Most essential insect behaviors, such as mate finding and host
location, are guided by odors. In nature, insects rarely encounter
odors as single compounds. Decisions regarding whether to progress
towards an odor source, or to abort, are probably based upon a
balance mechanism in which attractive and anti-attractive/repellent
inputs in the combined ‘odor bouquet’ are weighed against each
other. In the insect olfactory system, specific olfactory receptor
neurons (ORNs) constitute separate input channels, each detecting
compounds from different chemical and biological categories
(Andersson et al., 2009; Bengtsson et al., 2009; Larsson et al., 2001;
Mustaparta, 1975; Wibe and Mustaparta, 1996). ORN axons project
to the glomeruli of the primary olfactory center, the antennal lobe,
where integration of odor input takes place through lateral
interactions between glomeruli (Olsen and Wilson, 2008; Shang et
al., 2007; Silbering et al., 2008).
However, the organization of the insect peripheral olfactory
system allows for integration also at the level of ORNs or sensilla.
For instance, ORNs are often excited by some compounds and
inhibited by others (e.g. Andersson et al., 2009; Hallem and Carlson,
2006; Said et al., 2003), and responses to compound blends cannot
always be predicted based on the responses to the individual blend
constituents (Getz and Akers, 1997; Ochieng et al., 2002; Party et
al., 2009). Furthermore, insects typically have two or more ORNs
co-compartmentalized within a sensillum. The significance of such
colocalization is poorly understood, but the pairing rules seem very
strict in that specific ORNs are located in the same functional
sensillum type (de Bruyne et al., 2001; Ghaninia et al., 2007).
Selective ORN pairing might be an adaptation to refine the
perception of odor mixtures; for instance, both the spatiotemporal
resolution of volatile stimuli (Fadamiro et al., 1999) and compound-
ratio detection would be improved if the olfactory sensors are located
at the same point in space. Thus, neurons responding to compounds
that together constitute an ecologically important signal should then
often be found paired within the same sensillum. Good examples
are the pheromone ORNs that are colocalized with ORNs that
respond to pheromone antagonists (Cossé et al., 1998; Fadamiro et
al., 1999; Larsson et al., 2002; Wojtasek et al., 1998). ORN co-
compartmentalization might also provide the means for signal
modulation in the periphery, in that responses in neighboring neurons
could potentially affect the activity of each other (Getz and Akers,
1994). In fact, a theoretical model predicts the existence of passive
electrical interactions between colocalized ORNs (Vermeulen and
Rospars, 2004), but the effects of these interactions have not yet
been systematically characterized in insect sensilla.
Conifer-feeding bark beetles (Coleoptera: Curculionidae:
Scolytinae) constitute excellent models to study peripheral
integration of odor mixtures. They are among the most well-studied
insects in terms of behavioral responses to odor blends, such as
different combinations of ecologically relevant attractants and
inhibitors, and much is known about the peripheral detection of these
individual compounds (Andersson et al., 2009; Tømmerås, 1985).
The Journal of Experimental Biology 213, 3332-3339
© 2010. Published by The Company of Biologists Ltd
Peripheral modulation of pheromone response by inhibitory host compound in a
Martin N. Andersson1,*, Mattias C. Larsson1, Miroslav Blazenec2, Rastislav Jakus2, Qing-He Zhang1,†and
1Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden
and 2Institute of Forest Ecology, Slovak Academy of Sciences, 960 53, Zvolen, Slovakia
*Author for correspondence (email@example.com)
†Present address: Sterling International, Incorporated, 3808 N. Sullivan Road, Building 16, Spokane, WA 99216, USA
Accepted 30 June 2010
We identified several compounds, by gas chromatographic–electroantennographic detection (GC–EAD), that were antennally
active in the bark beetle Ips typographus and also abundant in beetle-attacked spruce trees. One of them, 1,8-cineole (Ci), strongly
inhibited the attraction to pheromone in the field. Single-sensillum recordings (SSRs) previously showed olfactory receptor
neurons (ORNs) on I. typographus antennae selectively responding to Ci. All Ci neurons were found within sensilla co-inhabited
by a pheromone neuron responding to cis-verbenol (cV); however, in other sensilla, the cV neuron was paired with a neuron not
responding to any test odorant. We hypothesized that the colocalization of ORNs had a functional and ecological relevance. We
show by SSR that Ci inhibited spontaneous activity of the cV neuron only in sensilla in which the Ci neuron was also present.
Using mixtures of cV and Ci, we further show that responses to low doses (1–10ng) of cV were significantly reduced when the
colocalized Ci neuron simultaneously responded to high doses (1–10m mg) of Ci. This indicated that the response of the Ci neuron,
rather than ligand–receptor interactions in the cV neuron, caused the inhibition. Moreover, cV neurons paired with Ci neurons
were more sensitive to cV alone than the ones paired with the non-responding ORN. Our observations question the traditional
view that ORNs within a sensillum function as independent units. The colocalization of ORNs might sharpen adaptive responses
to blends of semiochemicals with different ecological significance in the olfactory landscape.
