J Comp Physiol A (2003) 189: 563–577
DOI 10.1007/s00359-003-0434-y
O R I GI N A L P A P E R
M. Stranden Æ I. Liblikas Æ W. A. König
T. J. Almaas Æ A.-K. Borg-Karlson Æ H. Mustaparta
(–)-Germacrene D receptor neurones in three species
of heliothine moths: structure-activity relationships
Received: 13 January 2003 / Revised: 26 April 2003 / Accepted: 17 May 2003 / Published online: 25 June 2003

Springer-Verlag 2003
Abstract Specificity of olfactory receptor neurones plays
an important role in food and host preferences of a
species, and may have become conserved or changed in
the evolution of polyphagy and oligophagy. We have
identified a major type of plant odour receptor neurones
responding to the sesquiterpene germacrene D in three
species of heliothine moths, the polyphagous Heliothis
virescens and Helicoverpa armigera and the oligopha-
gous Helicoverpa assulta. The neurones respond with
high sensitivity and selectivity to (–)-germacrene D, as
demonstrated by screening via gas chromatography with
numerous mixtures of plant volatiles. Germacrene D
was present in both host and non-host plants, but only
in half of the tested species. The specificity of the neu-
rones was similar in the three species, as shown by the
‘‘secondary’’ responses to a few other sesquiterpenes.
The effect of (–)-germacrene D was about ten times
stronger than that of the (+)-enantiomer, which again
was about ten times stronger than that of (–)-a-ylangene.
Weaker effects were obtained for (+)-b-ylangene, (+)-
a-copaene, b-copaene and two unidentified sesquiterp-
enes. The structure-activity relationship shows that the
M. Stranden (&) Æ T. J. Almaas Æ A.-K. Borg-Karlson
H. Mustaparta
Neuroscience Unit, Department of Biology,
Norwegian University of Science and Technology,
7489 NO-Trondheim, Norway
E-mail: Marit.Stranden@bio.ntnu.no
Fax: +47-73-598294
I. Liblikas Æ A.-K. Borg-Karlson
Ecological Chemistry, Organic Chemistry,
Department of Chemistry,
Royal Institute of Technology,
SE-100 44 Stockholm, Sweden
I. Liblikas
Laboratory of Ecochemistry, Estonian Agricultural University,
Tartu EE-51005, Estonia
W. A. König
Institute of Organic Chemistry, University of Hamburg,
DE-20146 Hamburg, Germany
important properties of (–)-germacrene D in activating
the neurones are the ten-membered ring system and the
three double bonds acting as electron-rich centres, in
addition to the direction of the isopropyl-group
responsible for the different effects of the germacrene D
enantiomers.
Keywords GC-SCR Æ Germacrene D Æ Heliothine
moths Æ Olfactory receptor neurones Æ Sesquiterpenes
Abbreviations a-copaene: (tricyclo[4.4.0.0.]dec-3-ene,
1,3-dimethyl-8-(1-methylethyl)-) Æ b-copaene:
(tricyclo[4.4.0.0.]decane, 1-methyl-3-methylene-8-
(1-methylethyl)-) Æ GC: gas chromatograph Æ GC-MS:
linked gas chromatography mass spectrometry Æ
GC-SCR: linked gas chromatography single cell
recording Æ GD: germacrene D (1,6-cyclodecadiene,
1-methyl-5-methylene-8-(1-methylethyl-, E,E)) Æ
a-ylangene: (tricyclo[4.4.0.0]dec-3-ene, 1,3-dimethyl-8-
(1-methylethyl)-) Æ b-ylangene: (tricyclo[4.4.0.0.]decane,
1-methyl-3-methylene-8-(1-methylethyl)-)
Introduction
The sesquiterpene germacrene D (1,6-cyclodecadiene,
1-methyl-5-methylene-8-(1-methylethyl-, E,E)) (GD)
seems to play a significant role in host plant location
by females of the American tobacco budworm moth
Heliothis virescens (Fabricius) (Heliothinae; Lepidop-
tera; Noctuidae). Electrophysiological recordings from
single olfactory receptor neurones linked to gas chro-
matography and mass spectrometry have shown that a
major type of the antennal receptor neurones (80% of
the neurones recorded) respond with high sensitivity
and selectivity to GD (Røstelien et al. 2000a). The
behavioural significance of this sesquiterpene has been
studied in a two-choice wind-tunnel where mated
females could choose between a plant containing a low
release rate of (–)-GD dispensers and a plant with
564
control dispensers (Mozuraitis et al. 2002). An in-
creased attraction to and oviposition on plants with the
GD dispensers, suggested that GD has a positive effect
on mated H. virescens females, by acting either as an
attractant or as an attractant as well as a stimulant for
oviposition.
In the biosynthesis of many sesquiterpenes in plants,
e.g. copaenes and ylangenes, GD is an important
intermediate (Yoshihara et al. 1969; Cane 1990; Bülow
and König 2000). The two enantiomers of GD, formed
by separate enzymes (Schmidt et al. 1998), easily un-
dergo rearrangements, but the enantiomeric configura-
tion is kept as long as the chiral carbon atom is not
involved in the process (König et al. 1999). Many
plants contain both enantiomers, of which (–)-GD
seems to dominate in higher plants (Beechan et al.
1978; Niwa et al. 1980; Lorimer and Weavers 1987;
König et al. 1996; Bülow 1998; Schmidt et al. 1998,
1999; Bülow and König 2000; Stranden et al. 2002).
Interestingly, enantioselectivity has been demonstrated
for the GD receptor neurones in the Eurasian cotton
bollworm moth Helicoverpa armigera (Hübner) (He-
liothinae) (Stranden et al. 2002). Here, all GD neurones
respond with higher sensitivity to (–)-GD which has a
ten times stronger stimulatory effect than the (+)-
enantiomer.
The presence of significant numbers of GD receptor
neurones in H. virescens as well as in H. armigera raised
