Chem. Senses 27: 505-509,
2002
© Oxford University Press 2002
(-)-Germacrene D Increases Attraction and Oviposition by the Tobacco Budworm Moth Heliothis virescens
1 Norwegian University of Science and Technology, Department of Zoology, Neuroscience Unit, NO-7489 Trondheim, Norway 2 Royal Institute of Technology, Department of Chemistry, Organic Chemistry, Ecological Chemistry Group, SE-100 44 Stockholm, Sweden 3 Laboratory of Chemical Ecology, Institute of Ecology, Akademijos 2, LT-2600 Vilnius, Lithuania
Correspondence to be send to: Hanna Mustaparta, Norwegian University of Science and Technology, Department of Zoology, Neuroscience Unit, NO-7489 Trondheim, Norway. e-mail: hanna.mustaparta{at}chembio.ntnu.no
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Abstract |
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The sesquiterpene germacrene D (GD) activates a major type of olfactory receptor neuron on the antennae of the heliothine moths. In Heliothis virescens females, 80% of the recordings have shown activity of one neuron type responding with high sensitivity and selectivity to GD. With the aim of determining the behavioural significance of this sesquiterpene, we have used a two-choice wind-tunnel to study the preference of mated H. virescens females for host plants with and without (-)-GD added. Tobacco plants containing dispensers with low release rate of (-)-GD had a greater attractiveness than tobacco plants without this substance. In addition, a significant increase of oviposition was found on the plants with (-)-GD.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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Plants produce complex mixtures of volatile constituents, some of which are used as cues by insects to locate suitable hosts (Schoonhoven et al., 1998
). Females of the polyphagous tobacco budworm moth,
Heliothis virescens (F.) (Lepidoptera; Noctuidae; Heliothinae),
display positive anemotaxis to host-plant volatiles
(Tingle et al., 1990;
Mitchell et al.,
1991; Tingle and Mitchell,
1991), whereas caterpillar-induced plant volatiles, repel
conspecific females (De Moraes et
al., 2001). This species is found on a large number of
economically important plants, such as cotton (Gossypium hirsutum
L.), tobacco (Nicotiana tabacum L.), corn (Zea mays L.),
tomato (Lycopersicum esculentum Mill.), sunflower (Helianthus
annuus L.), as well as many wild plant species
(Matthews, 1991). To determine
which of the plant-produced compounds that are detected by insects, we used
gas chromatography linked to single cell recordings (GC—SCR) from
olfactory receptor neurons (ORNs) on the antennae of H. virescens
(Røstelien et al.,
2000a,b).
At least 17 types of ORNs, each of them specialized for a few structurally
similar plant compounds, have been found on the antennae of virgin females of
the tobacco budworm moth. One type of neuron appearing in 80% of all
recordings from ORNs (n > 80) responded with high sensitivity and
selectivity to germacrene D (GD; for structures, see
Figure 1)
(Røstelien et al.,
2000a). The large number of GD neurons on the antennae suggests
that the compound is of particular importance in the selection of plants for
nutrition and/or hosts for oviposition. The objective of the present study was
to determine the role of GD, either as an attractant or a repellent, in
host-plant location and oviposition by mated H. virescens
females.
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Material and methods |
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Insects
Heliothis virescens pupae were received weekly from a laboratory culture at Syngenta, Rosental, Switzerland. Pupae were sexed and kept in separate containers at constant conditions (22°C, 80-95% relative humidity, 14:10 h light: dark cycle, light phase starting at 8 p.m.). Every day, before onset of scotophase, the emerged females and males were placed in separate cylinders (diameter 10 cm, height 20 cm) with the tops covered by screens. The insects were provided with food (10% honey water) and pure water ad libitum. The following day, before onset of scotophase, adults were transferred to separate mating cylinders (one female and one male in each). Food and water were supplied and the insects were observed every 2 h under red light to confirm mating. The mated moths were sexed and the 3-day-old females were transferred to the wind-tunnel.
Plants
Seeds of tobacco were sown in the beginning of June and were grown indoors at room temperature and in natural daylight. The plants were used for the behavioural experiments at flowering stage; some plants had a few seed capsules. The tobacco plants contained no detectable amounts (<0.1 ng/µl) of GD when the GD receptor neurons were stimulated with fresh materials from the plants. This was the reason for selecting the tobacco plants for the behavioural experiments.
