Tracking parasitoids with the stable isotope 44Ca in agroecosystems

Agriculture, Ecosystems and Environment 118 (2007) 143–148 www.elsevier.com/locate/agee

Tracking parasitoids with the stable isotope

44

Ca in agroecosystems

Heike Wanner a, Hainan Gu a,1, Bodo Hattendorf b, Detlef Gu¨nther b, Silvia Dorn a,* a

Institute of Plant Sciences/Applied Entomology, ETH Zurich, Schmelzbergstrasse 9/LFO, 8092 Zurich, Switzerland b Laboratory of Inorganic Chemistry, ETH Zurich, Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland Received 16 January 2006; received in revised form 25 April 2006; accepted 1 May 2006 Available online 12 June 2006

Abstract Habitat management influences spatial and temporal distribution of parasitoids in farmland. The current work evaluates, for the first time, the potential of a novel marking technique using a calcium stable isotope (44Ca) under field conditions. In two subsequent trials, 44Ca-enriched Cotesia glomerata parasitoids were released into an organically managed cabbage field in a region known to harbor natural populations of this species. The trap plants infested with Pieris brassicae host larvae were distributed in the trial areas and collected 3 days after parasitoid release. Parasitism by released wasps was determined through calcium isotope analysis of the recovered caterpillars using inductively coupled plasma mass spectrometry (ICPMS). The spatial habitat use by female parasitoids was determined based on marked caterpillars on the trap plants. Both trials yielded relatively consistent results, showing that C. glomerata females dispersed over at least 50 m within 3 days. The total proportion of caterpillars parasitized by the marked wasps amounted to 32.4 and 24.4%, respectively, with no statistically significant difference. The potential of this approach for field investigations on habitat management and biological control is discussed. # 2006 Elsevier B.V. All rights reserved. Keywords: Cotesia glomerata; Displacement; Internal marking; Mark-release–recapture; Stable isotope

1. Introduction Parasitoids in agricultural habitats have to move between field margins offering shelter and food, and crop containing hosts. Therefore, information on the habitat exploitation by parasitoids in space is crucial for achieving an optimal reproduction success (Lewis et al., 1998), and knowledge on how far they can and actually do move will be the basis for successful augmentative planting of food sources (Corbett, 1998). Despite the fact that the significance of parasitoids for the biological control of insect pests is increasing rapidly (Copping, 2004), knowledge about their movement and spatial habitat use in the field remains very limited (Langhof et al., 2005), particularly in the presence of a native population. This dilemma can be attributed, at least partially, * Corresponding author. Tel.: +41 44 6323921; fax: +41 44 6321171. E-mail address: [email protected] (S. Dorn). 1 Present address: CSIRO Entomology, Black Mountain Laboratories, Canberra, ACT 2601, Australia. 0167-8809/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2006.05.005

to the lack of appropriate methods for marking parasitoids and/or tracking their movement in field studies (Lavandero et al., 2004). The current techniques suitable for monitoring the displacement of insects in ecosystems in the presence of a native population rely on the external marking of winged adults with paint or fluorescent powder (Corbett and Rosenheim, 1996) and different internal marking methods using trace elements (Gu et al., 2001; Lavandero et al., 2005) or proteins (Hagler et al., 2002a). Using rare stable isotopes as internal marking is a relatively new approach, with 15N being the only isotope used so far for marking parasitoids (Nienstedt and Poehling, 2000; Steffan et al., 2001). However, all these techniques, including the application of the stable isotope 15N, limit surveys to the released, marked individuals (Hagler and Jackson, 2001) and hence require their recapture, which can be a problematic point in mark-release–recapture experiments (Prasifka et al., 2001; Hagler et al., 2002b). The present study was conducted in a tritrophic study system consisting of cabbage plants (Brassica oleracea L.;

