Ability of European parasitoids (Hymenoptera) to control a new invasive Asiatic pest, Drosophila suzukii

Biological Control 63 (2012) 40–47

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Ability of European parasitoids (Hymenoptera) to control a new invasive Asiatic pest, Drosophila suzukii Stan Chabert a, Roland Allemand a, Mathilde Poyet a,b, Patrice Eslin b, Patricia Gibert a,⇑ a b

Université de Lyon, F-69000, Lyon; Université Lyon 1; CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France Laboratoire de Bio-écologie des Insectes phytophages et Entomophages, EA 4698 EDYSAN, Université de Picardie Jules Verne, 33, rue Saint Leu, F-80089 Amiens Cedex, France

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" We test the effectiveness of five

major European Parasitoids against Drosophila suzukii. " We found that only the two pupal parasitoids were able to develop on D. suzukii. " The three larval parasitoids exhibited no parasitism success. " D. suzukii exhibits high immune resistance capacity against Leptopilina species.

a r t i c l e

i n f o

Article history: Received 5 March 2012 Accepted 22 May 2012 Available online 30 May 2012 Keywords: Enemy release hypothesis Drosophila suzukii Host-parasitoid interactions Leptopilina heterotoma Asobara tabida

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a b s t r a c t Understanding the ecological factors involved in successful invasions is essential for choosing appropriate management measures. One mechanism recognized as often being essential for invasion success is for the invasive species to be less subject to attack by natural enemies. The spotted-wing drosophila, Drosophila suzukii (Matsumura, 1931) is an Asian pest of fruit crops that has recently appeared simultaneously in North America and Europe (2008). Here we investigate the effectiveness of European parasitoids of Drosophila in parasitizing D. suzukii. Of the five main European parasitoid species, only two pupal parasitoids with wide host ranges develop on D. suzukii. Two specialized larval parasitoids were unable to develop, presumably because of a strong immune response. The third specialized larval parasitoid rarely oviposited in D. suzukii. This confirms that host switching is often difficult for specialist parasitoids. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Recent years have seen drastic changes in the composition of biotic communities largely as a result of human activities (development of international trade and intercontinental transportation)

⇑ Corresponding author at: CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive, UCB Lyon 1, 43 bd du 11 novembre 1918, F-69622 Villeurbanne, France. Fax: +33 4 72431388. E-mail addresses: [email protected] (S. Chabert), Roland.Allemand@ univ-lyon1.fr (R. Allemand), [email protected] (M. Poyet), patrice. [email protected] (P. Eslin), [email protected] (P. Gibert). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.05.005

that have accelerated the movement of species, breaking down biogeographic barriers that had isolated populations for thousands of years (Williamson and Fitter, 1996; Mooney and Cleland, 2001). These changes can play an important role in the erosion of biodiversity and the disruption of the invaded ecosystems (Lodge, 1993). Some newly-introduced species become invasive, and have a considerable economic impact (Pimentel et al., 2000). A biological invasion can be defined as the successful establishment, development and maintenance of a species outside its native geographic range (Facon et al., 2006). Three phases are typically distinguished between the introduction of species and the stage of biological invasion (Sakai et al., 2001; Williamson, 2006): (1) the