the question of whether this neurone type is present in
other heliothine moths and whether the receptors have
become conserved with respect to specificity. We here
present results on the GD receptor neurones in the three
heliothine species, H. virescens, H. armigera and the
oriental tobacco budworm moth Helicoverpa assulta
(Guenée). By the use of gas chromatography linked to
single cell recording (GC-SCR) and gas chromatography
linked to mass spectrometry (GC-MS), we have analysed
response characteristics of these neurones with respect to
selectivity for GD and the structurally-related rear-
rangement products, in addition to the enantioselectiv-
ity. Applications of tetramethylrhodamine dextran to
the sensilla were performed to explore the projections of
the GD receptor neurones in the antennal lobe of the
brain.
Materials and methods
Insects
Adult females of H. virescens, H. armigera and H. assulta used in
the present study originated from laboratory cultures at Syngenta,
Basel, Switzerland, the Volcani Centre, Bet Dagan, Israel and Seoul
National University, South Korea, respectively. Some H. armigera
and H. assulta were obtained from laboratory cultures at the
Chinese Academy of Sciences, Beijing, People’s Republic of China.
They arrived as pupae and were kept as previously described
(Røstelien et al. 2000b; Stranden et al. 2002). When eclosed, they
were placed in separate boxes marked with eclosing date and given
honey water and pure water ad libitum. Insects aged 2–10 days were
used in the experiments.
Test samples
The sesquiterpene GD and the ‘‘secondary’’ substances were tested
as constituents in extracts, headspace samples and essential oils of
plant material as well as in isolated fractions (Table 1). Chemical
standards and reference samples were also included in the experi-
ments and are listed in Table 2.
Extracts and headspace samples
The procedure of extraction and dynamic headspace sampling was
carried out as described by Stranden et al. (2002) and Røstelien
et al. (2000b). In principle, purified air surrounding the plant inside
a glass container was sucked (flow below 40 ml min–1) through a
glass tube filled with the adsorbent [Porapak Q (80/100 mesh, Su-
pelco), Tenax TA (60/80 mesh, Chrompack) or a mixture of both
(1:1)]. Freshly cut plant materials were also used. The trapped
volatiles were eluted [with n-hexane (>99%), ethyl acetate (abso-
lute) or a mix of both (1:1), respectively] and used as test samples
on the receptor neurones (stored at –25C). The air was purified
through a carbon filter mounted in front of the glass tube. The
suction period lasted from 2 h to 3 days. Before each sampling
procedure the glass tube with the adsorbent was cleaned by rinsing
with N2 flow (10–30 ml min–1) and by heating to 200C with a
temperature rise of 12C min–1. The tubes were kept at 200C for
several hours until the adsorbent was clean. Alternatively, the tubes
with adsorbent were cleaned by repeatedly filling with solvent,
dried by N2 flow and activated by heat (200C) under continuous
N2 flow. The rinsed tubes were used for headspace sampling within
a few days.
Fractions
Fractions were isolated from essential oils of cubebe pepper, ylang-
ylang, orange and commercial TMP-turpentine by several parallel
series of medium pressure liquid chromatography (MPLC). The
procedure is described by Røstelien et al. (2000a) and Stranden
et al. (2002). Each separation of the polar part of the oils resulted
in about 150 fractions, which were analysed by GC-MS and pooled
with regard to the substances contained in each fraction, e.g. 22
fractions resulting from the orange oil. The fractions were used for
retesting the GD neurones in the GC-SCR experiments. Fractions
containing the largest amount of the active substances were further
separated for isolation. (–)-GD (97.5%) was isolated from the or-
ange oil by liquid chromatography on silica gel (SDS 60 A C.C 40–
63 lm) and eluted with hexane. By thermal treatment, (–)-GD
completely rearranged to a mixture of five major sesquiterpene
hydrocarbon constituents as described by Bülow and König (2000),
by heating up to 230C for 3 h in 10% hexane solution in a sealed
tube. The resulting solution was pre-fractionated by MPLC eluting
with hexane, giving a fraction of (+)-b-ylangene [(+)-(tricy-
clo[4.4.0.0.]decane, 1-methyl-3-methylene-8-(1-methylethyl)-)] and
(–)-b-copaene [(–)-(tricyclo[4.4.0.0.]decane, 1-methyl-3-methylene-
8-(1-methylethyl)-)]. Isolation of (+)-b-ylangene was carried out
using preparative GC on Pye Unicam GCV instrument, equipped
with 5.0 m · 8 mm glass column packed with 10% Carbowax
20 M on 0.125–0.160 mm Inerton AW (120 ml min–1 helium,
200C injector port temperature, 160C FID detector and 155C
column temperature). The isolated (+)-b-ylangene (99.5%) ap-
peared as one peak (i.e. enantiomerically pure) when tested on the
chiral column III (specifications given below).
Gas chromatography linked to single cell recording
The insect preparation and the electrophysiological recordings of
nerve impulses from single olfactory receptor neurones on the
antennae were carried out as described previously (Røstelien et al.
2000a; Stranden et al. 2002). Contact with single neurones was
made with a tungsten microelectrode positioned into the base of the
 565