Key words: Ips typographus, Coleoptera, Curculionidae, Scolytinae, single-sensillum recording, olfactory receptor neuron, colocalization,
co-compartmentalization, host selection, blend discrimination.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3333 Neurons interact in olfactory sensilla
Mass-attacks on Norway spruce [Picea abies (L.) Karst.] by the
European spruce bark beetle (Ips typographus, L.) are induced by
release of the male-produced aggregation pheromone, a mixture of
(4S)-cis-verbenol (cV) and 2-methyl-3-buten-2-ol (Schlyter et al.,
1987). Pheromone attraction is modulated by anti-attractant non-
host volatiles (NHVs) from leaves and bark of angiosperm plants
(Zhang and Schlyter, 2004). Mechanisms to prevent overcrowding
of host trees are thought to involve other semiochemicals, such as
verbenone, that appear in later attack phases. Verbenone synergizes
the inhibitory effect of NHVs in I. typographus (Zhang and Schlyter,
2003) and is used as a negative cue also by several other bark beetle
species (Lindgren and Miller, 2002; Schlyter and Birgersson, 1999).
Very little is known about the behavioral relevance of host
monoterpenes in I. typographus (Erbilgin et al., 2007) or the host
phenolics (Faccoli and Schlyter, 2007) and other less volatile host
compounds, such as sesquiterpenes.
It is known from single-sensillum recordings (SSRs) that several
ORN classes in I. typographus are tuned to host monoterpenes,
including two classes that are highly selective for para-cymene and
1,8-cineole (Ci), respectively (Andersson et al., 2009). All Ci neurons
(small-amplitude Bcell) were co-compartmentalized with neurons
responding to the pheromone component cV (large-amplitude Acell).
However, in other sensilla, the cV cell was paired with a different B
cell that did not respond to the test odorants. Observations suggested
that stimulating with Ci sometimes inhibited the cV cell, and this
inhibition appeared mainly in sensilla in which the cV and Ci cells
were co-compartmentalized, but the phenomenon was not thoroughly
surveyed (Andersson et al., 2009). The organization of cV and Ci
neurons in the two functional types of sensilla in I. typographus is
excellent for investigations of potential interactions between these
ORNs, the colocalization of which we now hypothesize has a
functional and ecological relevance. To test this hypothesis, we
characterized, by a combination of chemistry, behavior and
physiology, a semiochemical system involving both a behaviorally
attractive pheromone component and inhibitory plant volatiles.
In this study, we identified, by combined gas chromatographic
and electroantennographic detection (GC–EAD) (Zhang et al.,
2000), two (among others) EAD-active compounds, Ci and p-
cymene, that were abundant in spruce trees heavily attacked by I.
typographus, and demonstrated that Ci significantly inhibits
pheromone attraction in the field. We also detailed the inhibitory
effects of Ci on the cV pheromone neuron, comparing the two types
of sensilla by SSR using binary odor mixtures. The plant volatile
Ci inhibited the cV neuron primarily when it was paired with a Ci
neuron, also when the cV cell was simultaneously activated by its
ligand. Thus, coincident responses in colocalized neurons might
significantly influence odor perception, suggesting that the two
neurons do not act as independent units. Such colocalization effects
might sharpen adaptive responses to mixtures of semiochemicals
with different ecological origin and significance.
MATERIALS AND METHODS
Headspace sampling of attacked tree and fresh log
Headspace volatiles from the trunk of a Norway spruce (Picea abies)
tree heavily attacked by I. typographus for at least two weeks were
sampled in situ within a high-density polyacetate film (Look,
Terinex, England) enclosure by a battery-operated pump with an
activated charcoal filter tube in the air inlet (Zhang et al., 1999;
Zhang et al., 2000). The plastic film around the trunk at a level of
1.1–1.6m was sealed by a portable heat sealer on the side and
tightened to the trunk with tape at both open ends. The distance
between the film and bark surface was ca. 1cm. Volatiles (sampling
bark area ca. 0.45m2) were trapped on Porapak Q (50/80 mesh;
30mg in Teflon tube: 3mm?35mm) for 1.5h (airflow 300mlmin–1)
and extracted with 300ml diethyl ether (Fluka >99%).
Headspace volatiles from a non-attacked spruce log (25cm
diameter and 30cm long; freshly cut from a nearby healthy tree)
were collected using the same aeration procedure. The film was
replaced by a polyacetate cooking bag (35?43cm, Terinex,
sampling area ca. 0.24m2). All aeration extracts were kept at –20°C
before GC–EAD and GC–MS analyses.