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introduction of a number of founding individuals, (2) establishment (sometimes after a latency period), and finally (3) population growth. During the various phases of the invasion, individuals encounter new environmental conditions, and are generally subjected to strong selective pressures. Among the various key factors for a successful biological invasion, the ‘‘Enemy Release Hypothesis’’ (ERH) is commonly recognized as an essential mechanism. This hypothesis suggests that the success of an exotic species outside its native range is due to the absent or reduced effectiveness of its natural enemies (pathogens, predators, parasitoids) (Keane and Crawley, 2002; Blumenthal, 2006; Facon et al., 2006; Liu and Stiling, 2006). Within the community, each species has to face enemies that can be specialists or generalists with regard to other competing species. It is assumed that natural enemies in the area where the species is introduced need time to adapt before they can successfully reduce the ecological threat of a successful invader. Parasitoids are of particular interest for studying the mechanisms involved in eco-evolutionary processes in insects, because they develop in close association with their hosts, and are known to coevolve with them (Quicke, 1997). Parasitoids constitute a large group of insects (consisting mainly of Hymenoptera and Diptera), which develop inside or on the surface of other arthropods (usually insects), and consume the host tissues during their development. In insect communities, they play a critical role in controlling phytophagous populations, such as pest populations in agricultural and forest environments either as a natural control (Hawkins et al., 1997), or when they are used in biological control strategies (Greathead, 1986). Recently, the spotted-wing drosophila, Drosophila suzukii (Matsumura, 1931), a species belonging to the melanogaster group originally reported in Japan, has been observed elsewhere: in 2008 in North America and in Europe (Spain) (Calabria et al., 2010; Hauser, 2011). In 2010, its presence was confirmed in southern France, and it has subsequently spread rapidly throughout the country. D. suzukii is a pest of fruit crops with a very wide host range, and lives mainly on red fruits (raspberry, strawberry, cherry) (Lee et al., 2011). Although in the vast majority of fruit flies, larvae develop only in damaged or rotting fruit, D. suzukii is the one of rare Drosophila species which lays its eggs in sound fruit using its serrated ovipositor (Mitsui et al., 2006). Damage is then caused by larvae feeding on the pulp inside the fruits and berries. Subsequently, secondary fungal or bacterial infections may contribute to further fruit deterioration. Significant losses have been reported, particularly in the United States (California, Oregon, Washington), and following the first damage observed in Italy, D. suzukii was added to the watch list of the Organization for European Plant Protection Organization (EPPO) in January 2010. Despite the scale of the damage that can be produced by this species, there is a surprising lack of information available about its biology and its natural enemies, and in particular about parasitoids that could limit its expansion. To date, only one pupal ectoparasioid, Pachycrepoideus vindemmiae (=dubius, Hymenoptera: Pteromalidae) (Brown et al., 2011) has been successfully reared on D. suzukii. In a field-sampling study in Japan, three larval endoparasitoids were reported to develop on D. suzukii, Ganaspis xanthopoda (Hymenoptera: Figitidae) and two Asobara species, Asobara tabida (Hymenoptera: Braconidae) and Asobara japonica at an extremely low rate (Mitsui et al., 2007). In France, the communities of frugivorous Drosophila and their parasitoids involve several Drosophila species (mainly Drosophila melanogaster, Drosophila simulans, Drosophila immigrans, Drosophila subobscura), and five species of parasitoids, including three larval koinobiont and solitary endoparasitoids A. tabida, Leptopilina heterotoma and Leptopilina boulardi (Hymenoptera Eucoilidae), and two pupal ones P. vindemmiae and Trichopria cf drosophilae

