Enantiomeric specificity in a pheromone-kairomone system of two threatened saproxylic beetles, Osmoderma eremita and Elater ferrugineus

Article abstract of DOI:10.1007/s10886-007-9423-x

The scarab beetle Osmoderma eremita and its larval predator, the click beetle Elater ferrugineus, are threatened saproxylic beetles regarded as indicators of the species-richness of insect fauna of hollow deciduous trees. Male O. eremita produce the pheromone (R)-(+)-γ-decalactone to attract conspecific females, and this compound is also utilized by E. ferrugineus as a kairomone, presumably for detection of tree hollows containing prey. We have investigated enantiomeric specificity to γ-decalactone in this pheromone-kairomone system by electrophysiological and field trapping experiments. In single-sensillum recordings from male and female O. eremita, which used the (R)-enantiomer and the racemic mixture of γ-decalactone as odor stimuli, numerous olfactory receptor neurons (ORNs) responding to both stimuli were found. No neurons responded preferentially to the racemic mixture, showing that these beetles seem to lack receptors specific for the (S)-enantiomer. The enantiomeric specificity of ORNs was confirmed by gas chromatography-linked single-sensillum recordings where the two enantiomers in a racemic mixture were separated on a chiral column. Furthermore, in field experiments that used the (R)-enantiomer and the racemic mixture as lures, the attraction of O. eremita females corresponded to the amount of (R)-enantiomer released from lures with the (S)-enantiomer displaying no antagonistic effects. Trap catch data also suggested that the (S)-enantiomer is not a behavioral antagonist for E. ferrugineus. The odor-based system can be highly efficient in attracting the larval predator where trap catch in 1 yr almost equaled the total number of specimens collected in Sweden until 1993. Our study shows that racemic γ-decalactone could be used for cost-effective monitoring of both beetles.

Full text of DOI:10.1007/s10886-007-9423-x

J Chem Ecol (2008) 34:189–197
DOI 10.1007/s10886-007-9423-x
Enantiomeric Specificity in a Pheromone–Kairomone System
of Two Threatened Saproxylic Beetles, Osmoderma eremita
and Elater ferrugineus
Glenn P. Svensson & Mattias C. Larsson
Received: 5 October 2007 /Revised: 3 December 2007 /Accepted: 21 December 2007 /Published online: 31 January 2008
#
Springer Science + Business Media, LLC 2008
Abstract The scarab beetle Osmoderma eremita and its
larval predator, the click beetle Elater ferrugineus, are
threatened saproxylic beetles regarded as indicators of the
species-richness of insect fauna of hollow deciduous trees.
Male O. eremita produce the pheromone (R)-(+)-γ-deca-
lactone to attract conspecific females, and this compound is
also utilized by E. ferrugineus as a kairomone, presumably
for detection of tree hollows containing prey. We have
investigated enantiomeric specificity to γ-decalactone in
this pheromone–kairomone system by electrophysiological
and field trapping experiments. In single-sensillum record-
ings from male and female O. eremita, which used the (R)-
enantiomer and the racemic mixture of γ-decalactone as
odor stimuli, numerous olfactory receptor neurons (ORNs)
responding to both stimuli were found. No neurons
responded preferentially to the racemic mixture, showing
that these beetles seem to lack receptors specific for the (S)-
enantiomer. The enantiomeric specificity of ORNs was
confirmed by gas chromatography-linked single-sensillum
recordings where the two enantiomers in a racemic mixture
were separated on a chiral column. Furthermore, in field
experiments that used the (R)-enantiomer and the racemic
mixture as lures, the attraction of O. eremita females
corresponded to the amount of (R)-enantiomer released
from lures with the (S)-enantiomer displaying no antago-
nistic effects. Trap catch data also suggested that the (S)-
enantiomer is not a behavioral antagonist for E. ferrugi-
neus. The odor-based system can be highly efficient in
attracting the larval predator where trap catch in 1 yr almost
equaled the total number of specimens collected in Sweden
until 1993. Our study shows that racemic γ-decalactone
could be used for cost-effective monitoring of both beetles.