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Brassicaceae), the larvae of the cabbage white butterfly (Pieris brassicae L.; Lepidoptera: Pieridae) and the gregarious parasitoid Cotesia glomerata (L.) (Hymenoptera: Braconidae). This parasitoid is a gregarious endoparasitoid of Pieris larvae. The female wasps locate their hosts by chemical cues released by infested plants (Scascighini et al., 2005). Normally, they lay between 10 and 130 eggs into their hosts under field conditions, whereby clusters in excess of approximately 60 usually originate from superparasitism (Gu et al., 2003), which is frequently observed in the field (Tagawa, 2000). Under field conditions adult C. glomerata parasitoids live only 2–5 days (Laing and Levin, 1982), and the female parasitoids are usually not egg- but time-limited. The time budget to locate the preferred gregarious host P. brassicae, which occurs in rather rare but large clusters, may pose a limitation on its reproductive success (Vos and Hemerik, 2003; Gu et al., 2003). C. glomerata has been introduced to North America (Lemasurier and Waage, 1993) to control the solitary occurring caterpillars of the pest P. rapae, which typically infest a much higher percentage of crop plants than P. brassicae, a species not occurring in North America (Vos and Hemerik, 2003). The foraging range of C. glomerata on either host is unknown yet. Recently, an internal marking technique was developed for this parasitoid using the rare stable calcium isotope 44Ca (Wanner et al., 2006). Enrichment of the isotope 44Ca in parasitoids was achieved along the food chain, i.e. via their host larvae feeding on the 44Ca-enriched food plants. This process allowed incorporation of the stable isotope into the body tissues and fluids of the parasitoid, and thereby also into the eggs. Remarkably, the 44Ca-enriched female parasitoids could even transfer the marker through oviposition into their host larvae. However, experiments have been carried out under indoor conditions so far, and field data are still lacking. The present study was conducted to evaluate the potential of this novel technique at ecosystem level. For this purpose repeated field trials were carried out by releasing 44Caenriched C. glomerata parasitoids on a commercial organic vegetable farm in Switzerland, in an area known for native occurrence of this species (Wang et al., 2004). The objective of the research was to determine the suitability of this stable isotope marking technique, in particular to characterize the spatial habitat use of parasitoids and the resultant parasitism of host caterpillars on plants.

2. Material and methods 2.1. Marking procedure The parasitoids (C. glomerata) and the host insects (P. brassicae) used in the experiments came from laboratory colonies, which were derived from a Swiss population (Gu et al., 2001). Host caterpillars were reared on Brussels sprout plants (B. oleracea var. gemmiferra) in nylon gauze cages

(30 cm  30 cm  30 cm) at 21  1 8C, 60% r.h. and L16:D18. Cabbage plants were grown singly in pots with a standard soil mix (Optima: nitrogen 400 mg/l, phosphorus 200 mg/l, potassium 370 mg/l, calcium 220 mg/l, and magnesium 32 mg/l) at 26  3 8C and daylight in a greenhouse for 37 days before experimental use. C. glomerata parasitoids were enriched with the stable isotope 44Ca as follows. A 50 mM 44Ca solution was prepared using CaCO3 powder enriched with 44Ca to a level of 96.40% (Isoflex, San Francisco, USA) and ultra pure water. One drop of sub-boiled HNO3 was added to dissolve 44 CaCO3. Twenty cabbage plants were enriched with 44Ca by pouring 10 ml of the 50 mM isotope solution over the soil every second day. Additionally, the plants were given ultra pure water to prevent desiccation. Two days after the first enrichment, eight newly parasitized second-instar P. brassicae caterpillars were transferred onto each plant. The 44Ca-enriched plants were exchanged once for new ones during the rearing process. Enrichment of new plants started 2 days before they were used in experiments. After parasitoid egression from hosts, cocoon clusters were collected and kept in plastic boxes (120 mm  70 mm  30 mm) at the same conditions as described above until they were used for the field release 1 week later. Reliable marking of C. glomerata parasitoids enriched by this process was confirmed in an extended laboratory and greenhouse studies (Wanner et al., 2006). 2.2. Field experiments Field experiments were carried out in an organic cabbage field near Altikon (Canton Thurgau) in northern Switzerland (478340 N, 88520 E) in July 2004. No pesticide treatments were applied during the experiments, whilst outside of that period microbial insecticides based on Bacillus thuringiensis were used to control lepidopteran pests, as spontaneous parasitism was not sufficient. The experiment was conducted twice, referred to as T1 for trial 1 and T2 for trial 2 from here on, at an interval of 7 days on two different areas of 50 m  50 m, which were separated by a distance of 150 m within one large experimental field. The field measured 280 m in length and 65 m in width, was completely flat and was embedded in an intensively cropped, but diverse agricultural landscape. Its surroundings consisted of fallow land, a path separating the field from a hedge, and a paved road. Cabbage plants were planted in rows parallel to the long field edges, with 40 cm between the rows, and a distance of 25 cm between the plants within a row. The crop field comprised two cultivars of cabbage at two different growth stages each. They were planted in strips of different widths: (1) 27 m white cabbage plants (B. oleracea convar. capitata var. capitata f. alba) 15–20 cm high, (2) 10 m red cabbage (B. oleracea convar. capitata var. capitata f. rubra) 15–20 cm high, (3) 13 m white cabbage 30 cm high, and (4) 15 m red cabbage 50 cm high.

