For some of the behavioural traits studied,
A. rhopalosiphi De Stephani Perez males were more
susceptible to cold exposure than females. In other
insect groups, several studies have shown that
females are more cold- and heat-tolerant than males
(Ali et al. 1997; Anderson and Horsfall 1963).
However, explanations about this difference are
lacking. We propose here two main factors that
may explain these differences in the susceptibility of
the two sexes to thermal stress. The first one is the
haplodiploidy. Several studies using Apis mellifera
Linnaeus (Hymenoptera: Apidae) found that haploid
males were consistently less resistant to stress than
diploid females (Clarke et al. 1986; Smith et al.
1997), with some empirical evidence for ploidy
effects on parasite and pathogen resistance (O’Don-
nell and Beshers 2004) and pesticide stress resistance
(Carriere 2003). The second factor is sexual size
dimorphism. In parasitoid wasps, females are usually
larger than males (Godfray 1994). In cold tempera-
ture conditions, the insect metabolism relies exclu-
sively on body energy reserves, particularly on lipids
(Adedokun and Denlinger 1985; van Handel 1993).
As the fat reserves increase with size (Ellers et al.
1998; Rivero and West 2002), heavier mummies with
larger fat reserves should have a significant advantage
for survival at low temperatures.
To conclude, we found that a constant cold stress
has deleterious effects on some developmental traits
in A. rhopalosiphi De Stephani Perez, but it also has
important behavioural consequences, such as altered
mating behaviour or the recognition of odours in both
sexes. In the perspective of biological control using
cold-stored individuals, our results suggest that, after
14 days of cold storage, only a small proportion of
individuals, both male and female, are able to behave
optimally in the field after emergence. This would
seriously diminish aphid control in the field. There-
fore, we suggest that mummies should not be stored
for more than two weeks at a constant temperature
of 4�C before release. Moreover, since males and
females react differently to cold stress, this study
shows the importance of considering both sexes when
estimating cold-storage behavioural alterations on
parasitoids of this and other species. It was also
shown recently that thermal fluctuating regimes
during storage could alleviate the physiological
effects of cold storage (Colinet and Hance 2009;
Ismail et al. 2010). It would be interesting to verify if
this reduced damage induced by fluctuating regimes
also applies to the disturbances of behaviour.
Acknowledgments We thank D. Webb (Rennes University),
M. O’Neill and the three anonymous referees for their
constructive and valuable comments on the manuscript. We
also thank A. Bertrand for the assistance with some of
experiments on females. This paper is number BRC205 of the
Biodiversity Research Centre.
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