.
.
Keywords Osmoderma eremita Elater ferrugineus
.
.
.
.
Scarabaeidae Elateridae γ-Decalactone Sex pheromone
.
.
Kairomone Predator–prey interaction Single-sensillum
.
.
recording Olfactory receptor neuron Conservation
Introduction
Ever since the first insect pheromone was identified half a
century ago (Butenandt et al. 1959), the ultimate practical
goal of research on insect chemical ecology has been the
development of semiochemical-based strategies to control
populations of agricultural and forest pests (e.g., Cardé and
Minks 1995; Borden 1997; Foster and Harris 1997). In
contrast, surprisingly little effort has gone into the explo-
ration of the chemical ecology of threatened insects and the
potential to use infochemicals in conservation biology.
Monitoring with volatile attractants could be more efficient
than traditional survey methods in gathering ecological data
on focal species for conservation and in assessing species
diversity of threatened insect communities. Priority should
be given to assemblages that are difficult to detect and
monitor with currently available methods. One such group
is saproxylic beetles that live in hollow deciduous trees.
The severe fragmentation and isolation of this habitat in
recent centuries (Hannah et al. 1995) has resulted in the
associated insect fauna being endangered all over Europe.
Surveys of beetles that live inside tree trunks are often both
G. P. Svensson (*)
Department of Ecology, Ecology Building, Lund University,
SE-223 62 Lund, Sweden
e-mail: glenn.svensson@ekol.lu.se
M. C. Larsson
Department of Crop Protection Biology,
Swedish University of Agricultural Sciences,
P.O. Box 44, SE-230 53 Alnarp, Sweden

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J Chem Ecol (2008) 34:189–197
labor-intensive and time-consuming, and in many cases,
only a small fraction of suitable trees in an area can be
surveyed.
behavioral data. From an applied perspective, evaluating
the enantiomeric specificity in insect pheromone or kair-
omone systems is important for efficient semiochemical-
based monitoring. Because pure enantiomers are usually
more expensive than racemic mixtures (in the case of
commercially available (R)-(+)-γ-decalactone, currently
about 15 times as expensive, see the “Methods and
Materials” section), use of the latter could significantly
reduce the cost for monitoring. We, therefore, conducted
electrophysiological analyses and field experiments to
evaluate the effects of the (S)-enantiomer of γ-decalactone
on the behaviors of O. eremita and E. ferrugineus.
We recently initiated a research program to integrate
chemical ecology and conservation biology of saproxylic
beetles by using Osmoderma eremita Scopoli (Coleoptera:
Scarabaeidae) as a model. This beetle is strongly associated
with hollow deciduous trees (Luce 1996; Ranius and
Nilsson 1997) and it has become a major model for
ecological research on insects associated with this habitat
in Europe (e.g., Ranius 2001; Ranius and Hedin 2001).
Because of its role as an indicator for the species-rich
fauna of saproxylic insects, O. eremita has high conserva-
tion priority according to the European Union’s Habitat
Directive (Anonymous 1992), and it has recently been
intensively surveyed in Europe (Ranius et al. 2005). The
most well-known feature of O. eremita is its fruity, plum-
like or peach-like odor, which is emitted exclusively by
males. We identified the odor as (R)-(+)-γ-decalactone and
demonstrated its function as a sex pheromone used to
attract conspecific females (Larsson et al. 2003). We have
also shown that the likewise-threatened click beetle Elater
ferrugineus L. (Coleoptera: Elateridae), whose larvae prey
upon the larvae of O. eremita, utilizes (R)-(+)-γ-decalac-
tone as a kairomone to locate its prey (Svensson et al.
2004). The Osmoderma pheromone is produced in excep-
tionally large amounts and can even be detected inside tree
hollows by using traditional headspace sampling techniques
(Svensson et al. 2003).
