BioControl (2012) 57:349–360 DOI 10.1007/s10526-011-9396-0
Behavioural consequences of cold exposure on males and females of Aphidius rhopalosiphi De Stephani Perez (Hymenoptera: Braconidae) Delphine Bourdais • Philippe Vernon Liliane Krespi • Joan van Baaren
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Received: 29 September 2010 / Accepted: 7 July 2011 / Published online: 11 August 2011 Ó 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 (Hymenoptera: 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 environmental 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:
[email protected] P. Vernon e-mail:
[email protected] J. van Baaren e-mail:
[email protected]
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 Parasitoı¨des, Universite´ de Rennes I, Avenue du Ge´ne´ral Leclerc, 35042 Rennes cedex, France e-mail:
[email protected]
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 Ge´ne´ral Leclerc, 35042 Rennes cedex, France
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Denlinger 1991). Insect survival capacities and the mechanisms allowing them to increase their coldhardiness have been a central theme in the field of thermal biology and it is, thus, extensively documented (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 (Hymenoptera: 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 dirhodum 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’’ (Muratori 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;
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Sømme 1982). It decreases longevity (e.g. Colinet et al. 2006) and reproductive success acting, for instance, on male sterility (Amice et al. 2008; Carrie`re 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.
Behavioural consequences of Aphidius rhopalosiphi
Materials and methods Biological material Aphidius rhopalosiphi De Stephani Perez were collected less than one year before the experiments in cereal crops around Rennes (Lat. 48°060 1000 ; Long. -01°470 3900 ) (Brittany, France) and were reared in the laboratory on a mixed-age culture of S. avenae Fabricius originating from one parthenogenetic 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 photoperiod. 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 conditions (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
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individually for 24 h in small cages with honeydew and water before behavioural tests. Control individuals 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 reintroduction 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; Pe´rez-Maluf and Kaiser 1998; van Baaren and Ne´non 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
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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 observation, 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
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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 proportions) 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 behavioural 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 different 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 description 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 influence 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).
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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 Control used in Fig. 2 Mating
14 days of cold exposure No mating
Mating
No mating
28 days of cold exposure No mating
1a 1b
51.7 ± 7.5 1.7 ± 0.8
6 ± 2.8 8.8 ± 2.8
27.3 ± 4.2 0.4 ± 0.2
37.3 ± 5.5 1.9 ± 1.1
Phase 1: detection of the female The male walks continuously with its wings down The male walks continuously with its wings up (wing fanning)
8.9 ± 3.4 10.3 ± 4.0
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 The female moves when the male is on its back
2b
2.2 ± 0.3
0
1.3 ± 0.4
0
0
2c
1.2 ± 0.2
0
1.5 ± 0.4
0
0
2d
0.6 ± 0.3
0
1.2 ± 1.1
0
0
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
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
The male lowers itself on the female before the genitalia contact Phase 3: mate acceptance
Phase 4: copulation
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
7a
0.3 ± 0.2
2.1 ± 0.6
0
1.2 ± 0.4
0.2 ± 0.2
Other behavioural patterns Grooming: the wasp rubs its antennae and/or abdomen with its legs 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
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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 successfully) were considered to be the proportion potentially 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 hypothesis 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,
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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 difference (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 olfactometer 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
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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 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)
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).
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 selfsuperparasitism of around 30%. We observed an effect of cold stress duration on the rate of superparasitism (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).
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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)
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 succeeded 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,
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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
Behavioural consequences of Aphidius rhopalosiphi
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 statistically different at the 10% level (comparisons with Tukey’s HSD test)
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 phenomenon was also reported in other insect groups, such as Coleoptera (Ali et al. 1997) and Diptera (Bre´vault and Quilici 2000). Death after cold exposure may be due to a progressive blockage of metabolic activity
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(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 documented, 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 accompanied 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 demonstrated 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
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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’Donnell and Beshers 2004) and pesticide stress resistance (Carrie`re 2003). The second factor is sexual size dimorphism. In parasitoid wasps, females are usually larger than males (Godfray 1994). In cold temperature conditions, the insect metabolism relies exclusively 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. Therefore, 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
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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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