IOBC wprs Bulletin Vol 22(9), 1999
Herbivore-induced volatiles in the apple orchard ecosystem
Silvia Dorn and Alan HernSwiss Federal Institute of Technology, Zurich, Switzerland
Abstract: Plant-derived volatile chemicals may alter the behaviour of insects on the second as well as on the third trophic level. The plant itself via its chemical emission may play a key role in the regulation of its associated insect populations. In apple, Malus domestica, enhanced emission of volatile chemicals was found to be the result of herbivore feeding on leaves as well as on fruit. An attraction of natural antagonists to the odour blend was found in which a-farnesene was revealed to be a dominant component. The complex role of this terpenoid in the apple ecosystem is under evaluation. Direct feeding of a herbivore on the apple fruitlet triggered increased emission of a-farnesene, as we previously demonstrated for the European apple sawfly, Hoplocampa testudinea. The emitting source of the plant-herbivore complex was found not to be the feeding larva or its products but the injured apple fruitlet. Thus, the attractant principle was plant- and not insect-derived. Further studies focused on a herbivore of apple fruits of worldwide importance, the codling moth, Cydia pomonella. A pronounced sexual dimorphism was found in the response of adult moths to a range of a-farnesene dosages, indicating a stronger dependency of females on plant-derived volatiles. Possible implications for codling moth dispersal and infestation patterns are discussed.
Key words: Apple volatiles, a-farnesene, behaviour, Cydia pomonella, Hoplocampa testudinea, olfactometer
Scents in orchards may contain information for different groups of organisms within this ecosystem. The volatile chemicals in the atmosphere around the apple trees may contain information for male insects to locate conspecific females. This type of information is crucial within a given trophic level. Volatile chemicals may further contain information for female herbivorous insects to locate a suitable site for oviposition, and information for entomophagous insects to locate the habitat of their herbivorous host insect. This type of information is linking two or more trophic levels within the agroecosystem.
While several papers of this volume deal with the first type of semiochemicals, the pheromones, this contribution will focus on the second type, the allelochemicals. The orchard ecosystem is particularly attractive to study such questions. As a perennial system, it may have developed long-term strategies to regulate its associated insect populations. We will first exemplify the effect of herbicides-induced volatiles from leaves, then summarise the first study on herbivore-induced volatiles from fruits and finally focus on the role of a dominant volatile substance in the odour blend, E,E-a-farnesene.
Material and methods
Insects and plants
Apple seedlings were infested with the apple leafminer, Phyllonorycter sp. in an insectary. The braconid wasp, Pholetesor bicolor (Nees), was reared in some of the cages. Apple fruitles were naturally infested with the apple sawfly, Hoplocampa testudinea (Klug), under field conditions in spring. The codling moth, Cydia pomonella (Linnaeus) was obtained from a commercial breeding station in Switzerland and used for bioassays at an age of three days after emergence.
Dual choice bioassay
The Y-tube olfactometer bioassay method (Bertschy et al. 1997) was used to assess the insect's performance for one out of two odour sources. Purified, humidified air enters each arm of the olfactometer and flows over the respective odour source in an inserted glass chamber. The air leaves the olfactometer through the central tube. There, one insect is inserted at a time and has to move upwind as in nature. At the fork, it has to take a choice between the two odour sources. The choice response is analysed using a one sample Chi-square test.
Volatile collection and analysis
The volatile collection systems used under laboratory and field conditions are described by Boevé et al. 1996. Purified and humidified air passes through a glass tube (laboratory) or a PET bag (field), respectively, and is absorbed by a trapping filter consisting of Super Q. Standard methods were used for analysis by gas chromotography and gas chromatography - mass spectrometry.
Results and discussion
Herbivore-induced leaf volatiles
Leaves, the site of photosynthesis, are expected to strongly respond to environmental factors. Leaves respond not only to abiotic conditions such as light but also to specific biotic conditions such as herbivory (Dorn et al. 1999). Headspace analysis of apple leaves infested with the apple leafminer as compared to healthy leaves showed major quantitative differences. A prominent component that increased proportionally to the number of mines on the leaves was found to be E,E-a-farnesene (Lengwiler et al. 1994; Lengwiler et al. unpubl.). A natural antagonist of the apple leafminer, the braconid wasp Pholetosor bicolor, preferred odours of infested apple plants over odours of healthy plants in Y-tube olfactometer bioassay. Odours collected from infested plants with the volatile collection system, subsequently extracted with hexane and pippetted on filter paper, were preferred over the odour of the pure solvent (Lengwiler et al. 1994). This indicates an attractive effect of the volatile allelochemicals from herbivore infested leaves on the natural antagonist.