Table 1 Materials of different plant species used for stimulating the receptor neurones of heliothine moths. The sampling methods and
sources are indicated. The materials found to contain germacrene D are underlined

Plant material Sampling method (no. of samples) Source

Basil (Ocimum basilicum L.) Essential oil NMD (‘‘Norsk Medisinaldepot AS’’)
Bean (Phaseulos sp.) Headspace (2)
Canadian goldenrod (Solidago canadensis L.) Pentane extracts (3) Stranden et al. (2002)
Carrot (Daucus carota L.) Headspace(2)
Chilli pepper (Capsicum sp.) Headspace (12)
Cinnamon (Cinnamomum zeylanicum L.) Essential oil NMD
Clove (Syzygium aromaticum L.) Essential oil NMD
Clove bud (Syzygium aromaticum L.) Essential oil Aqua oleum
Commercial TMP turpentine Essential oil
Commercial TMP turpentine Fractions (5)
Cotton (Gossypium sp.) Headspace (16)
Cotton induced (Gossypium herbaceum L.) Headspace of plant with larvae S. Gouinguené and T. Turlings,
(Spodoptera littoralis B.) University of Neuchâtel, Switzerland
Cowpea induced (Vigna unguiculata L.) Headspace of plant with larvae S. Gouinguené and T. Turlings,
(Spodoptera littoralis B.) University of Neuchâtel, Switzerland
Cubebe pepper (Piper cubeba L.) Essential oil Dragoco
Cubebe pepper (Piper cubeba L.) Fraction (2) Røstelien et al. (2000a)
Cubebe pepper (Piper cubeba L.) Fraction Stranden et al. (2002)
Eastern red cedar (Juniperus virginiana L.) Essential oil F. Pagula, University of Maputo,
Mozambique
Fennel (Foeniculum vulgare Mill.) Essential oil NMD
Ginger (Zingiber officinale Rosc.) Hexane extract Stranden et al. (2002)
Hibiscus sp. Headspace (4)
Lemon (Citrus sp.) Headspace (2)
Lemon geranium (Pelargonium crispum L.) Headspace (2)
Lemon geranium (Pelargonium crispum L.) Pentane extract
Maize (Zea mays L.) Headspace (5)
Maize induced (Zea mays mays L., Delprim) Headspace of plant with larvae S. Gouinguené and T. Turlings,
(Spodoptera littoralis B.) University of Neuchâtel, Switzerland
Maritime pine (Pinus pinaster Aiton) Headspace Bichão et al. (2003)
Norway spruce (Picea abies L. ) Headspace Wibe and Mustaparta (1996)
Orange (Citrussp.) Headspace (8)
Orange (Citrussp.) Essential oil R.C. Treatt, germacrene D fraction
containing 40% germacrene D
Orange (Citrussp.) Fractions (22)
Paprika (Capsicum sp.) Headspace (2)
Peppermint (Mentha piperita L.) Essential oil NMD
Savin juniper (Juniper sabina) Essential oil F. Pagula, University of Maputo,
Mozambique
Sunflower (Helianthus annuus L.) Headspace (10)
Sunflower (Helianthus annuus L.) Pentane extracts (2) Stranden et al. (2002)
Tobacco (Nicotiana tabacum L.) Headspace (18)
Tomato (Lycopersicon esculentum Mill.) Headspace (4)
Wild briar (Rosea dumalis Bechst.) Headspace (15)
Yarrow (Achillea millefolium L.) Pentane extract Stranden et al. (2002)
Ylang-ylang (Cananga odorata Hook) Essential oils (2) Firmenich and Dragoco
Ylang-ylang fractions Fractions (8) A.-K. Borg-Karlson
Ylang-ylang fraction I with a- and b-ylangene Fraction Sina Escher, Firmenich, Switzerland
Ylang-ylang fraction II without a- and b-ylangene Fraction Sina Escher, Firmenich, Switzerland