Sexual communication in beetles is often achieved by
chiral pheromone compounds (e.g., Birch et al. 1980; Mori
et al. 1986; Leal 1991). When one enantiomer of a chiral
molecule is used for intraspecific communication, its
antipode may function as a behavioral antagonist to avoid
cross-species attraction, if the antipode is used by a closely
related species. For example, the sympatric scarabs Popillia
japonica Newman and Anomala osakana Sawada utilize
the (R)- and (S)-configuration of japonilure, respectively, as
sex pheromones, and strong reciprocal behavioral antago-
nism to the opposite enantiomer has been observed
(Tumlinson et al. 1977; Leal 1996). Similar stereochemical
discrimination has also been documented in insect predators
that eavesdrop on the pheromone communication systems
of their prey (Aldrich 1999), such as the clerid beetle
Thanasimus dubius F., which only responds to the (S)-
enantiomer of frontalin, the major enantiomer of the
pheromone of its prey Dendroctonus frontalis Zimmerman
(Payne et al. 1982, 1984).
Methods and Materials
Insects O. eremita is a large (25–35 mm) scarab beetle
living exclusively in hollow deciduous trees, mainly oaks
(Luce 1996). It is dependent on hollows with large amounts
of wood mold, i.e., a mixture of loose, rotten wood,
fragments of insects, fungi, and old bird nests. The
development time is normally 3 yrs (Ranius et al. 2005).
The species is distributed in most parts of Europe, but a
recent survey that included 33 countries indicated that it has
decreased in all areas (Ranius et al. 2005). Today, it has a
relict distribution and only occurs in isolated populations
in, e.g., woodland pastures. In Sweden, it is currently
known from only 130 sites (Antonsson et al. 2003), and
many populations occupy single isolated trees with few or
no suitable trees nearby, and may, therefore, be doomed to
extinction. E. ferrugineus is a large (15–25 mm) click
beetle and larval predator on several saproxylic beetles
associated with hollow trees, including O. eremita (Hansen
1966; Dajoz 2000). In Sweden, it is currently known from
only 25+ sites (Nilsson and Baranowski 1994). Ranius
(2002) found a strong correlation of occupancy between E.
ferrugineus and O. eremita with fragments of the predator
found almost exclusively in trees with fragments of its prey.
The development time is several years (Palm 1959).
According to Hansen (1966) and T. Tolasch (personal
communication) this species swarms during evenings and
nights, and its female-produced sex pheromone was
recently identified (Tolasch et al. 2007). The species is rare
in Sweden with only 147 adult specimens collected from
about 25 localities until 1993 (Nilsson and Baranowski
1994). It is considered endangered in the Swedish Red List
of threatened species (Gärdenfors 2005).
However, behavioral effects of nonpheromonal enan-
tiomers vary considerably among different insect species
(Mori 1998; Leal 1999), and more studies are needed to
understand the adaptive significance of these effects,
including mechanisms of enantiomeric perception and
Chemicals and Dispensers (R)-(+)-γ-Decalactone (97%
enantiomeric purity) was purchased from Sigma-Aldrich,
Sweden (catalog no.: W236012). Because the (S)-enantio-
mer of the compound is not commercially available, we
used a racemic mixture of γ-decalactone (catalog no.:

J Chem Ecol (2008) 34:189–197
191
W236004) to study the electrophysiological and behavioral
effects of the (S)-enantiomer. Dispensers for traps were
made from 2 ml glass vials loaded with 600 μl of neat (R)-
(+)-γ-decalactone or the neat racemic mixture. Cut strings
of cotton dental rolls (Celluron, Paul Hartmann, S.A.,
France) were inserted as wicks into the glass vials, and
dispensers were attached to traps via a metal hook.
Responses from ORNs were calculated as the total number
of spikes during 0.5 sec after the onset of stimulation minus
the number of spikes during 0.5 sec before the onset of
stimulation. The net response to an odor stimulus was
calculated by subtracting the response generated by the
control stimulation. In dose–response trials, net responses
for females and males were compared for stimulations with
the (R)-enantiomer and the racemic mixture at each dose
using paired t tests, and net responses for stimulations with
(R)-enantiomer were compared between the sexes at each
dose using unpaired t tests.
