Naturwissenschaften (2000) 87:216–219 Q Springer-Verlag 2000 SHORT COMMUNICATION Caroline Müller 7 Monika Hilker The effect of a green leaf volatile on host plant finding by larvae of a herbivorous insect Received: 19 October 1999 / Accepted in revised form: 28 February 2000 Abstract The role of a general green leaf volatile (glv) hydrolysis of glycosides, and oxidative degradation of in host finding by larvae of the oligophagous chrysome- leaf lipids to six carbon aldehydes, alcohols, and esters lid Cassida denticollis was investigated using a new of the latter (Karban and Myers 1989; Mattiacci et al. bioassay which takes into account the need for neonate 1995; Merkx and Baerheim Svendsen 1990; Visser and larvae of this species to climb fresh host plants from the Avé 1978). They have been shown to mediate informa- ground. A “stem arena” was designed in which plant tion between and within trophic levels. For example, stems of the host, tansy (Tanacetum vulgare), and stem herbivorous insects may be attracted by glv of their dummies (tooth picks), both wrapped in perforated fil- host plants (Mitchell and McCashin 1944; Schütz et al. ter paper, were offered to neonate larvae. The wrap- 1997; Visser and Avé 1978), and glv can modify re- ping allowed olfactory responses to be tested by pre- sponses of insects towards their aggregation or sex phe- venting access to contact stimuli of stems and dummies. romones (Dickens et al. 1990, 1992, 1993; Landolt and Larvae significantly preferred to climb the wrapped Phillips 1997; Light et al. 1993; Zhang et al. 1999). They tansy stems over dummies after a period of 15 min. The are also secreted by abdominal glands in some hetero- test glv, (Z)-3-hexen-1-ol, was not attractive when ap- pteran species and can act as pheromones or defensive plied to dummies. However, when the glv was applied devices (Aldrich et al. 1984; Hamilton et al. 1985), and to the bottom of the arena, the ability of larvae to dis- several parasitoid species of herbivorous insects use glv criminate between host stems and untreated dummies for host finding (Howse et al. 1998). was significantly enhanced. More larvae climbed wrap- This study examined whether a glv may influence ped host stems than dummies even within 5 min. While the host finding behavior of larvae of the leaf beetle numerous other herbivorous insects are known to be species Cassida denticollis Suffr. (Coleoptera, Chry- directly attracted by glv, this study shows that a singly somelidae). C. denticollis feeds on various Asteraceae offered glv on its own is unattractive to an herbivore such as Tanacetum vulgare L. (tansy), Achillea millefol- but enhances the herbivore’s ability to differentiate be- ium L., and Artemisia campestris L. Females lay their tween host and nonhost plants. eggs in spring on dry plant material that remains from annual plants of the previous year. Therefore neonate larvae must find fresh host plants by climbing the stems Introduction of green plants to reach the leaves for feeding. In our study (Z)-3-hexen-1-ol was tested as a representative of The importance of so-called general green leaf volatiles the glv’s known to be released by numerous plant spe- (glv) has been demonstrated in several interactions be- cies after leaf damage (Merkx and Baerheim Svendsen tween plants, herbivores, and antagonists of herbivor- 1990; Visser et al. 1979). The tested host plant tansy ous insects. General green leaf volatiles are well-known also releases (Z)-3-hexen-1-ol (gas chromatogra- to be released especially after leaf damage, subsequent phy–mass spectroscopy analysis by Müller, unpublished data). The amounts of glv are known to vary between plant species (Schoonhoven et al. 1998). Even within a species, amounts of glv released vary between individu- al plants depending on age, temperature, and extent of C. Müller 7 M. Hilker (Y) damage. We studied the following questions: (a) Do Institut für Biologie, Freie Universität Berlin, Haderslebenerstrasse 9, 14163 Berlin, Germany larvae of C. denticollis recognize host (tansy) stems by e-mail: hilker6zedat.fu-berlin.de their volatiles? (b) Does a glv [here: (Z)-3-hexen-1-ol] Fax: c49-30-83853897 affect larval ability to discriminate between host and 217 nonhost stems? (c) Is (Z)-3-hexen-1-ol itself attractive and three untreated wooden stem dummies were offered to the to larvae of C. denticollis? larvae in an alternating sequence. To keep conditions of this bioassay comparable to those used in the bioassay of the second experiment, 30 ml hexane were pipetted onto the center of the bottom of the stem