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One day before emergence of adult C. glomerata wasps, potted cabbage plants, each infested with five first-instar P. brassicae larvae, were distributed in the trial areas. These plants were arranged parallel to the edges of the trial areas in 10 rows consisting of 11 potted plants each. The first row was set up 5 m from the margin from which wasps were released, and the others every 5 m further into the trial area. The distance between potted plants within rows was also 5 m. A total of 110 plants were distributed per trial area. Trial areas were incorporated in the three strips with smaller plants of up to 30 cm in height, because in the remaining strip the plants were already too big to place the potted cabbage plants in between. To simulate natural emergence of wasps, cocoon clusters of 44Ca-enriched C. glomerata were attached to the abaxial side of leaves of potted cabbage plants with insect pins 1 day before the estimated emergence of the adult wasps. Three of these cocoon-bearing plants (with 400 cocoons each) were arranged at intervals of 12.5 m along an edge downwind from the trial areas since laboratory trials indicate that C. glomerata wasps perform upwind flights in response to volatile chemicals emitted from the herbivore infested cabbage plants (Gu and Dorn, 2001). The wind direction was determined as the mean value over 4 days prior to the set-up of the trial area. The cocoon clusters were obtained from host caterpillars feeding on the 44Ca enriched cabbage plants as described above. Of the 1200 parasitoid pupae used in each trial, approximately 1190 adult wasps emerged. From these wasps about 480 were expected to be female, since typically a sex ratio (males:females) of 3:2 is observed in the parasitoid strain used (H. Wanner, personal observation). Under field conditions, a single host larva parasitized by C. glomerata may yield as many as 120 emerging parasitoids (Gu et al., 2003). Therefore, the density of the parasitoid individuals released from the three plants was not exceptionally high. The time interval between the two trials was kept short enough to ensure a similar cropping environment for both trials, and long enough to eliminate the wasps released in a previous trial. C. glomerata wasps typically survive for 3–5 days under field conditions (Laing and Levin, 1982). Three days after parasitoid emergence, P. brassicae larvae were collected from potted cabbage plants and their position in the field was recorded. Subsequently, they were frozen singly in Eppendorf1 tubes at 80 8C for calcium isotope analysis. To examine the effect of climatic conditions on movement of C. glomerata and on host parasitism, air temperature, humidity, rainfall, wind speed and wind direction were measured on site with an automatic weather station (PC Radio Weather Station, Europe Supplies LTD, Hong Kong) during the field experiment. The climatic data were recorded every 10 min.

mass spectrometry (ICPMS) (Hattendorf et al., 2005). Prior to analysis, samples were dried at 70 8C for at least 48 h. They were transformed into a homogenous solution by digestion with 200 ml hydrogen peroxide and 400 ml subboiled nitric acid in a microwave autoclave (ultraCLAVE II, MLS GmbH, Leutkirch, Germany). Thirty-eight individual samples were digested at the same time. Two blank samples were prepared for each run of digestion to correct for background contamination of vessels and chemicals. The digested samples were diluted with ultra pure water to a total mass of 20 g. Furthermore, all samples and control blanks were spiked with indium, allowing correction for uncertainties during digestion and measurement procedure. Using the ICPMS Elan 6100 DRCplus (Perkin-Elmer, Norwalk, Connecticut, USA) the isotopes 40Ca, 43Ca and 44 Ca were measured. The 43Ca was measured to correct for variations of mass bias of the ICPMS. All 44Ca/40Ca isotope ratios were normalized using the following linear model of mass bias:

2.3. Isotope analysis of host larvae

where X¯ is the mean value of 44Ca/40Ca isotope ratios in the control samples, n is the degree of freedom (i.e., the number of control samples minus one), and SX¯ is the standard error of mean 44Ca/40Ca isotope ratio in the control samples.

