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.

References

Adedokun TA, Denlinger L (1985) Metabolic reserves asso-

ciated with pupal diapause in the flesh fly, Sarcophagacrassipalpis. J Insect Physiol 31:229–233

Ali MF, Abdel-Reheem EFM, Abdel-Rahman HA (1997)

Effect of temperature extremes on the survival and biol-

ogy of the carpet beetle, Attagenus fasciatus (Thunberg)

(Coleoptera: Dermestidae). J Stored Prod Res 33:147–156

Amice G, Vernon P, Outreman Y, van Alphen J, van Baaren J

(2008) Variability in responses to thermal stress in para-

sitoids. Ecol Entomol 33:701–708

Anderson JF, Horsfall WR (1963) Thermal stress and anoma-

lous development of mosquitoes (Diptera: Culicidae).

I. Effect of constant temperature on dimorphism of adults

of Aedes stimulans. J Exp Zool 154:67–107

Ardeh MJ, de Jong PW, Loomans AJM, van Lenteren JC

(2004) Inter- and intraspecific effects of volatile and

nonvolatile sex pheromones on males, mating behavior,

and hybridization in Eretmocerus mundus and E. eremicus(Hymenoptera: Aphelinidae). J Insect Behav 17:745–759

Arnqvist G, Nilsson T (2000) The evolution of polyandry:

multiple mating and female fitness in insects. Anim Behav

60:145–164

Bale JS (1987) Insect cold hardiness: freezing and supercooling—

an ecophysiological perspective. J Insect Physiol 33:899–908

Bourdais D, Vernon P, Krespi L, le Lannic J, van Baaren J

(2006) Antennal structure of male and female Aphidiusrhopalosiphi DeStefani-Peres (Hymenoptera:Braconidae):

description and morphological alterations after cold stor-

age or heat exposure. Microsc Res Tech 69:1005–1013

Brevault T, Quilici S (2000) Relationships between tempera-

ture, development and survival of different life stages of

the tomato fruit fly, Neoceratitis cyanescens. Entomol Exp

Appl 94:25–30

Campbell A, Frazer BD, Gilbert N, Gutierrez AP, Mackauer M

(1974) Temperature requirements of some aphids and

their parasites. J Appl Ecol 11:431–438

358 D. Bourdais et al.

123

Carriere Y (2003) Haplodiploidy, sex, and the evolution of

pesticide resistance. J Econ Entomol 96:1626–1640

Carriere Y, Boivin G (2001) Constraints on the evolution of thermal

sensitivity of foraging in Trichogramma: genetic trade-offs

and plasticity in maternal selection. Am Nat 157:570–581

Clarke GM, Brand GW, Whitten MJ (1986) Fluctuating

asymmetry: a technique for measuring developmental

stress caused by inbreeding. Aust J Biol Sci 39:145–154

Colinet H, Hance T (2009) Male reproductive potential of

Aphidius colemani (Hymenoptera: Aphidiinae) exposed to

constant or fluctuating thermal regimens. Environ Ento-

mol 38:242–249

Colinet H, Hance T, Vernon P (2006) Water relations, fat

reserves, survival, and longevity of a cold-exposed para-

sitic wasp Aphidius colemani (Hymenoptera: Aphidiinae).

Environ Entomol 35:228–236

David JR, Araripe LO, Chakir M, Legout H, Lemos B, Petavy

G, Rohmer C, Joly D, Moreteau B (2005) Male sterility at

extreme temperatures: a significant but neglected phe-

nomenon for understanding Drosophila climatic adapta-

tions. J Evol Biol 18:838–846

Ellers J, van Alphen JJM, Sevenster JG (1998) A field study of

size–fitness relationships in the parasitoid Asobara tabida.

