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A.S. Rott, J. Häckermann, N. Brand, A. Vallat & S. Dorn* ... of seasonal emissions of apple fruit volatiles on the host location behaviour of a parasitoid of the.
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Parasitoid exploitation of the seasonal variation in host plant volatile emission for herbivore location A.S. Rott, J. Häckermann, N. Brand, A. Vallat & S. Dorn* Institute of Plant Sciences, Applied Entomology, Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland Accepted: 29 January 2005

Key words: apple, Cydia pomonella, Hyssopus pallidus, olfactometer, apple volatiles, host location, Lepidoptera, Tortricidae, Hymenoptera, Eulophidae, integrated pest management

Abstract

Volatile compounds from the apple, Malus domestica Borkh. (Rosaceae), change considerably as the season progresses, and this is successfully exploited by the female codling moth Cydia pomonella L. (Lepidoptera, Tortricidae), as it searches for oviposition sites. In this study, we investigated the effect of seasonal emissions of apple fruit volatiles on the host location behaviour of a parasitoid of the codling moth larvae, Hyssopus pallidus (Askew) (Hymenoptera: Eulophidae). In dual choice olfactory bioassays, the behaviour of the parasitoid in response to apple cues was observed over the complete 2003 growing season. Our results show that codling moth infested apples evoked a strong response from the parasitoid at the beginning of the season, until July. Then, attraction dropped drastically, increasing again at the beginning of August. At the end of the growing season, just prior to harvest, infested apples hardly evoked any behavioural response. Interestingly enough, mid-season emissions of healthy apples were per se attractive to the parasitoid, and even preferred over volatiles from infested apples. Simultaneous volatile collections from healthy apples on twigs in the field were analysed throughout the season, showing that the overall quantity of headspace volatiles peaks at the beginning of June and mid-August. The seasonal volatile emission is correlated with the behaviour of the parasitoid during the fruit ripening stage. The results are discussed in relation to the use of H. pallidus as a potential biocontrol agent, in order to enhance current integrated pest management (IPM) programs.

Introduction Host location by the natural enemies of herbivores mainly relies on chemical stimuli, be it stimuli from the host habitat or food plant, or stimuli associated with the presence of the host and the host itself (Godfray, 1994). Numerous studies on tritrophic systems involving a natural enemy, such as a parasitoid, the herbivorous insect, and a plant, have shown that parasitoids use a variety of cues in deciding where to search and for how long (Vet et al., 1995; Vinson, 1998), and that chemical information from the plant is generally considered to play a pivotal role in the location, evaluation, and parasitism of a host by its parasitoid (see review by Dicke & van Loon, 2000; Mattiacci et al., 2000, 2001). Therefore, temporal changes in plant-derived chemical stimuli will affect interactions with herbivores as well as *Correspondence: Prof. Silvia Dorn, Institute of Plant Sciences, Applied Entomology, Swiss Federal Institute of Technology (ETH), Clausiusstrasse 25, CH-8092 Zurich, Switzerland. Tel.: +41 1632 3921, Fax: +41 1632 1171, E-mail: [email protected]

natural enemies. For example, the volatile blend emitted from apple fruit in situ changes drastically both in quantity and chemical composition as the season progresses (Bengtsson et al., 2001; Hern & Dorn, 2003). The greatest amounts of volatiles from apples were collected early and late in the season (Hern & Dorn, 2003). The codling moth, Cydia pomonella L. (Lepidoptera, Tortricidae), a major pest of apples, Malus domestica Borkh. (Rosaceae), uses these changing volatile profiles, including changing α-farnesene concentrations, to optimise apple location and its oviposition behaviour (Sutherland et al., 1977; Hern & Dorn, 1999, 2002). Because parasitoids use plant-derived chemical stimuli for host location, we postulated that a natural enemy of the codling moth, Hyssopus pallidus (Askew) (Hymenoptera: Eulophidae), will exploit this seasonal variation in apple volatiles for host location. For example, L. Mattiacci, C. Hausmann and S. Dorn (unpubl.) showed that more codling moth larvae were attacked in apples at an early and late stage of fruit maturation. Furthermore, recent studies of the host-searching behaviour of H. pallidus have shown that the volatile chemicals produced by damaged apples