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(Hymenoptera Diapriidae) (Allemand et al., 1999; Fleury et al., 2009). Most studies agree that Drosophila parasitoids induce a high rate of mortality in their host populations despite the fact that the level of parasitism varies depending on the breeding site, the local situation, and the season. The natural average rate of parasitism can reach about 90% at some sites in Southern France (Fleury et al., 2004). These high parasitism levels indicate that parasitoids may be one of the main factors of mortality in fly populations, and thus constitute selective force acting on their hosts. The aim of this paper is to test the ability of European frugivorous Drosophila parasitoids to develop in D. suzukii under laboratory conditions. The results should allow to estimate the potential role that these parasitoids may play to control this new invasive Asiatic Drosophila pest. 2. Materials and methods 2.1. Populations used Two D. suzukii females were wild-caught in October 2010 using banana traps in Sainte-Foy-lès-Lyon (lat 45.74°N, France) and used to establish a mass strain. This strain was mass reared at 21 °C, fed an artificial diet (David and Clavel, 1965), and maintained with a 12:12 LD photoperiod. Five European Drosophila parasitoid species were used in this experiment: three larval parasitoids, L. heterotoma and L. boulardi, A. tabida, and two pupal parasitoids P. vindemmiae and Trichopria cf drosophilae. Parasitoids were trapped in October 2010 in France (Rhône valley) using banana traps, and mass strains were kept in the laboratory at 21 °C on D. melanogaster with a 12:12 LD photoperiod. To avoid any possible sampling effect, two populations of each parasitoid species were tested (see Table 1). Each population was founded from 20 to 30 inseminated females. The additional experiment on the infestation behavior of A. tabida was done using the A1 strain collected in 1994 in Sainte Foyles-Lyon and reared on D. melanogaster larvae at 20 °C and LD 13:11. As a control, we used a Japanese thelytokous strain of A. japonica reared under the conditions described above. This strain was graciously provided by Professor J. van Alphen of Leiden University (The Netherlands). 2.2. Parasitoid effectiveness The temperature used for development was 25 °C, except for A. tabida, which was kept at 21 °C because of its thermal preference. The control, A. japonica, was allowed to develop at both 21 and 25 °C. D. suzukii females, between 5 and 18 days old, were allowed to oviposition at 21 °C (LD 12:12) on a banana medium (mix and heat: [20 g agar, 500 mL H2O], 400 g mashed banana, 50 g brewers’ yeast, 30 g flour, 20 g sugar, [4 g nipagine, 30 mL alcohol 70°], 550 mL H2O). Eggs were then counted and transferred in a vial containing standard artificial medium (David and Clavel, 1965). Because of the low fertility of D. suzukii in our laboratory conditions, for each parasitoid population, we set up two blocks of five vials each containing 60 host eggs (LD 12:12). After 24 h (to allow the eggs to hatch) or 5 days (to obtain pupae) for the larval and pupal parasitoids respectively, we added one mated female wasp (aged 4–7 days), fed with honey, with no previous experience of parasitism to each vial for 5 days. The vials were maintained until the flies and wasps had emerged. Parasitoid development was estimated on two groups of 10 vials (two blocks of five vials for each population) each containing 60 Drosophila eggs and one parasitoid female, as described above.

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Table 1 Geographical description of the different populations of parasitoids tested on D. suzukii, with indices (% ± SE) summarizing host-parasitoid interactions per population. DI: Degree of infestation, this measures the proportion of hosts that were successfully parasitized, SP: Success rate of parasitism, this measures the probability that an infested host will give rise to an adult wasp, TER: encapsulation rate after complete development this estimates the immune capacity of D. suzukii which allows it to survive to adulthood. NA: data non available. Species

Temperature/Population

A. japonica

21 °C 25 °C Igé (IG) Sablons (SA) Serrières (SE) Maison Neuve (MN) Ste Foy (SF) Sablons (SA) Sablons (SA) Eyguières (EY) St Etienne/Chalaronne (ST) Antibes (AN)