sensillum. The neurone was then tested for sensitivity to GD by
direct stimulation with the different mixtures, i.e. by blowing air
(3.3 ml s–1) through a cartridge containing a small sample of each
solution on filter paper. When the cell responded, the selectivity
was tested by injection of the various mixtures into the GC-column.
A split at the end of the column led half of the effluent to the GC-
detector and the other half over the insect antenna (100 ml min–1),
resulting in simultaneously recorded gas chromatograms and re-
sponses to the separated compounds. Two columns in parallel in
the GC allowed the neurone to be tested for the same mixture with
two different separation-patterns. Spike activity was recorded on
and analysed in the computer program Spike 2 (Cambridge Elec-
tronic Design Limited, Cambridge, Great Britain). It means that
the amplitude and waveform of all spikes in the recording were
compared, to find out whether they belonged to one or more
populations (templates). Some recordings of activity were made by
analogue tape recorder.
The following five column types were used: polar DB-wax
columns (J&W, 30 m, i.d. 0.25 mm, film thickness 0.25 lm), one
non-polar DB-5 column (J&W, 30 m, i.d. 0.25 mm, film thickness
0.25 lm) and three chiral columns; Cyclodex-b (30 m, i.d.
0.25 mm, film thickness 0.25 lm) (column I), heptakis-(6-O-t-bu-
tyldimethylsilyl-2, 3-di-O-methyl)-b-cyclodextrin (50% in OV1701,
25 m, i.d. 0.25 mm) (column II) (Schmidt et al. 1998; König et al.
1999) and octakis-(6-O-methyl-2,3-di-O-pentyl)-c-cyclodextrin
(80% in OV1701, 25 m, i.d. 0.25 mm) (column III) (König et al.
1990). In all experiments, one DB-wax column was used in parallel
with one of the other column types. Separations in the non-chiral
566
Table 2 Chemicals used in
standards and as reference Compound(s)/(purity)
samples in retesting the receptor
neurones of heliothine moths a-Bulnesene (84%)
(–)-ß-Caryophyllene (99%)
(–)-a-Copaene (95%) [containing 24% of the (+)-enantiomer
and 2% of (–)-a-ylangene]
b-Copaene
a-Cubebene
(+)-Cyclosativene (>99%)
b-Elemene
(+)-Germacrene D (10 lg/ll, 86%, 79% ee)
(–)-Germacrene D (0.1 lg/ll, 75%, 76% ee)
a-Guajene (94,3%)
a-Humulene=a-caryophyllene (>95%)
(+)-Longicyclene (97%)
(+)-Longifolene (>99%)
(+)-Longipinene (>99%)
a-Muurolene (95.8%)
c-Muurolene (54.5%)
(–)-a-ylangene [1 lg/ll of each compound, (–)-a-ylangene 22.6%,
(–)-a-copaene 36.7%, (+)-a-copaene 19.3%
and (+)-a-ylangene 20.5%]
columns were performed from the initial temperature 80C with an
increase rate of 6C min–1 to 180C, and a further increase rate of
15C min–1 to 220C. The enantiomeric separation of GD for the
chiral columns I, II and III was found to be optimal at the iso-
thermal temperature 130C, 125C and 110C, respectively. The
optimum enantiomeric separation of a-ylangene (tricy-
clo[4.4.0.0]dec-3-ene, 1,3-dimethyl-8-(1-methylethyl)-) and a-cop-
aene (tricyclo[4.4.0.0.]dec-3-ene, 1,3-dimethyl-8-(1-methylethyl)-)
was found to be isothermal at 70C for the chiral column II (no
baseline separation) and 85C for the chiral column III. Fraction I
and II of the orange oil were separated in the chiral column III at
the isothermal temperature 100C and 85C, respectively.
Direct stimulation
Direct stimulation with different concentrations of the reference
samples of the enantiomers was performed by blowing an air
stream (3.3 ml s–1) through the test cartridges and over the insect
antenna (Stranden et al. 2002). The sequence of stimulation was
from low to high concentrations, by alternating the tests of (–)-a-
ylangene, (+)-GD and (–)-GD. Each cartridge was tested twice in
the dynamic area. Purified air was blown over the antenna
(100 ml min–1) in the inter-stimulus intervals (1 min at low con-
centrations and a longer period at high concentrations, depending
on the response strength). Recordings and analysis of spike activity
was performed in the computer program Spike 2. To obtain dose-
response relationships, the response strength was plotted as num-
bers of spikes per 0.5 s for each concentration. The test samples
were diluted in hexane (>99%) in decadic steps down to 1 ng ll–1
for a-ylangene, down to 10 pg ll–1 for (+)-GD and down to
0.01 pg ll–1 for (–)-GD. Test cartridges with the enantiomers were
made by inserting a piece of filter paper in each tube on which 1 ll
of a dilution was applied. (–)-a-Copaene and (–)-b-caryophyllene
(Table 2) were used as standards to determine the concentrations of
a-ylangene and GD enantiomers, respectively.
GC-MS analyses
The active compounds were identified by GC-MS using a Finnigan
SSQ 7000 MS instrument linked to a Varian 3400 GC. Two of the
columns used in the GC-SCR experiments (DB-5 and chiral col-
umn III) were installed in the GC-MS to identify the active plant
volatiles and the active constituents in the different fractions.
Source
Hartlieb and Rembold (1996)
Fluka
Fluka
A.-K. Borg-Karlson
A.-K. Borg-Karlson
Fluka
A.-K. Borg-Karlson
Stranden et al. (2002)
Stranden et al. (2002)
Hartlieb and Rembold (1996)
Hartlieb and Rembold (1996)
Fluka
Fluka
Fluka
Hartlieb and Rembold (1996)
Hartlieb and Rembold (1996)
Bülow and König (2000)
Several DB-wax columns were used for the separation in GC-SCR
and GC-MS, each specimen with the same specification (given
above).
Staining of receptor neurones and confocal laser scanning
microscope
The electrophysiological recordings were followed by applying te-
tramethylrhodamine dextran (MW 3,000; Molecular Probes) to the
sensillum base in order to stain the neurone and visualise the
projection pattern in the antennal lobe. Fresh stain was prepared
by dissolving crystals in saliva until an intense purple colour was
obtained. A piercing action by the tungsten electrode was per-
formed to rupture the cell, in some cases resulting in strong firing
and death of the cell. A drop of the staining solution was positioned
at the recording site, allowing dye to enter the sensillum lymph
through the punctured cuticular wall at the base of the sensillum.
The preparation was kept humid in the dark (4C for a minimum of
3 days) allowing the dye to completely fill the cells by diffusion.
After the staining period the brain was dissected, fixed (2.5%
formaldehyde/0.135% glutaraldehyde in 0.1 mol l–1 phosphate
buffer, pH 7.4) and prepared for confocal laser scanning
c
Fig. 1 A Gas chromatogram of headspace volatiles of sunflower
(Helianthus annuus) separated in the polar (left) and the non-polar
GC column (right). Below is the simultaneously recorded activity in
a single receptor neurone (impulses s–1) of a female Helicoverpa
armigera. The neurone responded selectively to the sesquiterpene
(–)-germacrene D. B Gas chromatogram of a germacrene D
reference sample [90% (+)-germacrene D] separated in the chiral
column II and the activity of three neurones in females of H.
virescens, H. assulta and H. armigera, respectively. C Gas
chromatogram of an essential oil of ylang-ylang (Cananga odorata)
separated in the polar column and recorded activity of two
germacrene D neurones in H. virescens; neurone 1 (low sensitivity)
and neurone 2 (high sensitivity, same neurone as in B). For neurone
2 strongest response to (–)-germacrene D and weaker responses to
six other sesquiterpenes are shown; a-ylangene (1), a-copaene (2),
an unknown sesquiterpene (3) eluted in the end of linalool,
b-ylangene (4), b-copaene (5) and another unknown sesquiterpene (6)
eluted in the end of a-humulene. Below the waveforms of all spikes of
neurone 2 recorded in the selected period are presented as an overlay
(·3160 amplification), showing that they belong to one template
567
568