Chemicals
(-)-GD was isolated from an ylang-ylang (Cananga odorata Hook)
essential oil by MPLC as described previously
(Røstelien et al.,
2000a). Chemical purity of the compound exceeded 99.2% estimated
by gas chromatography using a DB-wax column (30 m, internal diameter 0.25 mm,
film thickness 0.25 µm). The enantiomeric purity of 99.9% was determined by
the use of a chiral column [25 m, internal diameter 0.25 mm, Heptakis
(6-0-t-butyldimethylsilyl-2,3-di-O-methyl)-ß-cyclodextrin
(50% in OV1701)] (Schmidt et al.,
1998; König et
al., 1999).
The dispensers (red rubber tubes) were loaded with 500 µg of (-)-GD
dissolved in 160 µl of hexane which was soaked into the walls from the
inside (18 x 30 mm). The rubber was left in a hood at room temperature
for 3 days to equilibrate the concentration inside the rubber and evaporate
the hexane. The tube was cut in six pieces and the release rate of (-)-GD from
all six parts was estimated by dynamic head-space (Porapak Q, 80-100 mesh,
99.999% nitrogen flow 20 ml/min) and gas chromatography (DB-wax column). The
dispensers were used in the tests when (-)-GD was released within the range of
80-300 ng/h. This amount was in a range matching the sensitivity of the GD
receptor neurons of H. virescens as measured by GC-SCR
(Røstelien et al.,
2000a). Control dispensers were loaded with 160 µl of hexane
only.
Two-choice wind-tunnel experiments
Behavioural tests were performed in a two-choice wind-tunnel (150 x
50 x 100 cm; Figure 2).
The lower parts (50 cm height) of the two largest walls and the floor were
made of Plexiglas, the upper parts of these walls as well as the end walls
were made of wire mesh and the ceiling of black mosquito net. Mesh size was
1.5 mm at the side walls and 2.5 mm at the end walls. Air was sucked through
the system by a tube (diameter 15.5 cm) connected to a fan. The inlet of the
tube was placed in the middle of the tunnel, 40 cm above the floor. The air
speed was 
15 cm/s, measured at the half distance between the tube and the
end walls. The flow rates and the form of the plumes were estimated by
introducing smoke obtained from TiCl4 (titanium tetra-chloride)
into the wind-tunnel. The airflow was mainly directed from the two ends of the
tunnel, with only weak flow from the sides. Two lamps (Osram Lumilux Plus Eco
L58W/21-830) placed 130 cm above the wind-tunnel provided some background
light in the room. To simulate dusk and provide uniform light allowing
observation without red light, a tent surrounding the wind-tunnel was placed
below the lamps.
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Two plants with similar total leaf area were placed in the tunnel, one in each end, 90 cm from each other and at the same distance from the air tube. A new pair of plants was used for each experiment that started at 10.00 a.m. Six rubber dispensers were uniformly placed on each plant, one plant with (-)-GD dispensers and the other with control dispensers. Two mated females were introduced into a release cylinder (diameter 10 cm, height 15 cm, made of wire mesh) fastened to a foot (10 cm). The cylinder was placed in the centre of the tunnel 15 cm below the air-tube. On the top, the cylinder had a platform (20 x 30 cm) with a centred opening for releasing the insects. The females were acclimated to the test conditions for 10 min before release. The observation of the behaviour started when the females left the platform. After the experiments, the females and the plants were removed from the experimental room, the wind tunnel was cleaned with hexane and the air-fan was turned to maximum capacity overnight. During the next day of experiment, the positions of the two plants [(-)-GD and control] were switched in the wind-tunnel. The data were taken in a protocol only from the females which demonstrated searching behaviour for a plant and/or visited both ends of the wind tunnel a few times. Females who stayed in a resting posture >20 min were not included.
The following experiments were conducted in the two-choice wind-tunnel with 3-day-old mated females.
Experiment 1: evaluation of the wind-tunnel
The preferences of the H. virescens females for one of the
wind-tunnel ends, each containing a single tobacco plant, were evaluated by
visually observing the behaviour for 1 h. The time (min) spent by the moths on
each side of the tunnel was measured. Eighteen females were tested, of which
six showed the searching behaviour.