GC–EAD and GC–MS analyses
A volume of 3ml of aeration samples was injected splitless into an
HP 6890 GC (Agilent, Palo Alto, CA, USA) containing a fused
silica column (HP-Innowax) with a 1:1 effluent splitter, allowing
simultaneous flame ionization
electroantennographic detection (EAD). Hydrogen was used as a
carrier gas. The column temperature was 40°C for the initial 2min,
rising to 200°C at 10°Cmin–1, and held for 2min. The outlet for
the EAD was inserted into a humidified air-stream (0.5ms–1) that
passed over an I. typographusantennal preparation. A glass capillary
indifferent electrode filled with Beadle–Ephrussi Ringer, and
grounded by means of a silver wire, was inserted into the severed
head of a beetle. A similar recording electrode, connected to a high-
impedance DC amplifier with automatic baseline drift compensation,
was placed in contact with the distal end of the antennal club. The
antennal signal was stored and analyzed on a PC equipped with an
IDAC-card and the program EAD v. 2.3 (Syntech, Kirchzarten,
Germany). A repeatable response was defined as a depolarization
of the antennal signal at the same retention time in three out of five
runs. The headspace samples were also analyzed by GC–MS on an
HP 6890 GC with an HP 5973 mass-selective detector (Agilent)
using the same type of GC column and conditions as described
above. Antennally active volatiles were identified by comparison
of retention times and mass spectra of standards.
detection (FID) and
Field trapping experiment
The behavioral effect of (±)-1,8-cineole (1,3,3-trimethyl-2-
oxabicyclo[2,2,2]octane; Ci; >99%, Aldrich) and p-cymene (1-
methyl-4-[1-methylethyl]benzene; >99%, Acros) on I. typographus
pheromone attraction was investigated using Lindgren multiple-funnel
traps (12 funnel size, Pherotech International, Delta, BC, Canada).
The minimum distance between traps was 50m and 15m between
traps and the nearest tree. Insects were collected when at least 30
beetles were caught in a pheromone-baited trap with the lowest catch.
Following collection, treatment positions were changed according to
a Latin square experimental design (Byers, 1991). The experiment
was conducted from June to August 2004 in clear-cuts situated within
spruce stands (80–90 years old) in the Polana Mountains in central
Slovakia. Traps were placed on SW slopes at altitudes of ca. 750m
above sea level. The standard commercial pheromone dispenser IT-
Ecolure was used as an attractant (Fytofarm, Bratislava, Slovakia).
IT-Ecolure is a wick-aluminium-foil-protected dispenser (Varkonda,
1996) filled with 3ml of pheromone mixture. The average release
rate is ca. 50mg/day under field conditions (Fytofarm). The
monoterpenes 1,8-cineole and p-cymene were filled in membrane
dispensers (Wilhelm Biological Plant Protection, Sachsenheim,
Germany) that consisted of a 4ml glass vial with a twisted cap that
was punched (diameter 12mm) and contained four laminated
permeable membranes (0.2mm thick, diameter 17mm). Baited vials
were positioned with the cap facing downwards, resulting in a uniform
evaporation. The estimated average release rate was ca. 50mg/day
under field conditions, as measured by weight loss over time.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Single-sensillum recordings with tungsten microelectrodes were
performed using standard equipment (Syntech) and well-established
experimental protocols. Details regarding insect rearing, insect
preparation, stimuli preparation and the odor delivery system have
been described previously (Andersson et al., 2009). Chemicals,
diluted in paraffin (product # 1.07162.1000, Merck, Darmstadt,
Germany), were applied in aliquots of 10ml on filter papers inside
Pasteur pipettes. Two doses (1 and 10ng) of (4S)-cis-verbenol (cis-
4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-ol; cV; 95%, Borregaard),
and two doses (1 and 10mg) of (±)-1,8-cineole (>99%, Aldrich)
were tested singly and as the four possible binary combinations. In
the latter case, the two compounds were applied on separate filter
papers inside the same pipette to prevent any potential compound
interactions that could have affected evaporation rates. The cV and
Ci cells are indifferent to p-cymene (>99%, Acros), but p-cymene
(10mg) was tested in binary combinations with both doses of cV (1
and 10 ng), as control stimuli. In pipettes containing single
compounds, a second filter paper loaded with paraffin solvent was
also present (i.e. all pipettes contained two filter papers). Stimuli
were delivered in random order and prepared before each
experimental session. Sensilla with the cV cell paired with a non-
responsive B cell (sensillum typeI) and sensilla containing the
colocalized cV and Ci cells (typeII) were both tested for responses.
Attempts were made to record from both sensillum types on all
individuals, and we succeeded in the majority of cases. Both males
and females were used in recordings. Sex separation was based on
external morphology (Schlyter and Cederholm, 1981) and confirmed
by dissection of genitalia.
For the field test, absolute catch was transformed according to
log(catch +1) and used as the variable for statistical processing.