Electrophysiology Single-sensillum recordings were per-
formed on both female and male O. eremita, but not on
E. ferrugineus, which is rare in Sweden. To restrain a
beetle, it was wrapped with parafilm, placed on a
microscope slide, and fixed with dental wax. To get access
to olfactory receptor neurons (ORNs), the three lamellae on
the antenna were held apart with thin metal pins. A
tungsten microelectrode or a thin silver wire was inserted
into the abdomen of the insect, serving as a ground
electrode, and a second tungsten microelectrode (electro-
lytically sharpened in KNO2 solution) was inserted into an
olfactory sensillum to establish contact with ORNs. The
recording electrode was connected to a ×2 gain probe
(Syntech, Hilversum, The Netherlands). A microscope with
up to ×500 magnification and a DC-3K micromanipulator
with a Piezo translator (PM10) (Märzhauser, Wetzlar-
Steindorf, Germany) were used to position the electrode.
Charcoal-filtered and humified air passed over the antenna
from a glass tube outlet at 10 mm distance from the
preparation. Odor stimulation of an ORN was achieved by
inserting the tip of a Pasteur pipette, containing defined
amounts of a stimulus, into a hole in the glass tube 10 cm
before the outlet. The pipette was linked to an air control
system (Syntech, Hilversum, The Netherlands), which
generated 0.5 sec air puffs through the pipette into the air
stream of the glass tube.
Coupled Gas Chromatography and Single-sensillum
Recordings To further test for the enantiomeric specificity
of ORNs to γ-decalactone in O. eremita, gas chromatog-
raphy linked to single-sensillum recordings (GC–SSR) was
performed with a chiral column to resolve the enantiomers
in the racemic mixture. A Hewlett-Packard 5890 Series II
Plus gas chromatograph was used, equipped with a Cyclo-
sil-B chiral column (30 m×0.25 mm i.d., 0.25 μm film;
J&W Scientific, USA). Hydrogen was used as carrier gas
(40 cm/sec), injector temperature was 220°C, and the
following temperature program was used: 60°C for 2 min,
10°C/min to 170°C, followed by 2°C/min to 200°C, and
then 10°C/min increase to 225°C. The transfer line
temperature was maintained at 225°C. As stimulus,
100 ng of the racemic mixture were injected into the GC.
Field Trapping Field bioassays were conducted in Bjärka-
Säby (58°16′N, 15°46°E) and Brokind (58°12′N, 15°40′E),
southeast Sweden, during July–August, 2006 and 2007.
The study area included five large stands of old hollow
oaks housing some of the largest populations of O. eremita
and E. ferrugineus in Sweden (Nilsson and Baranowski
1994; Ranius 2001; Antonsson et al. 2003). In 2006, two
different nondestructive trapping systems were used,
allowing beetles to be released alive after examination:
Lindgren funnel traps (Phero Tech, Delta, BC, Canada), and
custom-built traps consisting of two black plastic sheets
(25 cm height×30 cm width, 3 mm thickness) arranged in a
cross and attached perpendicular to a black plastic funnel
(upper diameter, 27 cm) leading down to a 5-l white plastic
container [for a picture of a trap with the same general
design, see Ruther et al. (2000)]. In 2006, 20 replicates of 4
traps were used. Each replicate contained two traps of each
type, baited with either 600 μl of the (R)-enantiomer or
600 μl of the racemic mixture. Custom-built traps were
about 1.5 times more efficient than Lindgren traps in catching
O. eremita (191 vs. 141 catches, χ2=7.53; df=1; P<0.01)
and E. ferrugineus (88 vs. 55 catches, χ2=7.62; df=1; P<
0.01). Thus, only custom-built traps were used in 2007.
