Agonists and antagonists of antennal responses of gypsy moth (Lymantria dispar) to the pheromone (+)-disparlure and other odorants

Article abstract of DOI:10.1021/jf904139e

Insects use the sense of smell to guide many behaviors that are important for their survival. The gypsy moth uses a pheromone to bring females and males together over long distances. Male moth antennae are equipped with innervated sensory hairs that selectively respond to pheromone components and other odors. Host plant odors, in particular, are detected by moths and sometimes cause an enhancement of the antennal and behavioral responses of the moths to their pheromone. Inspired by naturally occurring agonists and antagonists of insect pheromone responses, we have screened, by electroantennogram (EAG) recordings, a collection of compound sets and of individual compounds. We have detected interference of some compounds with the EAG responses of male gypsy moth antennae to the pheromone. We describe three activities: (1) short-term inhibition or enhancement of mixed compound + pheromone plumes, (2) long-term inhibition of pure pheromone plumes following a mixed compound + pheromone plume, and (3) inhibition of the recovery phase of mixed compound + pheromone plumes. Long-term inhibition was robust, decayed within 30 s, and correlated with the inhibition of recovery: for both activities clear structure-activity patterns were detected. The commercial repellent N,N-diethyltoluamide (DEET) was included for comparison. The most active and reproducible short-term inhibitor was a mixture of 1 -allyl-2,4-dimethoxybenzene and 2-allyl-1,3-dimethoxybenzene. The most active long-term inhibitors were a set of 1-alkoxy-4-propoxybenzenes, DEET, and 1-ethoxy-4-propoxybenzene. DEET was more specific in the olfactory responses it inhibited than 1-ethoxy-4-propoxybenzene, and DEET did not inhibit recovery, whereas 1-ethoxy-4-propoxybenzene did. Target sites for the three activities are discussed.

Full text of DOI:10.1021/jf904139e

3708 J. Agric. Food Chem. 2010, 58, 3708–3719
DOI:10.1021/jf904139e
Agonists and Antagonists of Antennal Responses of Gypsy
Moth (Lymantria dispar) to the Pheromone
(þ)-Disparlure and Other Odorants
,†
*
‡
E
RIKA
P
LETTNER
AND
R
EGINE
GRIES
†Department of Chemistry and ‡Department of Biological Sciences, Simon Fraser University, Burnaby,
British Columbia V5A 1S6, Canada
Insects use the sense of smell to guide many behaviors that are important for their survival. The
gypsy moth uses a pheromone to bring females and males together over long distances. Male moth
antennae are equipped with innervated sensory hairs that selectively respond to pheromone
components and other odors. Host plant odors, in particular, are detected by moths and sometimes
cause an enhancement of the antennal and behavioral responses of the moths to their pheromone.
Inspired by naturally occurring agonists and antagonists of insect pheromone responses, we have
screened, by electroantennogram (EAG) recordings, a collection of compound sets and of individual
compounds. We have detected interference of some compounds with the EAG responses of male
gypsy moth antennae to the pheromone. We describe three activities: (1) short-term inhibition or
enhancement of mixed compound þ pheromone plumes, (2) long-term inhibition of pure pheromone
plumes following a mixed compound þ pheromone plume, and (3) inhibition of the recovery phase
of mixed compound þ pheromone plumes. Long-term inhibition was robust, decayed within 30 s,
and correlated with the inhibition of recovery; for both activities clear structure-activity patterns
were detected. The commercial repellent N,N-diethyltoluamide (DEET) was included for compa-
rison. The most active and reproducible short-term inhibitor was a mixture of 1-allyl-2,4-dimethoxy-
benzene and 2-allyl-1,3-dimethoxybenzene. The most active long-term inhibitors were a set of
1-alkoxy-4-propoxybenzenes, DEET, and 1-ethoxy-4-propoxybenzene. DEET was more specific in
the olfactory responses it inhibited than 1-ethoxy-4-propoxybenzene, and DEET did not inhibit
recovery, whereas 1-ethoxy-4-propoxybenzene did. Target sites for the three activities are dis-
cussed.
KEYWORDS: Lymantria dispar; dialkoxybenzene; olfaction; antenna; insect; agonist; antagonist
INTRODUCTION
experiments (8-10). revealed that (þ)-disparlure, cis-(7R,8S)-
7,8-epoxy-2-methyloctadecane (þ)-1, is the main active compo-
nent of the sex attractant pheromone of L. dispar (Scheme 1). The
enantiomer, (-)-1, has been identified as a major component of
the pheromone of the nun moth, a closely related species (5, 6).
This enantiomer is not attractive by itself to either species, but
prevents upwind flight behavior in the gypsy moth, when pre-
sented with (þ)-1. The nun moth also uses (þ)-1 as a component
in its attractant pheromone, and enantiomer (-)-1 neither attr-
acts nor inhibits the nun moth (5,6). This discrimination between
blends of enantiomeric and other components has been proposed
as one mechanism for species differentiation (5, 6, 11, 12).