arena (Fig. 1). Materials and methods In the second experiment we studied whether the presence of the glv (Z)-3-hexen-1-ol affects the ability of neonate larvae to Insects find the host plant tansy. Larvae were offered the same arena with the same stems and dummies as in the bioassay of the first In May 1997 adults of C. denticollis were collected at ruderal sites experiment, but 30 ml of a solution of (Z)-3-hexen-1-ol in hexane in Berlin, Germany, and placed in plastic containers was applied onto the center of the arena. Two concentrations (0.3 (20!20!6 cm, Gerda, Schwelm, Germany) with a gauze lid and 1 ml/ml) were tested. (120 mm mesh). The bottom of each container was covered with In the third experiment we examined whether (Z)-3-hexen-1- filter paper and leaves of T. vulgare were offered as food plant. ol itself is attractive to the larvae. The responses of neonate (1- Egg batches (containing three to six eggs), which were laid on the day) and 5-day-old larvae were tested by offering three test stem filter paper, were transferred into glass vials (5!2.5 cm). Larvae dummies treated with (Z)-3-hexen-1-ol and three control stem were tested 5–7 h after hatching. Rearing conditions were 20 7C, dummies treated with hexane only in an alternating sequence. 75% relative humidity and a cycle of L16:D8. From a solution of (Z)-3-hexen-1-ol in hexane 15 ml was pipetted over the whole length of each test stem dummy at the concentra- tions of 0.3 and 1 ml/ml. General bioassay design Statistics To examine the role of (Z)-3-hexen-1-ol in the host finding be- havior of C. denticollis, a “stem arena” was designed in which Data were evaluated statistically by the Wilcoxon signed-rank test stems (2.5 cm) of the host plant tansy or wooden stem dummies for paired differences when comparing the numbers of larvae (tooth picks) with various volatile treatments were offered to lar- climbing test and control stems. The total numbers of larvae vae in an open petri dish (diameter 5.5 cm; Fig. 1). The bottom of climbing stems in experiment 1 at 5, 10, and 15 min were com- the petri dish was filled with soil covered by filter paper to fix the pared to the total numbers of larvae climbing stems at the same stems and dummies. To study responses of larvae to volatiles time intervals in experiment 2 by the 2!2 table x2 test. The same only, both plant stems and stem dummies were wrapped with per- test was used to compare the total number of (neonate) larvae forated filter paper tubes (diameter 0.5 cm) to prevent direct con- climbing stems in experiment 1 at 15 min and the total number of tact. Prior to each bioassay all solvents used were allowed to neonates (1 day old) climbing in experiment 3 at both the low and evaporate for 2 min. Stems were cut from the middle part of high glv concentration. The threshold value for significance was about 30-cm-high, growing plants and bioassayed immediately Pp0.05. after cutting. When starting the bioassay, 20 larvae were placed into the center of the stem arena. The numbers of larvae that had climbed the wrapped stems or stem dummies were recorded 5, 10, and 15 min after larvae had been placed into the arena. Larvae Results were not removed from a stem after climbing it. Instead, they were allowed to climb up and down more than one stem. Howev- When offered tansy stems and dummies, neonate lar- er, they did not move to another stem more than once within a vae of C. denticollis significantly preferred to climb period of 5 min. Each bioassay was replicated 20 times and con- ducted at 20 7C. their host plant stems after 15 min (Fig. 2A). Within 5–10 min some larvae climbed up both plant stems and stem dummies, but no preference was detectable within Experiments these short periods. In the first experiment we examined whether neonate larvae are The presence of the glv, (Z)-3-hexen-1-ol, in the ar- able to find their host plant stems by olfaction. Three tansy stems ena enhanced the larvae’s ability to differentiate be- tween host plant stems and dummies. At either low concentration (Fig. 2B) or high concentration (Fig. 2C) larvae distinguished significantly between host stems and stem dummies even after 5 min. In comparison, no such differentiation between tansy stems and dummies was observed after 5 and 10 min in the absence of addi- tional glv (Fig. 2A). When offered a choice of test dummies treated with (Z)-3-hexen-1-ol and dummies with solvent alone, the glv itself was not attractive to the larvae. At both glv concentrations neither neonate larvae nor 5-day-old larvae preferred to climb the treated stem dummies (Fig. 3).