J Anim Ecol 67:318–324

Farrell JA, Stufkens MW (1990) The impact of Aphidiusrhopalosiphi (Hymenoptera: Aphidiidae) on populations

of the rose grain aphid (Metopolophium dirhodum)

(Hemiptera: Aphididae) on cereals in Canterbury, New

Zealand. Bull Entomol Res 80:377–383

Godfray HCJ (1994) Parasitoids: behavioral and evolutionary

ecology. Princeton University Press, Princeton

Guerrieri E, Pedata PA, Romani R, Isidoro N, Bin F (2001)

Functional anatomy of male antennal glands in three

species of Encyrtidae (Hymenoptera: Chalcidoidea). J Nat

Hist 35:41–54

Hance T, van Baaren J, Vernon P, Boivin G (2007) Impact of

extreme temperatures on parasitoids in a climate change

perspective. Annu Rev Entomol 52:107–126

Hanna AD (1935) Fertility and toleration of low temperature in

Euchalcidia caryobori, Hanna (Hymenoptera, Chalcidi-

nae). Bull Entomol Res 26:315–322

Hay DB, Vinson SB (1971) Acceptance of Heliothis virescens(F.) (Lepidoptera, Noctuidae) as a host by the parasite

Cardiochiles nigriceps Viereck (Hymenoptera, Braconi-

dae). Anim Behav 19:344–352

Herard F, Keller MA, Lewis WJ, Tumlinson JH (1988)

Beneficial arthropod behavior mediated by airborne

semiochemicals. III. Influence of age and experience

on flight chamber responses of Microplitis demolitorWilkinson. J Chem Ecol 14:1553–1596

Hochachka PW, Somero GN (2002) Biochemical adaptation:

mechanism and process in physiological evolution.

Oxford University Press, Oxford

Isidoro N, Bin F, Colazza S, Vinson SB (1996) Morphology of

antennal gustatory sensilla and glands in some parasitoid

hymenoptera with hypothesis on their role in sex and host

recognition. J Hymenopt Res 5:206–239

Ismail M, Vernon P, Hance T, van Baaren J (2010) Physio-

logical costs of cold exposure on the parasitoid Aphidiuservi, without selection pressure and under constant or

fluctuating temperatures. BioControl 55:729–740

Langer A, Hance T (2000) Overwintering strategies and cold

hardiness of two aphid parasitoid species (Hymenoptera:

Braconidae: Aphidiinae). J Insect Physiol 46:671–676

Langer A, Stilmant D, Verbois D, Hance Th (1997) Seasonal

activity and distribution of cereal aphid parasitoids in

Belgium. BioControl 42:185–191

Lee RE Jr, Denlinger DL (1991) Insects at low temperature.

Chapman and Hall, London and New York

Lee RE Jr, Denlinger DL (2010) Rapid cold hardening:

ecological significance and underpinning mechanisms. In:

Denlinger DL, Lee RE Jr (eds) Low temperature biology of

insects. Cambridge University Press, Cambridge, pp 35–58

Levie A, Dogot P, Hance T (2000) Release of Aphidius rhop-alosiphi (Hymenoptera: Aphidiinae) for cereal aphid

control: field cage experiments. Eur J Entomol 97:527–531

Levie A, Vernon P, Hance T (2005) Consequences of accli-

mation on survival and reproductive capacities of cold-

stored mummies of Aphidius rhopalosiphi (Hymenoptera:

Aphidiinae). J Econ Entomol 98:704–708

Muratori F, Le Lannic J, Nenon J-P, Hance T (2004) Larval

morphology and development of Aphidius rhopalosiphi(Hymenoptera: Braconidae: Aphidiinae). Can Entomol

136:169–180

Ochieng SA, Park KC, Zhu JW, Baker TC (2000) Functional

morphology of antennal chemoreceptors of the parasitoid

Microplitis croceipes (Hymenoptera: Braconidae). Arthro-

pod Struct Dev 29:231–240

O’Donnell S, Beshers SN (2004) The role of male disease

susceptibility in the evolution of haplodiploid insect

societies. Proc Biol Sci 271:979–983

Perez-Maluf R, Kaiser L (1998) Mating and oviposition

experience influence odor learning in Leptopilina boulardi(Hymenoptera: Eucoilidae), a parasitoid of Drosophila.

Biol Control 11:154–159

Pierre J-S, Kasper C (1990) Analyse de la structure et de la

variabilite du comportement de ponte de l’hymenoptere

parasitoıde Aphidius uzbekistanicus Luz. dans son hote,

le puceron des cereales Sitobion avenae F. Apport de

l’analyse de donnees a la definition des item comporte-

mentaux. Biol Behav 15:152–168

Rinehart JP, Yocum GD, Denlinger DL (2000) Thermotoler-

ance and rapid cold hardening ameliorate the negative

effects of brief exposures to high or low temperatures on

fecundity in the flesh fly, Sarcophaga crassipalpis.