The moths perceive the pheromone through sensory hairs,
sensilla trichodea, on their feather-like antennae (13). Electro-
physiological studies with male gypsy moth antennae have
revealed that the gypsy moth has innervated sensory hairs that
respond only to (þ)-1 or only to (-)-1 (7). This means that the
moth detects both enantiomers of 1, distinguishes them, and
integrates the information in the brain. A practical consequence
of this enantiomer discrimination is that the number of moths
The gypsy moth, Lymantria dispar, is a pest of hardwood trees
in Europe, Asia, and North America. The moth begins its life as
egg masses deposited by flightless females, near the place where
the females emerged and were mated. Larvae hatch and feed on
the leaves of the tree where they eclosed (1). During epidemics
gypsy moth larvae have been known to completely defoliate large
areas of forest (2). The larvae pupate and enter diapause until the
next summer, when they emerge as adults. At this time, the female
moths produce a sex attractant pheromone, which they disperse
into the air. Male moths search for females many kilometers
upwind, using the pheromone plumes to orient (1, 2).
The structure ofthis pheromone was determined to becis-(7,8)-
epoxy-2-methyloctadecane (disparlure), by isolation of the com-
pound from ∼105 female gypsy moths (3,4). Further research, in
which the enantiomers of disparlure were tested against the
antennae of male gypsy moths (5-8) and in field trapping
*Author to whom correspondence should be addressed [e-mail
plettner@sfu.ca; telephone (778) 782-3586; fax (778) 782-3765].
pubs.acs.org/JAFC
Published on Web 03/01/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3709
Scheme 1. Gypsy Moth Sex Attractant Pheromone, (þ)-1, and Two Behavioral Antagonists
caught in pheromone-baited traps is highest with (þ)-1 of high
enantiomeric purity (g98% ee) (8). Thus, the pheromone plays a
central role in the reproduction of this moth species, and eaves-
dropping into this pheromone communication has been used in
attempts to control the moth.
reduce the amount of expensive (þ)-1 required, and antagonists
would render (þ)-1 unnecessary.
MATERIALS AND METHODS
Compounds. The synthesis of compounds tested is described in a
previous paper (23). The compounds can be grouped into five classes. One
class of compound was monoalkoxyphenols (2 series), the second class was
dialkoxybenzenes (3 series), the third class was monoalkoxy allyl phenols
(4 series), the fourth class was dialkoxyallylbenzenes (5 series), and the fifth
class was eugenol and alkyleugenols.
Pheromone-based control methods that have been tested for
the gypsy moth fall into three classes: (1) saturation of the air
with pheromone to mask the females and cause mating disrup-
tion, (2) trapping large numbers of males into strategically
placed traps, and (3) trapping samples of males in monitoring
traps and spraying the appropriate area with an insecti-
cide (2, 14). Of these three methods, the third one is widely used
to pre-empt outbreaks (14). The second approach (mass
trapping) has had only limited success because the areas in
which mass trapping is necessary, to have a significant impact,
are very large. The first approach (mating disruption) carries the
risk that large numbers of moths will be attracted to the treated
area by the applied pheromone from nearby nontreated
zones (14). For the gypsy moth, mating disruption is compli-
cated by the hydrophobicity of the pheromone, which makes
formulation and biodegradation difficult (14), and by the high
cost of (þ)-1. To implement mass trapping or disruption
schemes in heavily infested areas, it is thus of interest to develop
compounds that are not perceived by themselves but that either
enhance or interfere with the perception of the naturally emitted
pheromone plumes. These compounds should not be toxic or
bioaccumulate. In nature, host plant odors have been known to
synergize with pheromone responses (15-19) and nonhost plant
odors sometimes antagonize pheromone responses (20). Natur-
al (5) or synthetic pheromone mimics can also antagonize the
response of an insect to its pheromone (21, 22).
Here we describe the screening of pheromone agonists and
antagonists, using the gypsy moth as a model species. We have
investigated compounds that are synthetic mimics of phenol-
derived odorants (Scheme 2) (23). Phenolic compounds are found
in wood smoke with known insect repellent properties, making
them candidates for laboratory screening. We have screened a
collection of individual compounds and sets of two to five
compounds with two substituents, prepared previously (23). We
employed electroantennograms (EAG) as screening method. The
activities we detected in the EAG assays were short-term and
long-term inhibition or enhancement of the antennal response to
pheromone (þ)-1. Short-term inhibition or enhancement was
seen with mixed (þ)-1/compound stimuli, and long-term inhibi-
tion of pure (þ)-1 stimuli was seen after administration of mixed
(þ)-1/compound stimuli.