Twelve replicates were used, each containing 2 traps baited
with either 600 μl of (R)-enantiomer or 2×600 μl of racemic
Odor Stimuli Neat (R)-(+)-γ-decalactone and the racemic
mixture were diluted in hexane in decadic steps down to
concentrations to be used in electrophysiological experi-
ments. A test stimulus was then applied to a small piece of
filter paper inserted into the test pipette, and the solvent was
allowed to evaporate. Each pipette was loaded with 10 μl of
a stimulus solution, and the amount of compound loaded
into a pipette ranged from 100 ng to 100 μg. Pipettes
loaded with 10 μl of hexane served as controls. After
establishing contact with an ORN, it was stimulated with
both test odorants at the highest dose (100 μg) and the
control. If responses to the test odorants were not greater
than to the control, the neuron was classified as non-
responding. If the neuron responded to any of the test
odorants, a dose–response trial was often performed by
using the test stimuli at increasing concentrations. To
enable the receptor neuron to recover after odor stimulation,
at least 20 sec passed between each stimulation event.

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mixture, and a control trap without odorants. Traps were
suspended from oak branches at 2–4 m height and at least
10 m apart, and they were checked for the presence of
beetles every second day. The relative positions of traps
within replicates were changed every eighth day.
them, we did not determine the sex of most captured
individuals in 2006, except for a subset of 23 individuals,
mostly consisting of individuals found dead in the traps.
Results
In 2006 (but not in 2007), captured O. eremita were
marked with shallow marks drilled in the elytra (Ranius
2001), and E. ferrugineus were marked on the elytra with a
permanent marker pen to estimate the proportion of
recaptures, which comprised <10% of the total number of
captures in O. eremita and <4% in E. ferrugineus.
Statistical analyses on trapping data were based on the
total number of captures, including recaptures. For male
and female O. eremita and E. ferrugineus, χ2 analyses were
performed to check for the difference in trap catch among
treatments. We found no published information allowing
the sex of E. ferrugineus individuals to be distinguished
easily without genital examination, and to avoid killing
Electrophysiology Recordings were performed only from
sensilla on the inner lamella of the O. eremita antenna.
High-quality recordings were obtained from 234 sensilla of
30 females and 101 sensilla of 21 males. Based on their
spike amplitude, most sensilla appeared to contain two
ORNs, but odor stimulation always affected only one of
these neurons, whereas the other was nonresponsive to both
stimuli. In 88% of the recordings from both females and
males, the ORNs in the sensillum did not respond to the
(R)-enantiomer or racemic mixture of γ-decalactone. The
remaining sensilla (29 in females and 10 in males)
Fig. 1 a Single-sensillum record-
ings from the antenna of a female
O. eremita showing the response
of an ORN to the blank control
and to increased doses of (R)-
(+)-γ-decalactone. The bar
below the recordings indicates
the stimulus duration (0.5 sec).
b Dose–response relationships
of ORNs of female and male O.
eremita stimulated with the (R)-
enantiomer (R) or a racemic
mixture (R+S) of γ-decalactone.
For each dose, the same amount
of (R)-enantiomer was used in
both test stimuli. The response
to a stimulus is quantified as the
net number of spikes (number of
spikes during 0.5 sec after the
stimulation minus the number of
spikes during 0.5 sec before the
stimulation) minus the net blank
response. Error bars show the
standard error of the mean. The
asterisk indicates a significant
difference in spike activity be-
tween stimuli within the dose
(paired t test, P<0.05)

J Chem Ecol (2008) 34:189–197
193
contained a receptor neuron that responded to both test
odorants (Fig. 1a). No neurons were found that responded
primarily to the racemate, which would have indicated
specificity to the (S)-enantiomer. The electrophysiological
recordings revealed that γ-decalactone-sensitive ORNs
were sparsely distributed over the whole inner antennal
lamella of both sexes, but mainly found in the smooth area
close to the ventral edge of the lamella (cf. Larsson et al.