microscopy (Zeiss LSM 510). To ensure tissue transparency,
dehydration by a graded series of ethanol baths was followed by
immersing the brain into methyl salicylate (99% Merck). Stacks of
optical sections were analysed by one software (Zeiss LSM Image
Examiner v. 2.80.1123), and further processed with AMIRA (2.3,
TGS Europe S.A., France) in order to visualise selected structures
and projection patterns of olfactory receptor neurones in 3D.
Results
Out of 128 recorded receptor neurones responding to
plant volatiles in female heliothine moths, 69 (53%)
were classified as (–)-GD neurones. In the three species
the numbers were as follows; 37 of 64 (57%) recorded
neurones in H. virescens, 28 of 50 (56%) in H. armigera
and 4 of 14 (28.5%) in H. assulta. All recordings with
GD neurones appeared with spikes belonging to one
template amplitude and waveform when analysed
(Fig. 1C). The classification is based on results from
436 experiments by GC-SCR, and included one to 37
recordings from each neurone (Table 3). By screening
the samples of the various plant materials (Table 2),
GD was detected in about half of the species, both in
hosts (like sunflower and tomato for H. virescens and
H. armigera) and non-hosts (like Norway spruce), lis-
ted in Table 4. Twelve of these 16 plant species con-
taining GD were tested on the receptor neurones via
chiral columns. All showed the presence of (–)-GD
whereas the (+)-enantiomer was found in only three of
them (Canadian goldenrod, cubebe pepper and ginger).
Canadian goldenrod showed individual variations of
the enantiomeric ratio. Another interesting difference
was found for two strains of cotton and maize. In both
cases the induced samples (plants with larvae) of
one strain showed the presence of minor amounts of
(–)-GD whereas the sample of the other strain showed
no GD.
All neurones showed consistent responses to GD, i.e.
increased firing rate with the rise of the GC peak and a
slow decay during the decline of the peak resulting in
persistent firing after the peak. An example in Fig. 1A
shows a receptor neurone of H. armigera stimulated with
headspace volatiles of sunflower, both via a polar and a
non-polar GC column. The enantioselectivity, tested by
separation of GD on the chiral column II, showed that
the GD neurones in the three species responded to both
enantiomers, and significantly better to (–)-GD. As
shown in Fig. 1B, the response to (–)-GD at ten times
lower dose was stronger than to the (+)-enantiomer.
The sensitivity of the GD neurones varied, which
appeared by the different response strength to the same
concentration of (–)-GD, exemplified by neurones 1 and
2 in Fig. 1C. The neurones with low sensitivity showed
responses only to GD (neurone 1), whereas the high
sensitivity neurones responded to six other components
(appearing in the tests of ylang-ylang essential oil) in
addition to GD that elicited a much stronger response.
The two compounds eluting first and eliciting ‘‘second-
ary’’ responses, were found to be a-ylangene and
569
a-copaene when re-tested with the reference samples.
One reference sample contained both enantiomers of the
two compounds. The other, commercial (–)-a-copaene,
contained both enantiomers of a-copaene as well as 2%
(–)-a-ylangene. Separation of the two pairs of enantio-
mers on the chiral column III showed that (–)-a-ylan-
gene and (+)-a-copaene activated the neurone, whereas
the corresponding enantiomers had no effect (Fig. 2A).
The stronger effect of (–)-a-ylangene than of (+)-a-
copaene is shown in Fig. 2A, B. Stimulation with the
same amount of the two compounds elicited a marked
stronger response to (–)-a-ylangene (Fig. 2A). Thus, this
standard was used to test dose-response relationships for
(–)-a-ylangene, considering the effect of (+)-a-copaene
in the mixture to be less important. Figure 3 shows the
dose-response curves determined for a GD neurone in
H. virescens for (–)-GD, (+)-GD and (–)-a-ylangene.
The shifts of the curves show that the effect of (–)-GD
was about ten times stronger than the (+)-enantiomer,
which again was about ten times stronger than (–)-a-
ylangene. This was also demonstrated by the threshold
concentrations of the three compounds; below 1 ng for
(–)-GD, 10 ng for (+)-GD and 0.1 lg for (–)-a-ylan-
gene.
The other compounds eliciting ‘‘secondary’’
responses were identified as sesquiterpenes according to
mass spectra. However, two of them, nos. 3 and 6 in
Fig. 1C, could not be further identified because of the
lack of reference material. No. 3 co-eluted with, and was
masked by the monoterpene linalool that was not active.
The sesquiterpene no. 4 was identified as b-ylangene by
its typical mass spectrum that was obtained in the or-
ange oil fraction I (Fig. 4C). This fraction, separated in
the polar and the chiral column III, elicited a significant
response to the b-ylangene peak eluting before (–)-GD
that also elicited a response at the minute amount
present (Fig. 4A). The GC-MS analyses showed that
both enantiomers of b-ylangene were present in the
sample. However, only the latest eluted enantiomer
elicited a response. By co-injection of the reference
sample (+)-b-ylangene (obtained by thermal degrada-
tion of (–)-GD) and orange oil fraction I, the active
enantiomer was found to be (+)-b-ylangene. Confir-
mation of the identification of b-ylangene as the active
component was obtained by retesting with the ylang-
ylang fraction known to contain a- and b-ylangene. The
fifth active sesquiterpene, only present in the ylang-ylang
oil, was identified by GC-MS as b-copaene. The ses-
quiterpene no. 6 was present in both the ylang-ylang oil
and in the orange oil fraction II (Fig. 4B). In the ylang-
ylang oil it eluted from the polar column during the end
of the a-humulene peak. In the orange oil fraction II,
compound no. 6 eluted between the minor amounts of
b-ylangene and GD, all three eliciting responses. When
separating the orange oil fraction II on chiral column
III, two active compounds appeared in addition to
b-ylangene and (–)-GD (Fig. 4B), suggesting the pres-
ence of two enantiomers of no. 6. A mass spectrum
could only be obtained for the first-eluted component
570

Table 3 Number of neurones (N) recorded in each species (H. vi- a number of neurones (n) are given. Stimulation method by five
rescens, H. armigera and H. assulta) includes previously reported different GC column types as well as direct stimulation is indicated.
results by Stranden et al. (2002, indicated by an asterisk). The total Polar column=DB-wax, non-polar column=DB5 and chiral
numbers of responses (r) to each activating compound obtained in columns I, II and III

Activating compound H. virescens H. armigera H. assulta Stimulation method

(N=37) (N=28) (N=4)