44

All sampled caterpillars were analyzed for their Ca/40Ca isotope ratios using inductively coupled plasma

44

Ca

40 Ca

¼

I44 =I40 1 þ ð4=3ÞððS43 =S40 Þ  1Þ

where I = recorded intensity (cps), S = I/A, and A = the natural abundance of the isotope. All data were acquired in peak-jumping mode (i.e. measurement only at the centre of the mass peak) with a high scanning frequency to achieve a high signal correlation during sequential measurements. Ten replicates were measured for each sample and the average 44Ca/40Ca isotope ratios were used for data analysis. However, the samples that had a standard deviation >0.032 in their average 44Ca/40Ca isotope ratio were excluded due to the consideration of precision (i.e. 12 samples in the first replicate, 13 samples in the second and 10 samples from the control samples). Three more samples from the second replicate were excluded due to irregularities in the measured indium concentrations. 2.4. Identification of marked/parasitized host caterpillars For each trial, 120 control samples were taken from the same cohort of host caterpillars as was used in the field and at the same time as the sampling of field caterpillars took place, and they were analyzed for their isotope ratios. The upper 95% confidence limit of the mean 44Ca/40Ca isotope ratios of these control samples was used as a marking criterion to identify parasitized caterpillars (Wanner et al., 2006). This marking criterion (L2) was calculated as follows: L2 ¼ X¯ þ t0:05ð2Þ;n SX¯

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All samples with 44Ca/40Ca isotope ratios above the calculated L2 values were considered marked (i.e. parasitized). This method was shown to provide reliable identification of parasitized host larvae with an error less than 5%, as only 1 out of 28 caterpillars parasitized by enriched wasps could not be identified using the described marking criterion, and parasitism determined by isotope analysis was significantly and consistently correlated with parasitism assessed visually in dissected hosts. 2.5. Statistical analysis The mean wind direction was calculated and compared between the two trial weeks using the Watson–Williams test (Watson and Williams, 1956) for two mean angles. For comparing temperature, relative humidity and wind speed, their mean values for each of the 3 days from parasitoid release to sampling of host larvae were calculated, respectively, from raw data for each day, and they were analyzed for differences between the two trial weeks using Mann–Whitney U test. The numbers of parasitized larvae and of recovered caterpillars, and the number of parasitized larvae in strips of different cabbage varieties and sizes were compared between the two trial areas using x2-test. Whether the number of host larvae on cabbage plants influenced parasitism was tested using x2-test as well. Correlations between the number of host larvae and the number of parasitized larvae on the same plant were determined using Spearman’s rank correlation. The spatial autocorrelation, i.e. the tendency of neighbouring sample units to possess similar characteristics, was tested using Moran’s I, which computes as an index of covariation between different point locations the degree of correlation between the values of a variable as a function of spatial lags. This statistical method was applied for testing the spatial distribution of cabbage plants from which larvae were recovered as well as for the distribution of plants with parasitized larvae. The distribution of plants with recovered larvae was tested for spatial autocorrelation to investigate whether this factor affected the distribution of parasitism in the trial fields. The x2-test, Mann–Whitney U test and Spearman’s rank correlation were performed using SPSS 11 for Mac OS X, and the Morran’s I test was accomplished with crimeSTAT III (Ned Levine & Associates, Houston, Texas/National Institute of Justice, Washington, DC, USA).

3. Results Mean air temperature did not vary between the two trials (Mann–Whitney U test: Z = 0.218, d.f. = 2, P = 0.827), but differences between the maximum and minimum temperatures were greater during T1 than during T2 (Table 1). The relative humidity was higher during T2 (Z = 1.964, d.f. = 2, P = 0.05). Noticeable is the much higher amount

Table 1 Climatic variables measured with an automatic weather station on the study site during the two trials (T1 and T2) T1