Experiment 2: two-choice odour preference tests
The preference of the H. virescens females for a tobacco plant
with dispensers releasing (-)-GD versus a plant with control dispensers was
evaluated as in experiment 1. Twenty females were tested, of which eight
showed the searching behaviour. In all but two experiments, only one of the
two females introduced in the wind-tunnel showed active searching behaviour
toward the plants and was included in the protocol. In the two other
experiments, both females were included since both oriented toward the plants
without interfering with each other.
Experiment 3: oviposition preference tests
The oviposition preference of H. virescens females for a tobacco
plant with dispensers releasing (-)-GD versus a plant with control dispensers
was evaluated by allowing one of the two females tested in experiment 2 to
oviposit during the following scotophase. The number of eggs per plant was
counted. Six experiments were run.
Data analysis
The data were calculated as percentages and analysed with the Wilcoxon
matched pairs test (Sokal and Rohlf,
1995), using the computer program Statistica, for testing the
preference for control plant versus control plant (experiment 1) and the
preference for plant with GD versus control plant (experiments 2 and 3).
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Results and discussion |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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The females demonstrated active searching behaviour for the tobacco with (-)-GD dispensers by performing zigzag flight upwind towards the plant. They hovered for 2-4 s at a distance of a few centimetres in front of the leaves with the dispensers. They then landed on the leaf or hovered in front of another leaf. After a while, some females flew away from the plant and landed on the tunnel wall for a short time. Then they relocated the air stream and repeated the behaviour of upwind flight and hovering in front of the GD-enriched plant. Altogether, the behavioural tests (experiment 2) showed that mated 3-day-old H. virescens females spent a significantly longer time (74 ± 12% SD of the observed time, P < 0.02) in the side of the wind-tunnel with the tobacco plant containing the (-)-GD-releasing dispensers, as compared to the side with the control plant (Figure 3). In contrast, the females did not show preferences when presented with plants without GD dispensers, as described in experiment 1 (55 ± 17% SD of the observed time, P > 0.7; Figure 3). The greater attractiveness of the tobacco plant with dispensers releasing (-)-GD also resulted in an increase of the oviposition on these plants in all six experiments (Table 1). Most of the eggs (79 ± 15% SD, P < 0.03) were found on the (-)-GD-enriched plants (Figure 4). The presented data show that (-)-GD at low release rate, added to tobacco plants containing no GD, increases the attraction of gravid females to the plants. Whether (-)-GD acts synergistically with the volatiles released by the tobacco plant or acts alone as an attractant, remains to be tested. In addition, it will be interesting to determine whether (-)-GD also stimulates oviposition or if the increased number of eggs on the plant was a result of increased attraction to the plant.
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It is possible that (-)-GD is involved in one or in all of the three
contexts: location of food source; finding a host plant for oviposition; and
calling. The large number of very sensitive (-)-GD receptor neurons on the
female antennae indicates that (-)-GD can be detected at large distances,
being important in host-plant location. Furthermore, the presence of a
relatively large number of the same receptor neuron type on the male antennae
(S. Ulland and H. Mustaparta, unpublished data) suggests that (-)-GD acts as a
host-plant attractant for both sexes. This sesquiterpene is present in many of
the host plant species, but not in all, as with the tobacco plants
(Røstelien et al.,
2000a). Interestingly, it is the major component in leaf tissue
and one of the numerous volatiles in the flower tissue of the host plant
sunflower (Stranden et al.,
2002) (M. Ramirez and A.K. Borg-Karlson, unpublished data). These
findings indicate that (-)-GD might be involved in location of plants for
finding nectar, as well as host plants for mating and oviposition; this will
be studied in future experiments.
GD is a chiral compound present in several plant families, including
Asteraceae and Coniferaceae, host and non-host plants of H.
virescens, respectively. As in sunflowers, GD is common in leaves and is
also present in flowers of yarrow, pink (Dianthus spp.), mountain
tobacco (Arnica montana L.), goldenrod (Solidago virgaurea
L.), Canadian goldenrod (Solidago canadensis L.) and scented mayweed
(Matricaria recutita L.)
(Bülow, 1998) (J. Rohloff,
personal communication). The content of GD varies among plant species, from
pure (+)- or (-)-enantiomers (Figure
1) to the racemate, controlled by separate synthases, as shown in
Canadian goldenrod (Schmidt et
al., 1998). So far, it has been found that (-)-GD is the more
common of the two enantiomers. Pure (-)-GD (>99%) is found in the needles
of pine (Pinus L. spp.)