Germacrene D r=107 n=37 r=94 n=28 r=13 n=4 Polar and non-polar column
())-Germacrene D r=50 n=19 r=29 n=8* r=4 n=2 Chiral column I, II, III
())-Germacrene D r=22 n=4 r=15 n=2* r=4 n=1 Direct stimulation
(+)-Germacrene D r=37 n=17 r=15 n=7* r=3 n=2 Chiral column I, II, III
(+)-Germacrene D r=27 n=4 r=12 n=2* r=7 n=1 Direct stimulation
a-Ylangene r=50 n=19 r=32 n=19 r=5 n=3 Polar and non-polar column
())-a-Ylangene r=7 n=4 – – Chiral column II, III
())-a-Ylangene r=18 n=3 – r=3 n=1 Direct stimulation
b-Ylangene r=45 n=19 r=9 n=5 r=5 n=2 Polar and non-polar column
(+)-b-Ylangene r=3 n=2 r=5 n=1 – Chiral column II, III
Sesquiterpene no. 6 r=28 n=13 r=8 n=2 r=4 n=2 Polar column
a-Copaene r=11 n=4 – – Polar column
(+)-a-Copaene r=5 n=4 – – Chiral column II, III
b-Copaene r=9 n=7 – – Polar column
Sesquiterpene no. 3 r=3 n=3 – – Polar column

Table 4 Plant materials found to contain germacrene D and ‘‘secondary’’ substances by linked gas chromatography single-cell recording
(GC-SCR) in heliothine moths. The enantiomeric composition is given for the plant materials tested on the receptor neurones via chiral
GC columns

Plant materials containing germacrene D Enantiomers present ‘‘Secondary’’ substances present

Canadian goldenrod (Solidago canadensis L.) (+) and ()) individual 0

proportions
Commercial TMP turpentine ()) ())-a-ylangene
Cotton induced (Gossypium herbaceum L.) ())* 0
Cubebe pepper (Piper cubeba L.) (+) 35%, ()) 65% 0
Eastern red cedar (Juniperus virginiana L.) Not tested 0
Ginger (Zingiber officinale Rosc.) (+) 85%, ()) 15% 0
Lemon geranium (Pelargonium crispum L.) Not tested 0
Maize induced (Zea mays mays L., Delprim) ())* 0
Maritime pine (Pinus pinaster Aiton) Not tested 0
Norway spruce (Picea abies L.) ()) ())-a-ylangene
Orange (Citrus sp.) ()) ())-a-ylangene, (+)-b-ylangene,
sesquiterpene no. 6
Sunflower (Helianthus annuus L.) ()) 0
Tomato (Lycopersicon esculentum Mill.) Not tested 0
Wild briar (Rosea dumalis Bechst.) ())* 0
Yarrow (Achillea millefolium L.) ())* 0
Ylang-ylang (Cananga odorata Hook.) ()) ())-a-ylangene, (+)-a-copaene, sesquiterpene no. 3,
b-ylangene, b-copaene, sesquiterpene no. 6

*No detectable amount of the (+)-enantiomer

(Fig. 4D), as the amount of the second compound was
below the detection limit.
Altogether the (–)-GD receptor neurone type showed
high selectivity, by responding to (–)-GD and a few of
the structurally similar sesquiterpenes present among the
hundreds of compounds in the plant material tested. The
structure of (–)-GD in four different conformations, and
the four ‘‘secondary’’ substances identified are shown in
Fig. 5A, B, together with (–)-a-copaene which has no
effect. All seven compounds activating the GD receptor
neurone type were clearly demonstrated electrophysio-
logically in H. virescens. Shortage of individuals of H.
assulta and H. armigera limited testing of the ‘‘second-
ary’’ response characteristics. However, as shown in
Table 3, GD neurones in all three species showed
responses to (–)-a-ylangene, b-ylangene and the sesqui-
terpene no. 6 in addition to GD when stimulated with
the ylang-ylang essential oil and fractions of the orange
c
Fig. 2 A Gas chromatogram of a standard [called (–)-a-ylangene in
Table 2] containing (–)-a-ylangene, (–)-a-copaene, (+)-a-copaene
and (+)-a-ylangene separated in the chiral column III and the
activity of a receptor neurone in a H. virescens female. Bond-line
structures of the active compounds are shown. B Gas chromato-
grams of two concentrations of commercial (–)-a-copaene [(+)-a-
copaene (24%) and (–)-a-ylangene (2%)] injected in the polar
column and the activity of the same neurone in H. virescens as in
Fig. 1B. Responses are elicited only by the (+)-enantiomer of a-
copaene
571
572
Fig. 3 Dose-response curves of a receptor neurone in a H. virescens
female responding to (–)-germacrene D, (+)-germacrene D and (–)
-a-ylangene. The curves show the mean of two responses elicited by
repeated stimulation with the same cartridge [only one stimulation
with 0.1 lg (–)-germacrene D]. The shaded area marks the
spontaneous activity of the receptor neurone
oil. Thus, the ranking of active compounds was the same
in all three species.
Application of tetramethylrhodamine dextran to the
sensillum base after recording of the GD neurones were
made in seven recordings, in order to find out whether
this procedure would result in selectively stained
glomeruli of receptor neurones located in one sensillum.
No mass staining was obtained. One preparation of
H. assulta was successful, showing four selectively