Briefly, compounds were prepared from catechol (a), resorcinol (b), or
dihydroquinone (c) by alkylations and Claisen rearrangements. The
compounds derivedfrom catechol are labeled “a”, the ones from resorcinol
“b”, and the ones from dihydroquinone “c”. Groups were varied within a
set of six substituents: methyl 1, ethyl 2, propyl 3, butyl 4, isopentyl 5 and
allyl 6 (Scheme 2). Larger substituents were not explored, because we
needed the compounds to be sufficiently volatile for the electroantenno-
gram assays. Compounds and small sets of compounds (“minilibraries”)
are named in the same manner as in the synthesis reference (23).
Disparlure was from the following sources: (þ)-disparlure (Aldrich,
20
95%, [R]D þ0.8 (c 1.0, CHCl3), (-)-disparlure (gift from Dr. J. M.
Chong, University of Waterloo, ON, Canada to G. and R. Gries in 1996,
>98% ee), racemic disparlure, and dispar alkene (synthesized in E.P.’s
group, >98% pure cis and Z isomer, respectively), eugenol (vacuum
distilled (23)), 1-hexanol (Sigma-Aldrich, used as received).
Insects. Male moth pupae were obtained from the Insect Production
Service at the Canadain Forest Service’s Great Lakes Forestry Centre in
Sault Ste. Marie, Ontario (www.insect.glfc.cfs.nrcan.gc.ca) (We are grate-
ful to John Dedes for rearing the pupae.) Prior to 2006, their insect rearing
facility had a nondiapausing colony of gypsy moths, from which they
reared pupae. When the colony collapsed, they switched to frozen egg
masses from infested zones in eastern North America. Regardless of the
egg source, larvae were reared on sterile insect media (prepared in-house)
and allowed to pupate, and male pupae were shipped in cardboard
containers. Pupae were placed individually in small, round Ziploc contain-
ers (diameter ∼ 7 cm, height ∼ 4 cm) with a moist piece of filter paper
(Whatman) and a ∼1 cm diameter ventilation opening, covered with
mosquito netting, in the lid. Pupae were kept at 18-20 °C during light
(15 h) and at 16-18 °C during dark (9 h) periods.
Electroantennograms. Compounds were assayed by electroantenno-
gram (EAG) experiments. EAG traces were recorded as follows: a fully
dislodged antenna from a male gypsy moth was mounted on the end of a
reference electrode [a glass capillary with a silver wire, filled with buffered
saline (5 mM NaH2PO4, 10 mM Na2SO4, 4.5 mM KHCO3, 18 mM
MgCl2, 4 mM CaCl2, 6 mM KCl, pH adjusted to 6.8 using 5 mM
Na2HPO4)]. The tip of the antenna was severed with microscissors and
placed in the recording electrode, which was mounted on a micromani-
pulator. A constant stream of air, which had been purified (charcoal filter),
was blown over the antenna at a rate of 300 mL/min. Test samples were
delivered through a stimulus delivery tube with a side opening ∼2 cm from
the distal opening. The proximal end of the delivery tube was connected to
a controlled stimulus delivery apparatus (stimulus controller CS-O5,
Syntech Research and Equipment, Hilversum, The Netherlands). The
distal end of the delivery tube pointed at the antenna (∼1 cm from the
antenna). The side opening was connected to a cartridge fashioned from a
Pasteur pipet, which contained a small filter paper (Whatman no. 1) with
the stimulus. These pipet cartridges could be prepared a few days in
advance and stored at -80 °C wrapped in aluminum foil and in sealable
plastic bags. Sample cartridges were mounted once they had reached room
temperature. The stimulus delivery apparatus delivered a second stream of
air (a puff) through the Pasteur pipet at a velocity of 600 mL/min, at a
specified point in time, for 0.3 s. Three types of experiments were
conducted with this setup.
The activities of sets of compounds with one substituent
constant and the other varied were compared with the activities
of individual compounds with both substituents equal. Compar-
ing structure-activity patterns, we were able to find active
candidates and focus on these. DEET, a commercial insect
repellent (24), was included for comparison. The development
of pheromone olfaction agonists and antagonists is important for
practical applications as well as for understanding the insect
olfactory system. For example, agonists and antagonists could be
assessed for disorienting mate-seeking male gypsy moths. EAG
response of insects to their pheromone has been correlated to
upwind flight behavior (5, 6, 9, 12, 17, 21, 25). Agonists would

3710 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Plettner and Gries
Scheme 2. Compounds Screened in This Study and the Numbering System Used in the Description of the Synthesis of the Compoundsa
a Sets of compounds consisted of two to five compounds with the first group (R1) variable between five choices (methyl = 1, ethyl = 2, propyl = 3, n-butyl = 4, and isopentyl = 5)
and the second group attached (R2) constant. Group R2 could be choices 1-5 or allyl = 6. Eugenols were also tested here for three reasons: (1) eugenol has insect repellent
properties, (2) eugenol occurs in oak (26), a preferred host of L. dispar (1,2), and (3) the eugenol substitution pattern was not accessible through the Claisen chemistry we used to
generate the 4 and 5 series of compounds (23).