Physiol Entomol 25:330–336

Rivero A, West SA (2002) The physiological costs of being

small in a parasitic wasp. Evol Ecol Res 4:407–420

Roux O, van Baaren J, Gers C, Arvanitakis L, Legal L (2005)

Antennal structure and oviposition behavior of the Plu-tella xylostella specialist parasitoid: Cotesia plutellae.

Microsc Res Tech 68:36–44

Scott M, Berrigan D, Hoffmann AA (1997) Costs and benefits

of acclimation to elevated temperature in Trichogrammacarverae. Entomol Exp Appl 85:211–219

Sehnal F (1991) Effects of cold on morphogenesis. In: Lee RE

Jr, Denlinger DL (eds) Insects at low temperature.

Chapman and Hall, London and New York, pp 149–171

Shreve SM, Kelty JD, Lee RE Jr (2004) Preservation of

reproductive behaviors during modest cooling: rapid cold-

hardening fine-tunes organismal response. J Exp Biol

207:1797–1802

Behavioural consequences of Aphidius rhopalosiphi 359

123

Sigsgaard L (2000) The temperature-dependent duration of

development and parasitism of three cereal aphid para-

sitoids, Aphidius ervi, A. rhopalosiphi, and Praon volucre.

Entomol Exp Appl 95:173–184

Sinclair BJ, Vernon P, Klok CJ, Chown SL (2003) Insects at

low temperatures: an ecological perspective. Trends Ecol

Evol 5:257–262

Smith DR, Crespi BJ, Bookstein FL (1997) Fluctuating

asymmetry in the honey bee, Apis mellifera: effects of

ploidy and hybridization. J Evol Biol 10:551–574

Sømme L (1982) Supercooling and winter survival in terrestrial

arthropods. Comp Biochem Physiol Part A Physiol

73:519–543

Stilmant D, van Bellinghen C, Hance T, Boivin G (2008) Host

specialization in habitat specialists and generalists. Oec-

ologia 156:905–912

Tezze AA, Botto EN (2004) Effect of cold storage on the

quality of Trichogramma nerudai (Hymenoptera:

Trichogrammatidae). Biol Control 30:11–16

van Baaren J, Nenon J-P (1996) Host location and discrimi-

nation mediated through olfactory stimuli in two species

of Encyrtidae. Entomol Exp Appl 81:61–69

van Baaren J, Pierre J-S, Nenon JP (1993) Analysis of ovipo-

sition behaviour in the parasitoid Epidinocarsis lopezi(Hym.: Encyrtidae): comparison of flow charts on facto-

rial maps. BioControl 38:207–218

van Baaren J, Bonhomme A-S, Deleporte P, Pierre J-S (2003)

Behaviours promoting grouping or dispersal of mother

and neonates in ovoviviparous cockroaches. Insect Soc

50:45–53

van Baaren J, Outreman Y, Boivin G (2005) Effect of low

temperature exposure on oviposition behaviour and patch

exploitation strategy in parasitic wasps. Anim Behav

70:153–163

van Baaren J, Le Lann C, Pichenot J, Pierre J-S, Krespi L,

Outreman Y (2009) How could host discrimination abil-

ities influence the structure of a parasitoid community?

Bull Entomol Res 99:299–306

van Handel E (1993) Fuel metabolism of the mosquito (Culexquinquefasciatus) embryo. J Insect Physiol 39:831–833

Vinson SB (1981) Habitat location. In: Nordlund DA, Jones

RL, Lewis WJ (ed) Semiochemicals their role in pest

control. John Wiley and Sons, New York, pp 51–77

Wang X, Green DS, Roberts SP, de Belle JS (2007) Thermal

disruption of mushroom body development and odor

learning in Drosophila. PLoS ONE 2(11):e1125

Weselow RM (1972) Sense organs of the hyperparasite

Cheiloneurus noxius (Hymenoptera: Encyrtidae) impor-

tant in host selection process. Ann Entomol Soc Am

65:41–46

360 D. Bourdais et al.

123