© 2005 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 115: 199–205, 2005

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are utilised in long-range host location, while short-range orientation appears to be mediated by a combination of plant and herbivore-related chemical cues (Mattiacci et al., 2000; Gandolfi et al., 2003a,b). Cues that trigger successful foraging behaviour in H. pallidus are important, as the parasitoid females search for host larvae inside the fruit (Mattiacci et al., 1999), and successful searching behaviour can be directly linked to parasitism (Völkl, 1994). In this study, we elucidated the effect of seasonally emitted volatiles on the host habitat location of H. pallidus. This gregarious larval ectoparasitoid has the ability to enter a fruit infested with Cydia spec., paralyse the feeding caterpillar, and deposit its eggs on the host. After hatching, the larvae start feeding on the outside of the host caterpillar (Mattiacci et al., 1999; Tschudi-Rein & Dorn, 2001). Host handling and host feeding can take as long as 2 days, and the average clutch size is around 15 eggs with a strongly female-biased sex ratio (Zaviezo & Mills, 1999, 2000). Hyssopus pallidus has been identified as parasitizing apples infested with Cydia spec. in Swiss orchards (Tschudi-Rein et al., 2004). Currently, H. pallidus is not of economic importance as a biocontrol agent. However, due to its distinctive and unique ability to locate and parasitize the host inside the fruit, H. pallidus has the potential to be used for the parasitization of infested fruit in the field. This method would represent a promising strategy for preventing the emergence of the next generation of codling moths, and provide an interesting tool for enhancing existing integrated pest management (IPM) programmes. Here, we investigated the influence of seasonal chemical volatile emissions of the apple fruit, with an emphasis on the second trophic level (infested fruit vs. healthy), the foraging preference of the parasitoid over the growing season in particular, in order to evaluate the potential of H. pallidus as a biocontrol agent. The results will provide information on the importance of timing in the release of an inoculative biocontrol agent.

Materials and methods Rearing procedures

The C. pomonella adult moths, eggs, and larvae were reared in an insectary at 24 ± 2 °C, 60 ± 10% r.h., and under a L18:D6 photoperiod. The larvae were reared on apple: twice a week, two newly hatched C. pomonella were placed on each of 40 apples. Within 10–24 h, the larvae bored into the apple, and completed their development after 16–21 days. To prevent fifth instar larvae escaping after the last moult, the apples were covered with a plastic cylinder 1 week after infestation. To obtain the fifth instar larvae for rearing the parasitoids, the apples were carefully dissected after 18–21 days. The H. pallidus culture was maintained on fifth instar larvae of C. pomonella obtained from infested apples. For

parasitism, host larvae were offered to female parasitoids in glass vials (10 cm length; diam. 2.8 cm) at a ratio of one parasitoid per host. To simulate natural conditions, a small piece of fresh apple was added to the vials, and a droplet of honey was provided as food (Gandolfi et al., 2003a). New adults emerged from parasitized larvae 14–17 days later. Upon emergence, the parasitoids were transferred to a Plexiglass cage (25 × 25 × 5 cm) and maintained with water and undiluted honey. The apple used as a rearing medium, Malus domestica cv. Bohnäpfel, was obtained from a commercial organic farm and stored at 4 °C for up to 6 months. The apples for the bioassay (cv. Golden Delicious) were produced on an integrated farm. The branches with apples in the field were covered with a plastic bag during crop spraying. Choice experiments: Y-olfactometer

To determine the host location preference of H. pallidus, dual choice trials were conducted throughout the growing season with volatiles from healthy apples vs. blank or from healthy apples vs. infested apples. Infested apples were obtained by infesting freshly picked apples with one fourth or fifth instar larvae each and leaving them for 6 days at 24 ± 2C °C, 60 ± 10% r.h., and L18:D6. The olfactory response was measured in a glass Yolfactometer, which consisted of a Y-tube (1.8 cm diam.; length of common arm and two side arms = 22 cm) and two odour chambers (500 ml) (Hern & Dorn, 1999). These were connected to a flow meter, which allowed charcoal-filtered, moist air to enter at a constant rate of 230 ± 5 ml min−1 through each arm of the olfactometer. For the bioassay, 4– 6-day-old, mated females without oviposition experience were used. One female at a time was placed in the entrance of the Y-tube olfactometer and its position was recorded after 5 min. Behaviour was classified as no choice if the parasitoid remained in the common arm, and as choice for either the volatile bouquet of a healthy apple or blank (= control) if it had entered one of the arms in which the odour source was located (n = 25). The line for choice classification was set 3 cm behind the Y-junction. A plant was classified as ‘attractant’ when significantly more females chose this odour source vs. blank, and ‘repellent’ when the blank was significantly preferred in the dual choice bioassay, i.e., when the female wasps avoided the plant odour arm and instead entered the blank odour arm (e.g., Hern & Dorn, 1999, 2003). To avoid any asymmetrical bias in the set-up, the odour sources and their position were changed after five parasitoids had been tested. Each female was only tested once. At the end of the experiment, the Y-tube olfactometer was washed to remove all traces of odour sources. In a second choice experiment, the odour source offered was healthy apple vs. infested apple (n = 50). All bioassays