Treatments were compared with one-way ANOVA, and, to
compensate for unequal variances, Dunnett’s T3 was used as a post-
hoc test at 0.05. Sex separation was based on dissection of
genitalia and the sex ratio analyzed with 95% binomial confidence
intervals (95% CI) (Newcombe, 1998). Three hundred beetles, or
all individuals (if <300), per treatment were dissected.
From the SSRs, response curves of high temporal resolution were
obtained by counting (in Autospike 3.0, Syntech) the number of
spikes (action potentials) in the cV A cell in 200-ms bins, starting
1s before stimulus onset and ending 2s after onset. Clear excitatory
odor responses were recorded during a time-period of ca. 800ms.
For statistical comparisons of both excitatory and inhibitory
responses, the total number of spikes during this period was used,
after subtracting the number of spikes fired during stimulation with
control. Factorial ANOVA was used to compare the response of
the cV cell in the two sensillum types. Regression analysis on log-
transformed doses, log(dose+1), was used for comparisons within
a sensillum type. t-tests were used in pair-wise comparisons.
Cohen’s d was used as a measure of standardized effect size. In
addition, a d value was calculated from the adjusted R2obtained
from regression analyses (Nakagawa and Cuthill, 2007). According
to Cohen (Cohen, 1988), a d value above 0.8 is regarded as a large
effect. Tests were performed in SPSS 11.0 at 0.05.
GC–EAD and GC–MS analysis
GC–EAD analysis of the aeration samples from both a non-attacked
log and an attacked trunk of a spruce tree showed repeatable antennal
responses (in at least three out of five runs) to several compounds.
M. N. Andersson and others
In the non-attacked sample, responses were elicited by myrcene,
1,8-cineole/-phellandrene, -longipinene, 1-terpineol and 4-
allyanisole (Fig.1A; later-eluting compounds not shown). Responses
to the two major monoterpenes, -pinene and -pinene, were not
repeatable. In the aeration sample from the heavily attacked spruce
tree, EAD responses were again recorded to compounds present in
the fresh sample (including Ci), but several additional compounds
also elicited responses, including p-cymene, isolongifolene, the
pheromone component cis-verbenol and several unidentified
compounds (Fig.1B; later-eluting compounds not shown). 1,8-
cineole was ca. 20–30 times more abundant than cis-verbenol in
the head-space from the attacked tree. A minute amount of the other
essential pheromone component, 2-methyl-3-buten-2-ol, was
Retention time (min)
Fig.1. GC–EAD recording showing antennal responses (EADs) to
monoterpenes (FID) released by a non-attacked cut spruce log (A) and a
spruce tree heavily attacked by I. typographus (B). The onset of the
antennal response to the combined limonene/1,8-cineole/-phellandrene
peak in (A), and the 1,8-cineole/-phellandrene peak in (B), corresponds to
the part of the FID peaks that belongs to 1,8-cineole. Because the log was
cut, the total amount of monoterpenes is higher in (A), which results in
incomplete separation of 1,8-cineole/-phellandrene from limonene. *EAD
response is delayed in comparison with the retention time of terpinolene,
suggesting that the antennal response is to an unidentified trace
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3335Neurons interact in olfactory sensilla
detected in the monoterpene eluting range, but it did not elicit
repeatable EAD responses. Again, the antennal responses to the
major monoterpenes varied among the antennal preparations.
Field trapping experiment
A total of 32,363 I. typographus were caught in six replicates with
a highly significant overall variation among treatments (F5,3572.7,
P<0.001). The addition of 1,8-cineole to the pheromone strongly
reduced trap catch compared with pheromone alone (Fig.2; 88%
reduction, with a very high effect size; Cohen’s d–2.9 based on
log-transformed means). The reduction in trap catch by the presence
of p-cymene together with pheromone was weaker and not
significantly different from pheromone alone (50% reduction,
d–0.80). The proportion of males in the catch ranged from 19 to
33% but did not differ significantly between treatments (data not
shown). Catches in traps baited with 1,8-cineole or p-cymene alone
did not attract beetles, with close to zero catches like the blank
Sensilla classified as cV sensilla are found in two types. An A neuron
responding selectively to cis-verbenol is accompanied either by a
non-responding B neuron (sensillum typeI) or with a B neuron
responding to 1,8-cineole (typeII) (Fig.3, schematic drawing).
Dose–response curves for the cV and the Ci cells in typeII sensilla
have been published previously (Andersson et al., 2009). In typeI
sensilla, we now show that there was no general inhibition of the
cV cell by either dose of Ci. By contrast, the cV cell in typeII sensilla
was inhibited by both doses of Ci (Fig.4A). The difference between
sensillum types is obvious from the recorded spike trains (Fig.3).
The inhibition in typeII sensilla lasted for up to 1.4s and was
significantly stronger in typeII than in typeI sensilla during the
800ms time-slot (Fig.4A).