stained axon terminals in the antennal lobe (Fig. 6A). In
the reconstructed antennal lobe, each of the stained
terminals was located in one glomerulus, three of them
in glomeruli close to the entrance of the antennal nerve
(Fig. 6B). They appeared at a depth of 62.7, 56.8 and
56.8 lm, respectively, from the first slice, and extended
over 9.8, 23.5 and 27.4 lm, respectively. The fourth
stained axon terminal was in the ventro-medial antennal
lobe (depth 52.9 lm, extending 27.4 lm). The axon
terminal located in the most medial glomerulus at the
entrance of the antennal nerve, was stained more
strongly, filling a larger part of the glomerulus than the
others (Fig. 6B). A complete reconstruction of the
antennal lobe was not possible, since the borderlines
between the glomeruli could not be distinguished
throughout the whole lobe.
Discussion
The present results on the structure-activity relation-
ships of the (–)-GD receptor neurones, a major olfactory
neurone type in heliothine moths, contributes to the
understanding of two functional aspects of olfactory
receptor neurones. First of all, it provided insight into
the molecular properties that are important in the
interaction between a biologically relevant plant odorant
and a specific receptor neurone. Secondly, it demon-
strates the functional similarity of a particular type of
plant odour receptor neurone in related species of he-
liothine moths (H. virescens, H. assulta and H. armi-
gera). The sensitivity and selectivity of the neurones to
GD classified them as a distinct group (Fig. 1A). The
uniform spike amplitude and waveform indicated that
the responses originated from single neurones in each
recording. This is further supported by the correlation
between response strength to GD and the number of
secondary responses to the other compounds. The con-
sistently stronger enantiomeric effect, by a factor of 10,
of (–)- versus (+)-GD (Fig. 1B), suggests that all of the
GD neurones obtained in this study might be function-
ally similar, i.e. possessing the same type of receptor
proteins in the three species. Several parts of the mole-
cule may interact with the receptor and cause the dif-
ference in stimulatory effect. The different positions of
the isopropyl group in the enantiomers of the GD
molecule may prevent the (+)-enantiomer to fit opti-
mally into the receptor (Fig. 5A). This has previously
been suggested in the study of the GD neurones in
H. armigera (Stranden et al. 2002). Further evidence for
functional similarity of the neurones is provided by the
tests with ylang-ylang oil, where responses to the six
‘‘secondary’’ compounds showed the same ranking with
respect to activation; (–)-a-ylangene, (+)-b-ylangene
and sesquiterpene no. 6 having stronger effect than (+)-
a-copaene, b-copaene and sesquiterpene no. 3 (Fig. 1C).
This infers that the ten-membered ring system together
with the three double bonds acting as electron-rich
centres (Fig. 5B) are important for the molecule to fit
optimally into the receptor. There exist at least four
possible conformations of the ring, which may undergo
transformations at high temperatures. However, at
biological temperatures the flexibility of the ring system
is not known. The low effect of the tricyclic ylangenes
and the copaenes, may be due to the more rigid struc-
tures by the four-membered ring locking the two other
rings in perpendicular planes, as well as the presence of
only one double bond (Fig. 5A). It is difficult to inter-
pret the different effect of the enantiomers of copaene
and ylangene structures. To fully understand the stim-
ulatory effect of the active molecules, we need to know
the structure of the receptor protein including the amino
acids forming the receptor. It is interesting that the
c
Fig. 4 A Gas chromatogram of fraction I of orange (Citrus sp.)
essential oil separated in the polar and the chiral column III and the
activity of a single receptor neurone in H. virescens (same neurone
as Fig. 1B). The asterisk indicates the concentration below the
detection limit of the GC column (0.1 ng for the polar and 1 ng for
the chiral column). B Gas chromatogram of fraction II of orange
essential oil separated in the polar and the chiral column III and the
activity of a receptor neurone in H. virescens (same neurone as
Fig. 1B). C Mass spectrum of b-ylangene originating from orange
essential oil fraction I. m/z indicates the ratio of mass to the
number of charges on ions employed. D Mass spectrum of the
unidentified sesquiterpene no. 6 originating from orange essential
oil fraction II
573
574