A. tabida P. vindemmiae Trichopria cf drosophilae L. boulardi L. heterotoma

Lat. °N

46.39 45.32 45.32 44.43 45.74 45.32 45.32 43.69 46.15 43.58

Long. °E

4.74 4.77 4.76 4.30 4.79 4.77 4.77 5.03 4.88 7.12

From this known number of eggs, some of the Drosophila would die as a result of ‘natural’ mortality and an unknown proportion would be parasitized. There were three possible outcomes of this parasitism: the death of the drosophila, that of the parasitoid if the drosophila were able to resist to this parasitism, or the death of both species as a result of physiological inadequacy between both partners. To assess the quality of the development of D. suzukii in the absence of parasitoids (control vials), we estimated the developmental success from egg to adult at both 25 °C and at 21 °C. The values obtained at 25 °C were not significantly different whatever the age of the Drosophila parents (between 4 and 52 days old) (F(1,83) = 2.69, p = 0.105). We therefore pooled the values to increase the number of individuals (85 vials corresponding to 5100 eggs), and obtained a value of 48.2% ± 1.3 (n = 85) for T. The value of T used for the experiment with A. japonica at 21 °C, 62.7% ± 3.3 (n = 10), was obtained in another experiment with young drosophila mothers. We counted the total numbers of adults of Drosophila (di) and of parasitoids (pi) emerging from the vials for each female parasitoid. We scored two indices that summarize the host-parasitoid interactions (see for example Boulétreau and Fouillet, 1982; Boulétreau and Wajnberg, 1986; Gibert et al., 2010). First, the ‘Degree of infestation’ (DI) measures the proportion of hosts that were successfully parasitized, and is estimated as (T di)/T; T being the average number of emerging flies in the absence of the parasitoid. Second, the ‘Success rate of parasitism’ (SP) measures the probability that an infested host will give rise to an adult wasp, and this is estimated as pi/(T di). In some cases, pi > (T di); for these we set SP = 1. The biological significance of these two parameters is quite clear since the Degree of Infestation represents the probability of a given host’s being parasitized, and the Success rate of parasitism the probability that a parasitized host would give rise to a wasp. Moreover, when parasitized by parasitoids, many larvae of Drosophila species are able to defend themselves by surrounding foreign bodies with blood cells that will melanize and form a black capsule that kills the parasitoid by asphyxiation. In order to estimate the immune capacity of D. suzukii against parasitoids, we counted the number of adult flies with a capsule (dc). This enabled us to estimate the ‘encapsulation rate after complete development’ (TER) as the ratio dc/(T di + dc) (see Eslin and Prévost 2000 for instance). Because it is impossible to exclude the possibility that a Drosophila larva could be killed by the parasitoid’s oviposition without receiving a wasp egg, the degree of Infestation might be

Distance between the two populations tested (km)

120 70 46 180 340

No of replicates

DI

SP

TER

10 5 10 10 9 5 10 10 10 10 10 10

98 ± 0.8 91 ± 3.1 0 0 68 ± 8.5 50 ± 9.4 85 ± 5.1 69 ± 8.7 70 ± 4.9 63 ± 6.9 52 ± 7.7 83 ± 5.7

41 ± 8 71 ± 10.5 0 0 60 ± 11.2 53 ± 16.8 38 ± 11.8 76 ± 14.2 0 0 0 0

NA NA – – – – – – 45 ± 6.9 59 ± 10.8 68 ± 14.9 80 ± 8.9

overestimated and the success rate of parasitism and the encapsulation rate after complete development underestimated. 2.3. Behavioral experiment on A. tabida In the first experiment, a null Degree of infestation being observed for A. tabida, we performed a separate experiment on A. tabida alone to explore this further. Each A. tabida female (7 days old) used for this experiment was stimulated for 5 min with larvae of D. subobscura. D. subobscura is one of main natural hosts of A. tabida (van Alphen and Janssen, 1981). This protocol allowed us to select females that exhibited host-seeking behavior. The five female parasitoids selected were placed for one hour in a vial containing 20 Drosophila larvae (stage L2), either D. melanogaster or D. suzukii. Eight replicates were used. The behavior of the females (probing, oviposition) was recorded, and the number of larvae parasitized was determined after dissection. 2.4. Statistical analysis To compare the values of Degree of infestation, Success rate of parasitism and Encapsulation rate after complete development in the different populations (different temperatures for A. japonica) and blocks for each species separately, we used a hierarchical analysis of variance (blocks in populations) on arcsine (square root) transformed proportions with linear models on the statistical software R (version 2.10.1), after checking the normality of residuals and homoscedasticity with Shapiro’s and Bartlett’s test respectively. The transformed data enable to homogenize variances of proportions. 3. Results Estimations of the Degree of infestation, Success rate of parasitism and encapsulation rate after complete development (TER) for each species are summarized in Table 1. 3.1. A. japonica Our results for A. japonica (Fig. 1A and B) confirmed that this species was very effective at both temperatures, and of the six wasps tested it was the one that exhibited the highest level of Degree of infestation (DI > 90%). It was very successful (SP about 40– 70%), and performed slightly but significantly better at 25 °C than at 21 °C (F(1,12) = 7.04, p = 0.021 ; see Fig. 1).