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were done under 300 ± 20 × 10 Lux light intensity, at 22 ± 2 °C and 55 ± 5% r.h. Tests were conducted fortnightly throughout the apple-growing season, starting in mid-May until apple harvest in mid-September. Volatile collection and analysis

Volatiles were collected in situ throughout the season using radial diffusive sampling (Radiello model 3310, Rupprecht and Patashnick Co., Albany, USA) in 2003. Sampling occurred at eight sampling dates from 11:30 hours to 15:30 hours (end of May to end of August). Fifteen samples were taken on each sampling date from five apples selected by random sampling on each of three apple trees. The fruits and leaves to be sampled were visually inspected to ensure there was no apparent herbivore damage or disease. For each sample, the volatile compounds in the headspace of an apple fruit (including the twig and connected leaves) were collected directly on the tree with a Radiello sampler in a horizontal position inside a plastic bag (Nalophan, Kalle GmbH, Wiesbaden, Germany). The bag was tied securely around the branch of the apple. The adsorbent cartridge, which contained Tenax-TA, was placed inside the yellow diffusive body and screwed on a supporting plate attached near the apple. After sampling, the cartridges were placed in a steel tube (Unity Markes International Ltd™, Pontyclun, UK) and were analyzed by thermal desorption (Unity Markes International), which was connected to a Hewlett Packard GCMS instrument (GC 6890 Mass Detector 5973, Atlanta, GA). The cartridges were desorbed at 300 °C for a period of 5 min. Prior to desorption, each cartridge was given a purge flow for 3 min. The cold trap was packed with a 4 cm bed of Tenax-TA and a 2 cm bed of Carbopack B, and was held at −10 °C throughout the tube desorption process. The cold trap was subsequently heated at a rate of 60 °C per min to 300 °C, with a total cold trap desorption time of 3 min. The transfer line to the GC-MS was held at 150 °C. The GC column used was a phenyl methyl siloxane column (EC-5, Alltech, Deerfield, IL, USA; 30M; inner diameter of 0.25 mm and film thickness of 0.25 µm) fitted with a fused silica retention gap (HP1, HP Infochroma Ag, Zug, Switzerland; 5M 0.25 mm ID). The initial temperature was 50 °C for 5 min, then a 5 °C min−1 ramp to 300 °C (total run time was 65 min). The MS quadropole and MS source temperatures were 150 °C and 230 °C, respectively. Prior to sampling, the sorbent tubes were thermally conditioned for 45 min at 320 °C followed by 30 min at 335 °C with a flow of helium (60 ml min−1) through the trap. Identification of compounds was based on retention time, fragmentations and co-injection with standards. The spectra were compared to the NIST98 mass spectral database (National Institute of Standards and Technology,

Gathersburg, MD) and our own library of authenticated phytochemical compounds. Data analysis

The choice response of H. pallidus to the odour sources was analyzed using a one-sample χ2-test (Zar, 1998). Variation of the volatile emissions in relation to different sampling dates was analyzed using an ANOVA with repeated measures, with date as a within-subject factor. The sphericity assumption was not met, so the Huynh–Feldt correction was applied (Zar, 1998). Spearman’s non-parametric correlation was used to examine possible relationships between the percentage of H. pallidus showing a positive response to healthy apples and the variation in volatile emissions over the season.

Results Choice experiments

The results of the dual choice bioassays showed changes in odour preference with increasing ripeness of the apples (Figure 1). A significant repellent effect was noticed for volatiles emitted by the green fruits on 23 May (χ2 = 9.308, P