(Røstelien et al.,
2000a) and in the leaves of certain chemotypes of yarrow, as well
as in leaves and flower of sunflowers
(Stranden et al.,
2002) (M. Ramirez and A.K. Borg-Karlson, unpublished data). The
prominence of the (-)-enantiomer in the biological niche is also reflected in
the evolved specificity of the ORNs in heliothine moths. According to
GC—SCR, carried out in H. virescens as well as in
Helicoverpa armigera (Hubner) and Helicoverpa assulta
(Guenee), the ORNs responding to GD in these species belong to the same type,
all showing highest sensitivity for the (-)-form (M. Stranden, A.-K.
Borg-Karlson and H. Mustaparta, unpublished data). The dose—response
curves of the ORNs show an effect of (-)-GD 10 times higher than that of
(+)-GD (Stranden et al.,
2002). Thus, it is expected that both enantiomers of GD mediate
the same kind of message to these moth species. Furthermore, one can assume
that (-)-GD also acts as an attractant for the related species H.
armigera and H. assulta when present in the host plants.
Since tobacco plants are well known hosts of H. virescens, the
lack of GD in this species of plant, as shown in the GC—SCR studies of
heliothine moths, seems puzzling. It means that the presence of GD may not be
necessary for a plant to be used as a host by this species. Another
possibility is that the content of GD varies among strains of tobacco, as
shown for corn plants (M. Stranden and H. Mustaparta, unpublished data). On
the other hand, in the numerous analyses of volatiles produced by tobacco
plants, the presence of GD has not been reported
(Andersen et al.,
1988; Loughrin et al.,
1990). Other volatile constituents may certainly play an important
role in host location. Several components of plant volatiles—e.g.
E-ß-ocimene, E,E-
-farnesene,
(+)-linalool—in addition to GD, all activating particular types of ORNs
of heliothine moths, have been identified
(Røstelien et al.,
2000b) (M. Stranden, A.-K. Borg-Karlson and H. Mustaparta,
unpublished data). Obviously, a suitable host plant can be identified by the
ratio of the different volatile compounds released.
In another lepidopteran species, the codling moth Cydia pomonella
(L.) and in the two-spotted stinkbug Perillus bioculatus (F.),
antennal responses to GD have recently been reported by the use of GC linked
to electroantennogram recordings (GC—EAG)
(Weissbecker et al.,
2000; Bäckman et
al., 2001; Bengtsson
et al., 2001). Behavioural responses to GD have
previously been reported in only one lepidopteran species and two species of
beetles. For females of the pickleworm moths (Diaphania nitidalis
Stoll., Pyralidae), GD alone has been found to attract and increase
oviposition, but less effectively than the naturally produced leaf blend of
the host plant squash (Cucurbita pepo L.), containing 0.95% of GD
(Peterson et al.,
1994). Increased attraction to damaged, GD-enriched host plants
(Petasites paradoxus Retz., Adenostyles alliariae Gouan.)
has been found for the leaf beetle Oreina cacaliae (Schrank)
(Kalberer et al.,
2001). In contrast, a masking effect of (-)-GD on host attraction
is found for the cerambycid beetle Monochamus alternatus (Hope)
(Yamasaki et al.,
1997). Another interesting finding is the presence of (-)-GD in
volatiles collected from strawberry plants (Fragaria ananassa Duch),
on which pheromone producing strawberry blossom weevils (Anthonomus
rubi Herbst) were feeding (Innocenzi
et al., 2001). However, the behavioural effect of GD was
not studied in this case.
In conclusion, by the use of the two-choice wind-tunnel we have demonstrated that (-)-GD, released at low doses from dispensers placed on tobacco plants, increased attraction of and oviposition by mated females of the tobacco budworm moth H. virescens. Thus, the behavioural significance of the presence of a major olfactory receptor type with (-)-GD selectivity is demonstrated.
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Acknowledgments |
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The Norwegian Research Council (project No. 133958/420) provided the principal financial support for the project. We also acknowledge support from The Nordic Academy of Advanced Studies via a visiting professorship for A.-K.B.-K. (project no. 010434) and from the Swedish Institute (Visby programme). Professor Wilfried A. König (University of Hamburg, Germany) is gratefully acknowledged for providing the chiral column and the insect rearing team at Syngenta, Rosental, Switzerland for kindly providing the insect material. We thank Dr Robert Bielger for correcting the English.
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References |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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Accepted April 8, 2002
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