T2

Temperature, 8C Average (S.D.) Maximum Minimum

19.6  7.0 34.8 7.9

19.8  4.3 30.4 12.6

Relative humidity, % Average (S.D.) Maximum Minimum

60.2  23.5 93 20

75.2  16.2 95 38

Rainfall, mm Total Maximum within 10 min Minimum within 10 min

5.92 2.22 0

Wind speed, m/s Average (S.D.) Maximum Minimum

1.26  1.61 6.44 0

Wind direction, 8 Mean angle Mean angular deviation

269.38 62.59

36.26 10.65 0 0.92  1.32 5.86 0 51.94 71.96

of rainfall during T2 (Table 1), which could be mainly traced back to one intense rain incident on the third day of the trial. In both T1 and T2 it rained on day 1 and day 3, but not on day 2. The wind speed did not differ between the two trials (Z = 0.655, d.f. = 2, P = 0.513), but the wind direction significantly changed from west during the first trial to northeast during the second trial (Watson–Williams test: F = 405.74, d.f. = 902, P < 0.001). After a 4-day exposure the number of recovered caterpillars (Table 2) was significantly different between the two trials (x2-test: x2 = 34.545, d.f. = 1, P < 0.001), as was the number of plants from which they were recovered (x2-test: x2 = 19.832, d.f. = 1, P < 0.001). To distinguish the caterpillars parasitized by released wasps from non-marked caterpillars, the marking criterion L2 was calculated based on 44Ca/40Ca isotope ratios from the respective control samples. It amounted to 2.1279962 in T1 and to 2.110462 in T2. Remarkably, the frequency of host caterpillars parasitized after the 3-day exposure to the released parasitoids (Table 2) was not significantly different between trials (x2-test: x2 = 2.540, d.f. = 1, P = 0.111). However, the frequency of potted cabbage plants with parasitized caterpillars was significantly different (x2-test: x2 = 7.327, d.f. = 1, P = 0.007). On many plants only a proportion of caterpillars was found parasitized. The number of caterpillars recovered from a plant significantly correlated with the number of caterpillars found marked on the same plant (Spearman’s rank correlation: T1: rS = 0.260, P < 0.001; T2: rS = 0.371, P < 0.001). Further, in T1 the density of caterpillars on a plant influenced whether parasitism occurred on it or not (x2-test: x2 = 19.725, d.f. = 4, P = 0.001), but this was not the case in T2 (x2 = 8.778, d.f. = 4, P = 0.067).

H. Wanner et al. / Agriculture, Ecosystems and Environment 118 (2007) 143–148 Table 2 Quantification of host caterpillars distributed on trap plants in the two trials and of parasitism in Pieris brassicae caterpillars, based on samples taken 3 days after 44Ca enriched Cotesia glomerata parasitoids were released into the field T1

T2

N of trap plants with caterpillars Initially distributed Recovered with caterpillars Parasitized

110 93 (85%)a 53 (57%)b

110 63 (57%)a 22 (35%)b

N of host caterpillars on trap plants Initially distributed Recovered Parasitized

550 220 (40%)a 72 (32%)b

550 131 (24%)a 32 (24%)b

a b

Percentage of number initially distributed. Percentage of number recovered.

3.1. Spatial distribution of parasitism In T1, plants with marked caterpillars showed no spatial autocorrelation within the trial area (Morran’s I: Z = 0.592, P > 0.05), neither did the plants from which caterpillars were recovered (Z = 0.650, P > 0.05). Similarly, in T2, cabbage plants with marked caterpillars were not spatially autocorrelated (Z = 1.082, P > 0.05), but plants from which caterpillars were recovered were spatially autocorrrelated (Morran’s I: Z = 3.790, P = 0.001). Interestingly, the different cropping systems did not influence parasitism rate in either trial (x2-test: T1: x2 = 2.444, d.f. = 2, P = 0.295, T2: x2 = 1.205, d.f. = 2, P = 0.547), as neither crop cultivar nor plant size influenced whether a caterpillar on a given potted cabbage plant was parasitized or not.