Leaves are the source of photosynthetic products; apples are the sink. The question arose whether the apple fruits respond to herbivory at all. We were the first group to address the question of how fruits would react to herbivore feeding regarding emission of volatiles.
Herbivore-induces apple volatiles
Herbivore infestation had a major impact on the quantitative release pattern of volatiles from apple fruitlets: Apples infested with sawfly larvae feeding inside the fruit emitted higher quantities of chemicals into the headspace (Boevé et al. 1996). This becomes most obvious for E,E-a-farnesene, trans-b-ocimene and 2-phenylethanol. Focusing on a-farnesene, which is its major source of origin?
Although the feeding last-instar larvae contain active glands, almost no a-farnesene was detected in their secretion. The faeces contained a small amount of this compound (Boevé et al. 1996). Since they are deposited on the apple surface, it is likely that they are slightly contaminated by apple volatiles. Thus, this terpenoid does not seem to be a product of the sawfly but a product of the apple. It seems to a large extent to be a herbivore-induced volatile.
The emission of large amounts of E,E-a-farnesene from infested apple as well as from infested leaves triggered numerous questions: What are its ecological implications? It could have effects on a wide array of insects in apple orchards. How could its effect be modified? Is it the dosage which is crucial, or are there further odour components in the blend which determine, in concert with E,E-a-farnesene, the response of a given insect species? We tested the hypothesis that its effect on insects is dosage dependent, and used a herbivorous insect of worldwide importance, the codling moth Cydia pomonella, as the test species. Similarly to the previously described apple sawfly, its larvae feed within the apple, and its faeces become visible at the apple surface. We chose a range of dosages corresponding, at the lower end, to the estimated concentration in the headspace of a few healthy apples, and at the upper end to a few dozens of sawfly-infested apples. This estimate is based on the release of E,E-a-farnesene from apples as measured in the first half of the apple growing season, corrected by a factor of 10 based on a recent paper by Raguso and Pellmyr (1998). They recovered approximately one tenth of the original amount of compounds in a known blend using a dynamic headspace entrainment system.
The codling moth adults were given the choice between E,E-a-farnesene and the solvent hexane in a Y-tube olfactometer. The degree of responsiveness was regularly ca. 50%, irrespective of sex.
Fig. 1 and 2 represent the responding moths, the bars indicating the proportion of moths choosing a-farnesene. Female codling moths, mated and ready to oviposit, were significantly attracted to low dosages of E,E-a-farnesene (Fig. 1). Over 75% of the responding females preferred the odour of the terpenoid at the two lowest dosages tested over the odour of the solvent. Medium dosages were neutral; the females showed no preference. The highest dosages tested were significantly avoided by the females. Thus, the type of response of the mated female moths was found to be dose-dependent.
Fig. 2 depicts the response for mated males. Their behaviour pattern looks quite different. Over a wide range of dosages they showed no preference at all. Their response was neutral. Only at the highest dosage tested a preference was noted.
Figure 1. Attraction of Cydia pomonella females (N = 50) to increasing dosages of E,E-a-farnesene in a Y-tube olfactometer. The synthetic preparation contained 77% E,E-a-farnesene and 20.7% Z,E-a-farnesene, and both isomers were shown to have similar efficacy. Asterisks indicate significant preferences for a-farnesene when attraction is above 0.5, and significant avoidance below 0.5, at P < 0.001 (**) and P < 0.001 (***), respectively.
Thus, in conclusion, we found a marked sexual dimorsphism towards E,E-a-farnesene in the codling moth. Females showed preferences in a dose-dependent manner. Males did not show any preference up to a high threshold value. This indicates a stronger dependency of females from this plant-derived allelochemical. Males, on the other hand, strongly respond to the pheromones from their conspecific females.
For mated female codling moths, Wearing and Hutchins (1973) described for low dosages a stimulatory effect of a-farnesene on oviposition and for high dosages similarly to the high dosages applied in our system a disappearance of this stimulation. Thus, both findings consistently indicate that low dosages may be signalling favourable and high dosages suboptimal or unfavourable conditions for codling moth females.
Plant-derived volatiles may alter the behaviour of female insects at the second as well as on the third trophic level. The plant itself may play a key role in the regulation of its associated insect population.
Figure 2. Attraction of Cydia pomonella males (N = 50) to increasing dosages of E,E-a-farnesene. Asterisks indicate significant preference at P < 0.01 (**).
We thank Dr. Roman Kaiser, Givaudan-Roure, Dübendorf, Switzerland, for the synthesis of a-farnesene and for providing us with many reference samples for GC and GC-MS analysis.
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