Stimulating with cis-verbenol at a lower dose (1ng) elicited a
weak response in the cV cells (Fig.3). The response was significantly
stronger in typeII than in typeI sensilla (t21–2.12, P<0.05; Cohen’s
d–0.94; Fig.4B). Synchronous stimulation with cV and Ci clearly
reduced the cV response in a dose-dependent manner. The reduction
was found only in cV cells co-compartmentalized with Ci cells, as
demonstrated by factorial ANOVA showing a significant interaction
between sensillum types and stimuli (F2,603.43, P<0.05; cV/p-
cymene stimulus excluded from analysis; Fig.4B). In some typeII
sensilla, the cV cell responded to the cV:Ci (1ng:10mg) stimulus
just as if the cis-verbenol was absent (Fig.3). Thus, the response of
the pheromone cell was in such cases shut down to a level lower
than or similar to the blank control. In both types of sensilla,
combining 1ng cV with 10mg p-cymene had no effect on the cV
response (Fig.4B). The B cells in both types of sensilla were, on
average, not affected by cV stimulation (the few spikes in the B
cells in Fig.3 upon cV stimulation are due to random fluctuations
in background activity).
The higher dose (10ng) of cV elicited a clear response in both
sensillum types (Fig.3). Similar to the low (1ng) cV dose, the
response to the higher dose also seemed stronger in typeII sensilla
than in typeI, as indicated by the relatively large effect size
(Cohen’sd–0.75), but the difference was not significant (t21–1.81,
P>0.05; Fig.4C). At the even higher screening dose (10mg) of cV,
Mean catch ± s.e.m.
Ph + p-Cymene
Fig.2. Field data showing inhibition of response to synthetic pheromone
(Ph) when combined with the two EAD-active compounds (1,8-cineole or p-
cymene). A blank and the two compounds alone are included as controls.
N6 number of replicates (trap rotations). Treatments with the same letter
are not significantly different by Dunnett’s T3 post hoc test at 0.05 of
Type IType II
cV 10 ng
cV 1 ng
Ci 10 µg
cV 1 ng + Ci 10 µg
Fig.3. Top: schematic drawing of ORN pairing in the two functional
sensillum types. In typeI sensilla (left column), the cis-verbenol (cV) A
neuron is paired with a non-responsive Bneuron. The typeII sensilla (right
column) contain a cV Aneuron and a 1,8-cineole (Ci) B neuron. SSR
traces: the cV neuron clearly responds to the higher 10ng cis-verbenol
dose (first row). The response threshold is close to the lower 1ng dose
(second row). The cV response is stronger in typeII sensilla. TypeI sensilla
are unaffected by Ci (10mg), whereas, in typeII sensilla, Ci elicits a
powerful excitatory response in the Bneuron, whereas the cV Aneuron
simultaneously is inhibited (third row). The cV neuron in typeI sensilla
responds to the binary cV:Ci mixture (1ng:10mg) in a manner similar to the
response to 1ng cis-verbenol alone, whereas the cV neuron in typeII
sensilla does not respond to cis-verbenol during the Ci response in the
Bneuron (fourth row). Note: in this recording, not a single spike was
elicited in the cV cell in typeII sensilla by the cV:Ci mixture. However,
shutdown of the cV neuron by this stimulus was typically not complete.
Horizontal bars indicate the 0.5s stimulation period.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
the response did not differ between the sensillum types (average
response typeI: 157Hz, N12; typeII: 161Hz, N7; P>>0.05).
At the 10ng cV dose, the binary mixtures of cV and Ci reduced
the cV response in a dose-dependent manner. In contrast to the low
cV dose, factorial ANOVA showed no interaction between sensillum
types and stimuli (F2,610.35, P>0.05; cV/p-cymene stimulus
excluded from analysis) and regression analyses demonstrated
significant response reductions in both types of sensilla. However,
the significance levels from the regression analyses, as well as the
effect size measures, differed between sensillum types, indicating
a stronger effect in typeII sensilla (Fig.4C). The addition of 10mg
M. N. Andersson and others
p-cymene did not significantly alter the response to 10ng cV in any
type of sensillum, although a tendency was found in sensilla without
We show that the antennae of I. typographus respond to volatiles
from healthy host tree logs and to other volatiles released primarily
from heavily attacked trees. More EAD-active compounds were
found in the spruce that had been under attack for two weeks, in
comparison with a non-attacked log, which indicates an important
multi-component ‘unsuitable host signal’ that might regulate bark
Ci 1 µg
Ci 10 µg
/800 ms ± s.e.m.
Number of spikes/
00.4 0.8 1.21.6 2.02.4 2.8
00.4 0.8 1.21.6 2.02.4 2.8
cV 1 ng
cV 1 ng Ci 1 µg
cV 1 ng Ci 10 µg
s ± s.e.m.