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3.2. Local pupal parasitoids As we have already said, two populations were tested for each species, and experiments were done on two successive blocks (five vials per block). Both pupal parasitoids effectively parasitized D. suzukii, but the outcomes were very variable. For P. vindemmiae, a significant block effect was found for the Degree of infestation for both populations (t = 2.25, p = 0.048 for MN ; t = 3.18, p = 0.01 for SE ; see Fig. 2A and C). The values of Success rate of parasitism averaged 57%, and there was no significant difference between blocks or populations. For Trichopria cf drosophilae, the Degree of infestation was significantly different between the different blocks (F(1,17) < 29.5, p < 0.001) and populations (F(1,16) < 5.92, p = 0.027), with higher values obtained for SF than for SA (84.8% vs 69.1%). Success rate of parasitism was found to be significantly different between the two populations (F(1,16) < 5.74, p = 0.029; see Fig. 2b), with SF having a lower Success rate of parasitism than SA (38.2% vs 76.3%) (see Fig. 2B and D). 3.3. A. tabida A. tabida seemed to be unable to parasitize D. suzukii (DI = 0). In the laboratory, we noticed that on D. melanogaster the A. tabida females displayed more oviposition behavior when they were not isolated. We therefore performed another experiment on D. suzukii following the same procedure, but this time with four females placed together in the vial. This confirmed the null Degree of infestation observed in the previous experiment in three replicates. In Japan A. tabida has been found emerging from D. suzukii pupae (Mitsui et al., 2007), so we performed a separate behavioral experiment to confirm our results. We observed that when in close contact with D. suzukii, A. tabida females almost never tried to

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oviposit inside the larvae: oviposition was observed only three times (out of a total of 160 larvae), so that only 1.25% of D. suzukii larvae were parasitized by A. tabida. Under the same conditions, in a control with D. melanogaster as host 225 ovipositions were observed, resulting in 81.25% of larvae infested by A. tabida, most of which were superinfested.

3.4. Leptopilina species For the two Leptopilina species, we observed a high level of Degree of infestation that averaged 67% in L. boulardi, and reached 95% in one population of L. heterotoma (AN). We found a significant block effect (t = 2.58, p = 0.02) for this population. No significant difference was found between the populations for L. boulardi. For L. heterotoma, the two populations were significantly different, with higher values obtained in AN than in ST. Nonetheless, the Success rate of parasitism was null for both populations of both species, showing that these species could not develop on D. suzukii (Fig. 3). This unexpected result could be explained by the high immune response produced by D. suzukii against these two species, as shown by the high values of the encapsulation rate after complete development, which averaged 74% for L. heterotoma and 52% for L. boulardi. For this parameter, we observed a significant block effect for L. boulardi in the EY population (t = 2.54, p = 0.02). No significant differences were found between populations for the two species. We performed another experiment following the same procedure, but placing together four Leptopilina females in the vial (three replicates). For L. boulardi, we confirmed the null Success rate of parasitism, but for L. heterotoma (AN) three adults emerged (from 180 eggs), indicating that although it is difficult for this species to develop on D. suzukii, it is physiologically possible.

Fig. 1. Results of the parasitism of D. suzukii by A. japonica at two developmental temperatures. (A) Percentage of emerging Drosophila that escaped parasitism (di) and the percentage of parasitoids emerging as adults (pi). (B) Mean ± se of the Degree of infestation (DI) and Success rate of parasitism (SP). At 21 °C two blocks of five replicates were used while only one block of five replicates was used at 25 °C. For each replicate, 60 eggs were exposed to one parasitoid female. Different letters indicate a significant difference between blocks (p < 0.05);  and NS indicate significant (p < 0.05) and non-significant differences between the two temperatures, respectively.

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Fig. 2. Results of the parasitism of D. suzukii by the two pupal parasitoids: (A and C) parasitism by P. vindemmiae – (B and D) parasitism by Trichopria cf drosophilae. For each species, two populations were tested at 25 °C with two blocks per population, and five replicates of 60 eggs and one parasitoid female per block. (A and B) shows the proportion of emerging Drosophila that escaped being parasitized (di) and the percentage of parasitoids emerging as adults (pi). (C and D) shows the mean ± se of Degree of infestation (DI) and Success rate of parasitism (SP). Different letters indicate a significant difference between blocks (p < 0.05);  and NS denote significant (p < 0.05) and nonsignificant differences between the two populations, respectively.