Fig. 5 A Structures of five

compounds (two different
rotations) activating the (–)-
germacrene D receptor
neurones and of (–)-a-copaene,
having no stimulatory effect.
The approximate activity
threshold for each compound is
given. Dark lines behind the
plane. B Structures of (–)-
germacrene D in four different
conformations, indicating the
flexibility of the ten-carbon ring

Fig. 6A, B A frontal view of the antennal lobe (medial side to the
right) of a H. assulta female after applying tetramethylrhodamine
dextran to the base of a sensillum with a (–)-germacrene D receptor
neurone. Section in confocal laser scanning microscope (depth
66.8 lm from the frontal slice) (A) and 3D reconstruction of the
antennal lobe in Amira (B) showing the stained axon terminals in
the four glomeruli
identified compounds with ‘‘secondary’’ effect on the
GD neurones are rearrangement products of GD, as
shown in laboratory experiments. In acidic solution GD
easily rearranges to, for example, a-ylangene, a-copaene
(König et al. 1999) and thermally it rearranges to, for
example, b-ylangene, b-copaene (Bülow and König
2000). Formations of the copaenes and ylangenes can be
explained by rearrangements for the two conformations
of ())-GD with crossed double bonds (Fig. 5B).
The considerations made here are based on the indi-
cations from studies of insects as well as vertebrates that
one olfactory receptor neurone possesses one type of
membrane receptor (Bozza et al. 2002; reviewed by
Vosshall et al. 2000; Mombaerts 1999). Whether each
neurone expresses a single or several subtypes of a
receptor protein has still to be verified in molecular
biological studies. Interestingly, in H. virescens antennae
seven types of putative receptor proteins have been
identified, of which one type was expressed in a partic-
ularly large number in both females and males, and
575
among the identified receptors no co-expression in single
neurones was found (Krieger et al. 2002). We speculate
as to whether the type expressed in a particularly large
number could be the (–)-GD receptor protein. The
varying sensitivity of the (–)-GD neurones within a
species seen in the present study (e.g. in H. virescens,
from which most recordings were made) (Fig. 1C), may
reflect the number of receptor proteins expressed in each
neurone. The correlation between higher sensitivity and
increased number of ‘‘secondary’’ responses with similar
ranking of effects, supports the presence of a single type
of receptor protein in the (–)-GD neurones of the he-
liothine moths. The different sensitivity of the (–)-GD
receptor neurones explains why a- and b-ylangene and a-
and b-copaene were found to be inactive in this neurone
type in a previous study (Røstelien et al. 2000a). The
biological importance of different sensitivity of neurones
belonging to one type might be ascribed to detection of
concentrations. Recruiting less sensitive neurones would
be an efficient way to detect higher concentrations of a
particular odorant, in addition to the increased firing
rate of each neurone. This has also been discussed for
pheromone receptor neurones in heliothine moths
(Mustaparta 2002).
The ‘‘secondary’’ responses to plant odorants raise
the question of whether these odorants play a significant
role in host finding. The high sensitivity and large
number of the neurones indicates that the moths detect
(–)-GD over large distances. One may argue that the less
effective sesquiterpenes (Fig. 3) may not be detected in
nature. However, when the insect is sitting on the plant,
the concentration of the compounds may be high en-
ough for detection. On the other hand, most of the
‘‘secondary’’ components were not found in the host
plants, but in the non-hosts ylang-ylang, Norway spruce
and orange (Table 4). Thus, it is possible that these
compounds do not play a significant role in the host
location. The behavioural effect of these components has
not been studied. Our hypothesis is that high concen-
trations of these compounds may elicit the same
behaviour as (–)-GD. Attraction to (–)-GD is only
shown for mated females of H. virescens (Mozuraitis et
al. 2002). Interesting questions are whether it elicits the
same behaviour in the two other species, i.e. whether it
has the same effect on the behaviour of the oligophagous
H. assulta as compared to the polyphagous H. virescens
and H. armigera. Since (–)-GD is widely distributed in
higher plants, including typical hosts and non-hosts of
heliothine moths, additional constituents are obviously
important in host location. So far, most is known about
hosts and non-hosts for important crop plants (e.g.
cotton, tobacco and maize), while only scarce knowledge
exists about wild plant species as hosts of heliothine
moths (Fit 1989; Matthews 1991).
The results obtained in the present study demonstrate
the importance of using GC-SCR to identify biologically
active plant odorants. The large variety of plant mate-
rials tested containing hundreds of volatile constituents
have resulted in consistent responses to GD and six
576
other related sesquiterpenes in the three insect species.
Direct stimulation with these plant samples only might
have led to the conclusion that these receptor neurones
of heliothine moths are broadly tuned. Furthermore the
GC-SCR is particularly important in identification of
minor constituents as well as enantiomers of biological
significance. For instance, in direct stimulation of the
neurones, the response elicited by the (+)-GD sample
might have been exclusively caused by the 10% impurity
of (–)-GD (Fig. 3). Thus, the GC-SCR experiments
using the chiral column II were necessary to show the
relative effect of the two enantiomers (Fig. 1B). The
other example in this study was the stimulation with
commercial (–)-a-copaene, in which the 2% impurity of
(–)-a-ylangene and the 24% of (+)-a-copaene elicited
the response, whereas (–)-a-copaene had no effect
(Fig. 2B). Since many sesquiterpenes are unstable and
not commercially available, GC-SCR is particularly
helpful in resolving the question about biologically rel-
evant odorants in plant mixtures.
An interesting question in olfaction is whether one
functional type of olfactory receptor neurones project to
a particular glomerulus in the antennal lobe of the brain.
Both in insects and vertebrates molecular biological
studies have shown that olfactory receptor neurones,
expressing the same type of receptor proteins, project in
specific glomeruli (one or two) (Vosshall 2001; Mom-
baerts 1999). The question here is in which of the 62
ordinary glomeruli of the female heliothine moths (Berg
et al. 2002) the (–)-GD neurones project. The pre-
liminary result of one stained preparation of H. assulta,
showed that selective staining of a few axon terminals
could be obtained by applying dextran to the sensillum
base. Whether all four axons belonged to the same
sensillum remains to be tested in more specimens.
Morphological studies of heliothine moths have shown
the presence of one to four receptor neurones in single
sensilla (Faeravaag 1999). However, in the electrophys-
iological recordings of (–)-GD neurones, spikes belong-
ing to one template have consistently appeared
(Røstelien et al. 2000a; Stranden et al. 2002; this study).
Whether these sensilla contain one or more neurones, of
which some might have been silent, is uncertain. The one
stronger stained axon terminal, filling a larger part of the
most medial glomerulus close to the antennal nerve, may
be the projection of the (–)-GD neurone, since this
neurone was active during the staining procedure
(Fig. 6A, B). Interestingly, in optical recordings (calcium
green), activity in a glomerulus located in a corre-
sponding position in the antennal lobe of H. virescens
females was found during stimulation with (–)-GD
(>99.2% pure, 99.9% enantiomerically pure) (Skiri et al.
2002). Further optical recordings and electrophysiolog-
ical experiments combined with staining may reveal
whether one or more glomeruli receive information
about (–)-GD, and whether the position of the glomeruli
corresponds in the three species.
In conclusion, by the use of the GC-SCR and GC-
MS, we have described structure-activity relationships of
one type of plant odour receptor neurones in three
species of heliothine moths. The neurones show the same
properties in the three species; by responding strongest
to (–)-GD, being ten times more effective than (+)-GD.
The weaker effect of the ylangene and the copaene
enantiomers show that the important property of the
(–)-GD is the ten-membered ring system and the three
double bonds acting as electron-rich centres, as well as
the direction of the isopropyl group.
Acknowledgements This paper is dedicated to Prof. em. Bertil
Kullenberg on the occasion of his 90th birthday. The Norwegian
Research Council (project no. 133958/420) provided the principal
financial support for the project. We also acknowledge the support
from The Nordic Academy of Advanced Studies via the visiting
professorship for Dr. Anna-Karin Borg-Karlson (project no.
010434) and the support from the Swedish Institute (project in
ecological biochemistry, the New Visby Program). The Insect
Rearing Team at Syngenta, Basel, Switzerland, Prof. Ezra Dun-
kelblum at the Agricultural Research Organisation, the Volcani
Centre, Bet Dagan, Israel, Prof. Kyung Saeng Boo at Seoul Na-
tional University, South Korea, and Prof. Chen-Zhu Wang at the
Chinese Academy of Sciences, Institute of Zoology, Beijing, Peo-
ple’s Republic of China, are all acknowledged for providing insects.
Dr. Sandrine Gouinguené and Dr. Ted C. J. Turlings, Laboratory
of Animal Ecology and Entomology, University of Neuchâtel,
Switzerland kindly provided the induced plant odours, Dr. Franz-
Josef Hammerschmidt, Dragoco Gerberding & Co. AG, Holzm-
inden, Germany provided the ylang-ylang oil for fractioning, Sina
Escher, Firmenich, Switzerland the ylang-ylang fractions and Fe-
lisberto Pagula, University of Maputo, Mozambique, the essential
oil of Eastern red cedar. We thank Prof. Linda White, Department
of Neuroscience, NTNU for improving the language of the man-
uscript.
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