Number of spikes/800 ms
cV 10 ng
cV 1 ng
Ci 1 µg
Number of spikes/200 ms ± s.e.m.
cV 10 ng Ci 1 µg
cV 10 ng Ci 10 µg
er of spikes/800 ms ± s.e.m.
0 0.4 0.81.21.62.0 2.42.8
Ci 1 µgCi 10 µg
cV 1 ngcV 1 ng
Ci 10 µg
cV 1 ng
pC 10 µg
cV 10 ng cV 10 ng
Ci 1 µg
cV 10 ngcV 10 ng
Ci 10 µg pC 10 µg
Type IType II
Fig.4. Temporal response characteristics of the cis-verbenol (cV) A cell in typeI (left column) and typeII (mid column) sensilla to single odorants and binary
mixtures. Arrows indicate onset of stimulation, and blue lines indicate the 800ms integration interval. Right column: total response of both sensillum types
during the integration interval, after subtracting the blank response. N10–12 for all stimuli and sensillum types. (A)Stimulating with only 1,8-cineole (Ci)
inhibited the cV cell only in typeII sensilla (factorial ANOVA: F1,3915.0, P<0.001). (B)Responses to cV and binary cV–Ci mixtures at the low (1ng) cV dose.
Ci reduces the cV response only in typeII sensilla. Bold line (right column) indicates significant regression at P<0.01 (F1,3211.2; R20.26, d1.27). There
was no effect on the cV response by the presence of p-cymene (pC; P>>0.05 for both sensillum types). (C)Responses to cV and binary cV–Ci mixtures at
the high (10ng) cV dose. Bold line (right column) indicates a highly significant regression at P<0.01 (F1,337.60, R20.19, d0.88). Narrow line indicates
significant regression at P<0.05 (F1,305.28, R20.15, d0.74). A nonsignificant tendency for inhibition of the cV response by p-cymene was observed in
typeI sensilla (typeI, P>0.05; typeII, P>>0.05). For clarity, the response to the cV/p-cymene stimulus is shown only in the right column in (B) and (C).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3337Neurons interact in olfactory sensilla
beetle density on attacked trees. As Ci was present in both attacked
and non-attacked samples, its inhibitory properties presumably
represent a quantitative rather than a qualitative effect; preliminary
results indicate that the amount of Ci increases after attack by I.
typographus, as newly attacked trees release approximately four
times more Ci than non-attacked trees [N6 (C. Schiebe, P.
Brodelius, G. Birgersson, J. Witzell, P. Krokene, J. Gershenzon, A.
Hammerbacher, B. S. Hansson and F.S., personal communication)].
The amount of Ci released from the attacked tree was 20–30 times
larger than the amount of cV. In our field test, the release ratio
between the pheromone and Ci was 1:1, but, as the pheromone
contains 2-methyl-3-buten-2-ol and cV in a ca. 50:1 ratio, the cV:Ci
ratio in the field test was similar to the ratio released from the
attacked tree. In contrast to earlier reports on the kairomonal activity
of mainly (–)--pinene on I. typographus (Erbilgin et al., 2007;
Hulcr et al., 2006; Jakus and Blazenec, 2003), a mixture of EAD-
active host monoterpenes, -pinene, -pinene and p-cymene, was
unattractive in itself, but interrupted the pheromone response of the
closely related larch bark beetle I. subelongatus(Zhang et al., 2007).
More work is surely needed to determine the behavioral roles of
these antennally active volatiles from healthy and attacked hosts.
The abundance of host monoterpene-selective ORNs on the I.
typographus antennae (Andersson et al., 2009) suggests that beetles
are able to distinguish spruces based on their odor profiles, which
vary among trees of different genotypes and physiological states
(Keeling and Bohlmann, 2006). Terpenoid compounds are involved
in constitutive and induced defenses of both conifers and
angiosperms (Keeling and Bohlmann, 2006; Mumm et al., 2008) –
insects feeding on such plants would likely benefit from selecting
hosts with low terpenoid content. Indeed, several monoterpenes are
toxic and lethal to bark beetles (Everaerts et al., 1988; Raffa and
Smalley, 1995) and to their symbiotic fungi (Raffa et al., 1985).
Although not vectored by bark beetles, inoculation of Norway spruce
with pathogenic fungus Heterobasidion parviporum resulted in an
increased content of 1,8-cineole (Zamponi et al., 2007). In addition,
p-cymene was the most effective of all tested terpenes in reducing
growth of coniferous decay fungi (De Groot, 1972). It is therefore
likely that both Ci and p-cymene are involved in the induced defense
of conifers against bark beetles and/or their symbiotic fungi.