2001, 2003). In dose–response trials, ORNs of females and
males showed equal sensitivity to (R)-(+)-γ-decalactone
(for all doses, P>0.05; Fig. 1b). However, a small but
significant difference in spike frequency was observed for
ORNs of females when stimulated with the (R)-enantiomer
compared to the racemic mixture at the highest dose (t=
2.96, df=6, P<0.05; Fig. 1b). Single-sensillum recordings
coupled with gas chromatography confirmed that the γ-
decalactone-sensitive ORNs indeed only responded to the
(R)-enantiomer (Fig. 2). There was a clear separation of the
two compounds on the chiral column with the (R)-
enantiomer eluting 8.5 sec earlier than its antipode, which
allowed the γ-decalactone-sensitive receptors to recover
between stimulation events.
respectively; χ2=2.59; df=1; P>0.05; Fig. 3a). In 2007,
122 captures of females in odor-baited traps were observed
(Fig. 3b). No significant difference in trap catch was
observed between traps baited with the (R)-enantiomer
and those baited with the racemic mixture (with the same
amount of (R)-enantiomer) (56 vs. 66; χ2=0.82; df=1; P>
0.05; Fig. 3b). Seven and three males were captured in
attractant-baited traps with (R)-enantiomer and the racemic
mixture, respectively, and three females were found in
control traps.
E. ferrugineus: In 2006, 143 captures of 139 individuals
were observed (Fig. 3a). The majority of trapping events
(60%) included single individuals, whereas up to six beetles
were found in a trap on other occasions. Similar to O.
eremita females, significantly more insects were caught in
traps baited with the (R)-enantiomer compared to those
baited with the racemic mixture with half the amount of
(R)-enantiomer (88 vs. 55; χ2=7.62; df=1; P<0.01). The
subset of 23 beetles used for sex determination contained
22 females and only 1 male. In 2007, only six females were
trapped (three for each treatment, data not shown in
Fig. 3b).
Trap Catch O. eremita: In 2006, 332 females were caught
in odor-baited traps with significantly more caught in traps
baited with the (R)-enantiomer compared to those baited
with the racemic mixture (with half the amount of (R)-
enantiomer) (200 vs. 132; χ2=13.93; df=1; P<0.001;
Fig. 3a). Also, 87 captures of males were observed, but
no difference was observed in the number of captures
between odor treatments (51 vs. 36 for R and R+S,
Discussion
In this study, we show both electrophysiological and
behavioral evidence for enantiomeric anosmia in the
pheromone communication system of O. eremita. Record-
ings from single sensilla showed that both sexes have
Fig. 2 Example of enantiomer-specific response to γ-decalactone in
the ORN of a female O. eremita using GC–SSR. The upper trace
shows the spike activity of the ORN, the middle trace shows the FID
response to the two enantiomers separated on a chiral column, and the
lower trace shows the spike activity of the ORN calculated as the
number of spikes per second. After responding to the (R)-enantiomer,
the ORN recovers almost completely and no second activity peak is
observed during stimulation with the (S)-enantiomer

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J Chem Ecol (2008) 34:189–197
Fig. 3 a Trap catch of O.
eremita and E. ferrugineus in
2006 using traps baited with
600 μl of (R)-enantiomer (R) or
600 μl of the racemic mixture
(R+S) of γ-decalactone (χ2 test:
**P<0.01, ***P<0.001). b
Trap catch of O. eremita in 2007
using traps baited with 600 μl of
(R)-enantiomer (R) or 2×600 μl
of the racemic mixture (2×R+S)
of γ-decalactone, and control
traps (χ2 test: ***P<0.001)
ORNs specific to the (R)-enantiomer of γ-decalactone, the
compound produced by conspecific males. No neurons
tuned to the opposite enantiomer were found in the sensilla
investigated. In dose–response trials that used the (R)-
enantiomer and the racemic mixture as stimuli, the spike
frequency of female ORNs was significantly higher for the
pure (R)-enantiomer than for the mixture (with half the
amount of (R)-enantiomer) at the highest dose (100 μg),
also indicating that those neurons do not respond to the (S)-
enantiomer (Fig. 1b). These observations were confirmed
by using coupled gas chromatography and single-sensillum
recordings where the two enantiomers were resolved on a

J Chem Ecol (2008) 34:189–197
195
chiral column: the neurons always responded to the first-
eluting (R)-enantiomer and never to the later-eluting (S)-
enantiomer (Fig. 2).
anosmic to the nonpheromonal enantiomers, entirely lack-
ing receptors for their detection (Larsson et al. 1999, 2001;
Leal 1999), which seems to be the case also for O. eremita.