4. Discussion The aim of this paper was to investigate the effectiveness of French local Drosophila parasitoids against the pest D. suzukii under standard laboratory conditions. We showed that the three larval parasitoid species tested are not able to parasitize successfully D. suzukii. Only two parasitoid pupae were able to grow in this new host. In nature, female parasitoids respond to a hierarchy of physical and/or chemical stimuli that lead them to their potential host (Doutt,1959; Godfray, 1994), and so this study constitute a first approach in laboratory of the importance of the Enemy Release Hypothesis which predicts that (i) specialized enemies of the introduced species are absent in the invaded region, (ii) host switching of specialized enemies from the native species to the introduced species is rare and (iii) finally, generalist enemies are more effective against native species than invasive ones (Keane and Crawley, 2002). Our results match under laboratory conditions the predictions of the Enemy Release Hypothesis as only pupal parasitoids, which are known to be generalists, were effective against D. suzukii,

whereas the larval parasitoids were unable to parasitize this new host, showing that host switching is difficult for the more specialized local parasitoids. The successful parasitism obtained with pupal parasitoids is not surprising, especially in the case of P. vindemmiae, which is the most generalist of the five parasitoids used in this experiment, and has been reported to attack over 60 fly species, including many tephritid fruit flies and several Drosophila species (Wang and Messing, 2004). This species is widely distributed, and has been found in America, Africa and Europe (Carton et al., 1986). The successful parasitism of the two French populations of Pachycrepoideus found on D. suzukii (57%) in this study is of the same magnitude as had already been reported on D. melanogaster (Delpuech et al., 1994). Trichopria cf drosophilae is a more specialist species and is able to develop on many frugivorous Drosophila (Carton et al., 1986). For this species, we found significant differences between populations with regard both to the degree of infestation and the success of parasitism. The two populations used were collected at the same time and at a distance of only 50 km, so these differences are difficult to explain, but suggest possible genetic variability.

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Fig. 3. Results of the parasitism of D. suzukii by the two Leptopilina species. (A and C) Parasitism by L. boulardi – (B and D) Parasitism by L. heteroroma. For each species, two populations were tested at 25 °C with two blocks per population, and five replicates of 60 eggs and one parasitoid female per block. (A and B) shows the proportion of emerging Drosophila that escaped being parasitized (di–dc) and of infested flies that exhibited an immune response (dc). (C and D) shows the mean ± se of Degree of infestation (DI) and encapsulation rate after complete development (TER). Each letter indicates significant differences between blocks (p < 0.05);  and NS denote significant (p < 0.01) and non-significant differences between the two populations, respectively.

In France, these two pupal parasitoids correspond to less than 20% of the parasitoid community (Fleury et al., 2009). However, this could have been underestimated as a result of the experimental procedures used, which depends on the time of exposure in the field and very little is known about their capacity to control natural populations of Drosophila. With regard to the larval parasitoids, our results demonstrate that the French populations of the two Leptopilina species were not able to develop in D. suzukii (no Leptopilina adult parasitoids obtained from 1200 D. suzukii eggs for both species). This result is not very surprising for L. boulardi, which is able to develop on only a few frugivorous Drosophila species, including D. melanogaster and D. simulans (Barbotin et al., 1979; Carton et al., 1987; Carton and Nappi, 1991; Fleury et al., 2004), and also in the tropical species Drosophila yakuba (Dubuffet et al., 2008). This parasitoid species has been recorded in Mediterranean and intertropical climates (Barbotin et al., 1979; Chabora et al., 1979; Hertlein, 1986; Nordlander, 1980; Carton et al., 1991; Allemand et al., 2002). In contrast, L. heterotoma is clearly the most generalist parasitoid of the three larval parasitoids investigated. Its successful development has been recorded on numerous Drosophila species (Drosophila