Because the 1,8-cineole and cis-verbenol neurons are not always
colocalized within the same sensilla, we have a unique model for
studying intra-sensillum interactions between a pheromone
compound and a plant-derived behavioral antagonist. There is much
evidence suggesting that insect decision making in host choice is a
matter of weighing relative ratios of different plant compounds,
possibly from different sources. Compartmentalization of neurons
in sensilla might reflect this task (Bruce et al., 2005) as it probably
favors coincidence detection, which in turn would improve the
accuracy of compound ratio discrimination of odor mixtures. In
nature, I. typographus frequently encounters cV and Ci in
combination as cV is released when a spruce tree is attacked. Along
with our behavioral data, the colocalization of the neurons suggests
that the binary mixture constitutes an ecologically significant signal,
assuming that selective pairing of ORNs is adaptive and not due to
chance. Some male moths have a remarkable ability to distinguish
odor filaments from different sources with extremely high
spatiotemporal resolution (Fadamiro et al., 1999; Witzgall and
Priesner, 1991). It was suggested that such amazing feats depend
on ORNs being located within the same sensillum (Fadamiro et al.,
1999), which is common among pheromone agonist/antagonist
neurons in Lepidoptera (Baker et al., 1998; Larsson et al., 2002).
Similar arrangements are found also in Coleoptera (Wojtasek et al.,
1998), but, so far, not in bark beetle pheromone neurons (Andersson
et al., 2009; Mustaparta et al., 1977; Mustaparta et al., 1980).
Consistent with behavioral observations, asynchronous arrival of
pheromone constituents disrupts the spiking pattern of projection
neurons in the antennal lobe, with resulting effects on the temporal
aspects of odor coding (Christensen and Hildebrand, 1997).
The system of cis-verbenol and 1,8-cineole in I. typographus is
different from the pheromone agonist/antagonist organization in
other insects; the compounds are of animal and plant origins,
respectively, and unlikely to be involved in reproductive isolation.
It would, rather, be a means to judge the fitness value of an integrated
odor stimulus comprised of elements from conspecifics and potential
hosts. It would nevertheless be of interest to investigate the effects
of spatial separation of pheromone and 1,8-cineole and compare the
effect of spacing with that of another anti-attractant compound (e.g.
verbenone), detected by an ORN not colocalized with the cV cell.
The colocalization of one pheromone-responding ORN with one
that responds to a plant odor is also different from that shown by
most other insects. Typically, pheromone ORNs are located in
specific morphological sensillum types, whereas ORNs responding
to plant odors are found in other sensilla (Hansson et al., 1986;
Hansson et al., 1999; Larsson et al., 2001) (but see Said et al., 2003).
The pairing of host odor- and pheromone-responsive ORNs in I.
typographus suggests that host localization, involving both the
pheromone and host-derived compounds, is an integrated system
that does not segregate between different classes of semiochemicals.
Signal modulation between receptor neurons at the peripheral
level is another factor that could influence grouping of neurons in
sensilla. Indeed, our results indicate response interactions between
neighboring ORNs as the inhibition of the cV cell was much more
pronounced when a colocalized Ci cell responded. Neurons within
contact chemosensilla of the grasshopper Schistocerca americana
have also been shown to interact, in that a single spike in the small-
spiking cell resulted in an extended spike interval in the colocalized
large-spiking cell (White et al., 1990). Contacts between sensory
cells in thermo-/hygroreceptive sensilla styloconica have been
found in, for example, the silkmoth Bombyx mori (Steinbrecht, 1989)
and were hypothesized to be involved in the antagonistic response
characteristics of these cells. However, contacts such as electrical
or chemical synapses have not been found between insect ORNs.
A non-synaptic mechanism that could explain the inhibition is
the passive electrical interaction that occurs between ORNs that are
colocalized in sensilla (Vermeulen and Rospars, 2004). According
to the model, excitatory responses in an ORN can hyperpolarize a
neighboring cell that is insensitive to the stimulus and in close
contact. The model also suggests that pairing of ORNs with similar
odor tuning might reduce the amplitude of the receptor potentials
and thus lower the sensitivity of the cells (Vermeulen and Rospars,
2004). If signal modulation unavoidably occurs between receptor
neurons at the peripheral level, pairing of ORNs with differentiated
response profiles would be expected as it would circumvent an
overall reduction in olfactory sensitivity. To our knowledge, ORNs
with largely overlapping response spectra have rarely been found
within the same sensillum (but see Hansen, 1984), although it
theoretically could improve discrimination between chemically
In our case, the strong excitatory response in the Ci cell might
change the receptor potential of the cV cell so that a higher cV dose
is required to produce the same number of action potentials. If so,
the inhibitory interaction would be expected to be reciprocal – that
is, a strong cV response should inhibit the Ci cell. However, we
were unable to investigate this owing to the fact that the small-
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3338 M. N. Andersson and others
amplitude spikes of the Ci cell were completely obscured by a strong
response of the large-spiking cV cell. In a previous study, we found
that, when the methyl-butenol B cell responded, the colocalized A
cell was strongly inhibited (Andersson et al., 2009). Unfortunately,
no excitatory responses from this A cell were recorded to any odorant
tested. In addition, it would be of great interest to identify the
ligand(s) for the non-responsive B cell that is paired with the cV
cell in typeI sensilla, perhaps by means of GC-coupled SSRs
(Stensmyr et al., 2001), to investigate whether responses in this cell
also inhibit the cV cell. Functional characterization of this ORN in
conjunction with behavioral testing of the ligand would be desirable
in order to consolidate the relevance of ORN interactions. In fact,
inhibition of Acells when Bcells respond seems to be a common
phenomenon (Blight et al., 1995; Hallem et al., 2004; Larsson et
al., 2001), but it has not been systematically addressed.