When the nonpheromonal enantiomer in a racemic mixture
cannot be detected, the expected outcome might be a
slightly lower attraction to the racemate than to the pure
pheromone, considering that the release rate of the
attractant is only 50% as high in the racemic mixture.
The high numbers of E. ferrugineus observed in
attractant-baited traps in 2006 suggest that this click beetle
may have higher populations than previously recognized.
Scientists used to dealing with pest insects may not
consider 139 beetles impressive, but it is close to the total
of 147 specimens documented in Swedish collections until
1993 (Nilsson and Baranowski 1994) and is certainly many
times more than entomologists have previously encountered
during a lifetime. Earlier investigations of only eight E.
ferrugineus individuals found in attractant-baited traps
indicated that both males and females might be attracted
to the O. eremita pheromone (Svensson et al. 2004). This
study, with a larger sample analyzed, showed that the
pheromonal signal is exploited almost exclusively by
females, presumably to find trees containing O. eremita.
The much lower number of E. ferrugineus captured per trap
in 2007 suggests that populations of this predator fluctuate
much more than populations of its prey.
Many trees suitable for saproxylic beetles cannot be
analyzed with traditional methods, such as wood mold
sampling or pitfall trapping, because of the characteristics
of their hollows. For example, only about 25% of the oaks
in the current study areas that potentially harbor O. eremita
and E. ferrugineus have been investigated because other
hollows have been too far up in a tree to be reached by a
ladder or the cavity has been too deep to place a trap in the
wood mold (Hedin and Mellbrand 2003). With a majority
of suitable habitat patches not investigated, estimates of
population sizes of focal species may be severely biased,
which may in turn affect conservation strategies. With
efficient nondestructive trapping systems available, we can
now gather detailed ecological data about these species that
have not been possible with previously available methods.
Attractant-baited traps placed outside tree hollows will only
catch those individuals that have left their natal tree and
should be used as a complementary method to pitfall traps
and other methods to increase the accuracy of population
size estimates and analyses of dispersal rates and dispersal
distances. Odor traps may also be used for targeted efforts
to rediscover species at localities where they have not been
observed for sometime. Our data show that attractant-based
monitoring could be a powerful tool for conservation
purposes, and more research should be focused on the
chemical ecology of threatened insects to develop new
strategies for their preservation.
Field trapping data were in concordance with electro-
physiological data, and the attraction of both O. eremita
females and its predator E. ferrugineus to attractant-baited
traps was correlated with the amount of (R)-enantiomer
released from lures. In 2006, significantly more captures of
O. eremita females and E. ferrugineus were observed in
traps with pure (R)-enantiomer compared to the racemic
mixture, releasing half the amount of (R)-enantiomer. In
2007, no difference in trap catch of O. eremita females was
observed when both treatments released the same amount
of (R)-enantiomer. Thus, there is no inhibitory effect of
adding the (S)-enantiomer to the O. eremita pheromone.
From a practical point of view, the choice of lure is not
critical, and the much cheaper racemic mixture can be used
if the lure constitutes a significant part of the trap costs.