busckii, Drosophila funebris, Drosophila hydei, Drosophila kuntzei, D. melanogaster, Drosophila obscura, Drosophila phalerata, D. simulans, D. subobscura and Drosophila willistoni) and related genera (Chymomyza or Scaptomyza) (Jenni, 1951; Carton et al., 1986; Janssen, 1989; Gibert et al., 2010). L. heterotoma exhibits a wide holarctic distribution (Nordlander, 1980; Carton et al., 1986; Janssen et al., 1988; Hardy and Godfray, 1990; van Alphen et al., 1991; Mitsui et al., 2007; Fleury et al., 2009; Gibert et al., 2010), and is thus sympatric with D. suzukii in Japan. For these two larval parasitoids, we observed a significant rate of effective encapsulation by D. suzukii, reaching 52% for L. boulardi and 74% for L. heterotoma. A. tabida is able to develop in a few Drosophila species, including D. subobscura, D. obscura and D. melanogaster, but can also develop on D. tristis (Janssen, 1989; Kraaijeveld and Van Alphen, 1995). Its geographic range includes the northwest of America (Hoang, 2002), Japan (Mitsui et al., 2007) and Europe (Carton et al., 1986). For this species, we found a null degree of infestation. This was rather surprising because this species has been reported amongst emerging parasitoids from field sampling of D. suzukii in Japan, but at a very low rate: in only one individual out of over 1152 D. suzukii pupae collected in the field (Mitsui et al., 2007).

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Additional experiments that we performed on the behavior of A. tabida females showed that this species almost never tried to oviposit on D. suzukii. Overall, only three of the 160 D. suzukii larvae were infested by A. tabida. One possible explanation of the difference between our results and those of Mitsui et al. is that local European populations of A. tabida have still not adapted to this exotic new host. One way to test this hypothesis, which would fit in with the predictions of the enemy release hypothesis, would be to compare the parasitism efficiency of Asian and European populations of A. tabida under the same conditions. In our experiment, we used two French populations for each parasitoid species in order to avoid a possible strain or sampling effect that could have interfered with our conclusions. In most cases we found concordant results for the two populations. A slight but significant difference was observed for the Degree of Infestation and SP in Trichopria cf drosophilae, which is difficult to explain since the two populations used were separated by only 46 km. More interestingly, a highly significant difference was found for the two populations of L. heterotoma, separated by 340 km, with the higher Degree of infestation found for the population from further south. This result confirms in the findings of previous studies revealing considerable latitudinal genetic differentiation between populations of this area, with greater investment in reproduction in southern populations (Fleury et al., 2004; Ris et al., 2004). Finally we must highlight a significant block effect in many cases that could be related to a problem in the quality of the development of D. suzukii. As stated in Section 2, we used a modified medium enriched with banana for rearing the flies, because this species has very low fecundity on standard medium (but with an average of four eggs laid per day and per female on banana medium). However, development was carried out on a standard medium that may be not have been ideal for developing this species, and in future experiments this should be improved. 5. Conclusions In conclusion, our results are consistent with the enemy release hypothesis that predicts that enemies specialized on native species will be less effective against invasive species. This was indeed what we found for the three major Drosophila larval parasitoids present in France. In contrast, pupal parasitoids, which are much more generalist, seemed to be able to develop as well in this new host as in local ones. To really conclude on the role of the enemy release hypothesis on D. suzukii success, our results should be complemented by studies under natural conditions. It would indeed be interesting to test whether pupal parasitoids are able to meet D. suzukii and to have a control on population density of this pest. Concerning larval parasitoid larvae, it seems unlikely that these species may have a better success in the wild than in optimal laboratories conditions. In order to understand whether the poor development of larval parasitoids, especially that of L. heteroroma that lives in sympatry with D. suzukii in Japan, is due a genetic differentiation between French and Asian populations, it would be interesting to test the parasitic capacity of Asian populations of this species on D. suzukii. Investigations of the physiological mechanisms responsible for the high immune capacity of D. suzukii would also be relevant. Acknowledgments This work is part of the ANR CLIMEVOL funded by the Agence Nationale de la Recherche. We are also grateful to the INRA centers of Avignon and Antibes for allowing us to collect insects. We thank J. Martinez and M. Henry for their valuable advice about the statis-

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