To our knowledge, our study is the first to investigate interactions
between ORNs in sensilla where spikes from the individual neurons
could be distinguished. However, the idea of ORN interaction is
not novel. In honeybee sensilla placodea, neurons within a sensillum
responded to odors in a coordinated manner, suggesting that
responses of individual ORNs are not independent (Getz and Akers,
1994). Unfortunately, as these sensilla contain 18–35 ORNs, spikes
from individual neurons were not distinguishable but could only be
grouped into three or four ‘response units’, which makes the
interpretation of the results less straightforward compared with our
observations. Other studies have also shown that receptor neuron
responses to binary mixtures cannot always be predicted based on
the responses to individual constituents, and that the mixture
interaction can be both inhibitory and synergistic (Getz and Akers,
1995; Getz and Akers, 1997; Ochieng et al., 2002; Party et al., 2009).
In our case, it was clear that the inhibition of cV neurons in
response to binary cV–Ci mixtures occurred exclusively in typeII
sensilla at the low (1ng) cV dose. The inhibition in both sensillum
types at the higher (10ng) cV dose suggests that two different
mechanisms could be operating, one being the interaction between
neurons and the other possibly an effect of a second compound
present at a high concentration. An inhibitory interaction would be
expected if the rate of molecules entering the cuticular pores is
limited or if there is competition for, for example, odorant-binding
proteins (OBPs) within the sensillum lymph. Possibly, the different
degree of inhibition by Ci could also be explained by inherent
differences between the Aneurons or between the two types of
sensilla. Interestingly, the cV neurons in typeII sensilla also
demonstrated a greater sensitivity to cV than the ones in typeI
sensilla. The response to 1ng cV was almost twice as strong in
sensilla containing both the cV and Ci neuron, and a similar, albeit
not significant, tendency was found at the 10ng dose. We do not
know why this difference exists, but it might be explained for
instance by differences in olfactory receptor gene expression rates
or differences in the sensillum environment, such as OBPs or other
factors. Alternatively, cis-verbenol-sensitive neurons of typeI and
typeII sensilla might express odorant receptors (ORs) with slightly
different response profiles. Molecular characterization of I.
typographus ORs is required to distinguish between these
It is uncertain whether the recorded physiological inhibition of
Ci is of ecological relevance as the Ci needs to be present at a ca.
1000 times higher dose than the cV in order to elicit detectable
inhibition. Our head-space collection from the attacked tree indicated
that the ratio between cV and Ci is ca. 1:20–1:30, but our sample
size is too small to draw any definite conclusions. In addition, the
excitatory inputs from the cV and Ci cells provide the means for
central integration, which probably explains some (or most) of the
anti-attractant effect of Ci.
In conclusion, the host monoterpene 1,8-cineole inhibits the
attraction of I. typographusto its aggregation pheromone, and highly
specific ORNs for the compound are present on the antennae. Insects
typically group pheromone ORNs in sensilla that are distinct from
the ones that contain ORNs for plant compounds, which makes our
system deviant from this general rule. In particular, the inhibitory
interaction that occurs between the pheromone and host odor ORNs
is the first one described for any insect. Although the ecological
consequences of the peripheral inhibition in isolation could not be
established, it is clear that an inhibitory interaction occurs in the
periphery and that the cV cells differ in sensitivity depending on
which type of sensilla they are housed in. Our observations thus
question the traditional view that ORNs within a sensillum act as
independent response units.
LIST OF ABBREVIATIONS
gas chromatographic–electroantennographic detection
gas chromatography–mass spectrometry
olfactory receptor neuron
We thank Muhammad Binyameen and Elisabeth Marling for assistance in bark
beetle rearing. We also thank Sylvia Anton and Michel Renou (INRA, Versailles)
for useful input on the manuscript. This study was funded by FORMAS, project #
230-2005-1778, ‘Semiochemical diversity and insect dynamics’, and by the
Linnaeus-program ‘Insect Chemical Ecology, Ethology, and Evolution’ (ICE3).
M.C.L. was also funded by the Crafoord foundation and the Trygger foundation.
This study was supported by the Centre of Excellence ‘Adaptive Forest
Ecosystems’, ITMS: 26220120006, the Research & Development Operational
Programme, funded by the ERDF.
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