4. Discussion Field trials demonstrated that stable isotope marking with Ca is a powerful tool to directly study dispersal and parasitism patterns of the gregarious parasitoid C. glomerata in cabbage crops. For the first time, stable isotope marking was used successfully to trace parasitism at ecosystem level. Marking with the stable isotope 44Ca in field experiments has proven to offer several advantages over more conventional marking techniques, which are typically applied for marking minute insects, including parasitoids. Firstly, unlike the other described external and internal marking techniques, the isotope marker was transferred from the parasitoid to the host, thereby enabling direct tracing of parasitism in the field. The marker remained detectable after a 3-day period of field exposure. Secondly, studying parasitoid movement based on parasitized host larvae was only possible so far, when a natural parasitoid population was removed by application of insecticides (Langhof et al., 2005). However, insecticide sprays cannot guarantee a complete elimination of the natural population, since a few parasitoids might survive insecticide sprays or escape from the sprayed field, and later immigrate into the trial area from 44

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outside. Furthermore, it is not known to which extent insecticide applications might also affect subsequently released parasitoids. The present study was conducted in a region with natural occurrence of this parasitoid species, with parasitism rates of 55–70% on plants with different densities of caterpillars (years 2000–2004, H. Gu, Q. Wang, Y. Zhou, personal observations). The novel 44Ca-marking technique allowed tracing of parasitism by focusing on marked C. glomerata females and their progeny in the egg stage. Thereby, the net displacement of these females could be tracked from the field edge where they had emerged to the trial area where they parasitized hosts. Our model parasitoid species is well known for its superparasitism (Tagawa, 2000; Gu et al., 2003), and both naive and experienced female wasps are willing to attack hosts that have been parasitized before by conspecifics, with reports of host larvae being parasitized up to five times (Gu et al., 2003). Thus, superparasitism will largely rule out the option that the native population will influence the distribution of parasitism by the released wasps. The present results documented clearly that C. glomerata is a robust biological control agent, consistent even under variable biotic environments. Female wasps of C. glomerata effectively parasitized caterpillars in the field without being influenced by different cultivars and crop growth stages. This finding contrasts previous observations in Trichogramma spp. egg parasitoids, where dispersal was dependent on the shape and the densities of the canopies (Bigler et al., 1997). In the latter case, however, dispersal was mediated by wind. Consistently in both trials, assessment of parasitism showed that C. glomerata females were able to disperse over the entire trial area of 2500 m2 within 3 days. Surprisingly, no gradient in parasitism depending on distance from release site was observed, as a random distribution was noted. Even when the caterpillars were not randomly distributed, which was the case in the second trial, the spatial pattern of parasitism remained random. The total number of parasitized caterpillars was not significantly different between the two field trials. However, the proportion of plants that bore parasitized caterpillars was lower in the second trial, where the number of caterpillars and the number of plants from which they were recovered was also lower. A reduced number of feeding caterpillars presumably result in lower emissions of volatiles (Mattiacci et al., 2001), and therefore in a weakened attractiveness for the released parasitoids, which locate their hosts by means of chemical cues (Scascighini et al., 2005). Chemically mediated orientation of the parasitoids might have been affected further by altered climatic conditions in the second trial. The mean wind direction changed, probably making the odor-guided upwind location of host caterpillars more difficult within the trial area. Additionally, heavy rainfall in the second trial might have washed off some caterpillars, leading to reduction in the volatile blend emitted from the plants (Vallat et al., 2005). Furthermore, rainfall might have reduced parasitoid activity in the second trial.

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The 44Ca marking method is cost effective with calculated material costs in the order of US$ 3000 to mark the approximately 960 female wasps, which were released during the two trials, and to analyze the 335 recovered and 240 control caterpillars. These costs are paid off by the unique advantages over other methods used in markrelease–recapture studies on parasitoids, such as tags, dyes, trace elements or protein markers. Marking with the stable isotope 44Ca could be reliably used to track parasitoids at ecosystem level in space and time. It holds the potential for application in determining the influence of habitat management measures, such as augmentative planting of flowering plant strips at field edges, on the dispersal pattern and the efficacy of C. glomerata and possibly other gregarious parasitoids in biological control. Furthermore, with the 44Ca-marking technique colonization of new crop fields can be studied, as well as recolonisation from the surroundings after an insecticide application, which can lead to an initial suppression of parasitoid populations (Holland et al., 2000).

Acknowledgements We thank Silke Hein for helpful discussions, and we thank her, Anja Rott and Kathrin Tschudi-Rein for comments on an earlier version of this manuscript. We also acknowledge Maja Hadian, Isabell Peters and Marco Hinnen for support with fieldwork, and Urs Guyer and Marion Schmid for their help with plant rearing. This study was supported by a TH grant (ETH Zurich) to HG, DG and SD.

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