Effects of nonpheromonal enantiomers on insect phero-
mone attraction range from attractive to neutral to strong
antagonistic effects (Mori 1998). The lack of inhibitory
effects on the attraction of O. eremita and E. ferrugineus by
the antipode of their pheromone/kairomone is in stark
contrast to many other cases where the presence of the
wrong enantiomer, sometimes at concentrations of only a
few percent, results in strong inhibition of response to the
pheromone. This phenomenon is common in many insect
groups that use chiral pheromones, including scarab
(Tumlinson et al. 1977; Leal 1996; Tolasch et al. 2003)
and other beetles (Birch et al. 1980; Levinson and Levinson
1999; Lacey et al. 2004), gall midges (Hillbur et al. 2001),
and moths (Szöcs et al. 1993; Larsson et al. 2002). The
generally accepted explanation for this strong antagonistic
effect is that it prevents cross-attraction of sympatric,
usually congeneric, species that use the antipode as a
pheromone component. Electrophysiological investigations
of insect olfactory receptor neurons have demonstrated
special sensory adaptations for avoiding enantiomers
produced by heterospecifics. Species that use chiral
pheromones often possess dedicated olfactory receptor
neurons that respond selectively to each enantiomer, also
when only one of these constitutes a pheromone component
(Okada et al. 1992; Larsson and Hansson 1998; Larsson
et al. 2002; Wojtasek et al. 1998).
In several scarab species, racemic mixtures do attract
significant numbers of beetles, indicating that these species
are indifferent to the presence of nonpheromonal enan-
tiomers (Leal 1999). Absence of biological activity to
nonpheromonal enantiomers has been demonstrated in the
scarabs Anomala octiescostata Burmeister and A. cuprea
Hope, for which the racemic mixture and pure enantiomer
are equally attractive (Leal 1999). Electrophysiological
investigations have shown that both species appear to be

196
J Chem Ecol (2008) 34:189–197
Acknowledgment We thank Björn Brage, Marie Garstad, Therese
Lindström, and Niclas Berg for the field assistance, and Fredrik
Schlyter for lending the Lindgren funnel traps. This project was
supported by Stiftelsen Eklandskapet i Linköpings kommun, Lunds
Djurskyddsfond, Bjärka-Säby stiftelsen, and the Magnus Bergvall
Foundation.
LARSSON, M. C., LEAL, W. S., and HANSSON, B. S. 1999. Olfactory
receptor neurons specific to chiral sex pheromone components in
male and female Anomala cuprea beetles (Coleoptera: Scara-
baeidae). J. Comp. Physiol 184:353–359.
LARSSON, M. C., LEAL, W. S., and HANSSON, B. S. 2001. Olfactory
receptor neurons detecting plant odours and male volatiles in
Anomala cuprea beetles (Coleoptera: Scarabaeidae). J. Insect
Physiol 47:1065–1076.
LARSSON, M. C., HALLBERG, E., KOZLOV, M. V., FRANCKE, W.,
HANSSON, B. S., and LÖFSTEDT, C. 2002. Specialized olfactory
receptor neurons mediating intra- and interspecific chemical
communication in leafminer moths Eriocrania spp. (Lepidoptera:
Eriocraniidae) . J. Exp. Biol 205:989–998.
LARSSON, M. C., HEDIN, J., SVENSSON, G. P., TOLASCH, T., and
FRANCKE, W. 2003. Characteristic odor of Osmoderma eremita
identified as a male-released pheromone. J. Chem. Ecol 29:575–587.
LEAL, W. S. 1991. (R,Z)-5-(−)-(Oct-1-enyl)oxacyclopentan-2-one, the
sex pheromone of the scarab beetle Anomala cuprea. Natur-
wissenschaften 78:521–523.
LEAL, W. S. 1996. Chemical communication in scarab beetles:
reciprocal behavioral agonist–antagonist activities of chiral
pheromones. Proc. Natl. Acad. Sci. U. S. A 93:12112–12115.
LEAL, W. S. 1999. Enantiomeric anosmia in scarab beetles. J. Chem.
Ecol 25:1055–1066.
LEVINSON, A., and LEVINSON, H. 1999. Inhibition of sexual attraction
and mating by pheromone enantiomers in male Lasioderma
serricorne. Naturwissenschaften 86:138–140.
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