Experiment 1. This experiment explored the agonistic or antagonistic
activity of the synthetic aromatic compounds (Scheme 2) with the gypsy
moth pheromone (þ)-1. The pheromone was kept constant at 100 ng/
cartridge, and six puffs were recorded for each replicate: (i) clean air, (ii)
pure (þ)-1 (100 ng on the cartridge), (iii) (þ)-1 (100 ng) and the compound
(1 μg on the cartridge, mixed with the pheromone), (iv) (þ)-1 (100 ng) and
the compound (10 μg), (v) (þ)-1 (100 ng) and the compound (100 μg), and
(vi) pure (þ)-1 (100 ng). The following four parameters were measured
with this experiment, using the various phases of the EAG signal
(Figure 1):
the first pure pheromone puff (ii)
LTI
¼ 100 ꢀ ðd
-dðnetÞðviÞÞ=d
ðnetÞðiiÞ
ð3Þ
ðcompoundÞ
ðnetÞðiiÞ
(d) percentage inhibition of the recovery period (RI(compound)) of
the mixed puff with the highest dose of the compound (v) (eq 6).
The height of the recovery (hyperpolarization), r(puff) above the baseline
is usually ∼20% of the total deviation of the signal from the baseline
(r(puff) þ d(puff), e.g., Figure 1). For many of the mixed puffs, the
proportional height of the recovery was either greater or lesser, depending
on the compound. The proportional height of the recovery for the first
pure (þ)-1 puff (ii) is
(a) net depolarizations, d(net) (in mV), of puffs ii-vi, d(sample), corrected
for the depolarization with clean air, d(air)
R
¼ 100 ꢀ r =½rðiiÞ þ dðiiÞꢁ
ð4Þ
ðpuff iiÞ
ðiiÞ
d
ðnetÞ
¼ d
-d
ðairÞ
ð1Þ
ðsampleÞ
and the proportional height of the highest dose mixed puff (v) is
(b) percentage short-term inhibition for each compound (STI(compound)
)
of puffs iii-v, relative to the first pure pheromone puff (ii)
R
¼ 100 ꢀ r =½rðvÞ þ dðvÞꢁ
ð5Þ
ðpuff vÞ
ðvÞ
STI
¼ 100 ꢀ ðd
-dðnetÞðcompoundÞÞ=d
ð2Þ
ðcompoundÞ
ðnetÞðiiÞ
ðnetÞðiiÞ
The relative change in the recovery is
(c) percentage long-term inhibition for each compound (LTI(compound)
of the pure pheromone puff that followed the last mixed puff (vi) relative to
)
RI
ðcompoundÞ
¼ 100 ꢀ ½R
-Rðpuff vÞꢁ=R
ðpuff iiÞ
ð6Þ
ðpuff iiÞ

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3711
Figure 1. EAG assay for the compounds tested in experiment 1 (see text): (A) typical trace for compound 3c{2,3}; (B) typical trace for compound 3a{4,4}.
The puffs are numbered i-vi (see Materials and Methods) and were (i) air, (ii) pure pheromone (þ)-disparlure, (þ)-1 (100 ng), (iii-v) disparlure mixed with
the test compound, the latter at three different doses, or (vi) pure (þ)-1. Depolarizationsfromthe restingpotential arelabeledd; hyperpolarizationsseenduring
the recovery phase after a puff are labeled r. Subscripts refer to the puff number; calculations done are shown under Materials and Methods.
Experiment 2. The decay of the long-term inhibitory effect was
investigated with the strongest long-term inhibitor, set 3c{3,1-5}, and
the strongest, most consistent short-term inhibitor, set 5b{1,1}. One
hundred micrograms of the inhibitor was premixed with different doses
of (þ)-1, and the following seven puffs were aimed at the antenna: (i) clean
air, (ii) pure (þ)-1 at the overall dose being tested (10, 50, 100, 500, or
1000 ng), (iii) the mixed puff with (þ)-1 (test dose) and (100 μg of) 5b{1,1},
(iv) the mixed puff with (þ)-1(testdose) and (100 μg of) 3c{3,1-5}, (v-vii)
pure (þ)-1 at the dose being tested, administered sequentially, always
allowing the antenna to recover to baseline before a new puff. This recovery
(2) The second objective of this experiment was to determine whether
the oak volatiles, methyleugenol and 1-hexanol, can enhance the antennal
response to (þ)-1 and whether the mixed pheromone/plant odorant
stimulus can be inhibited by compound 3c{2,3} or DEET. The puff order
was (i) air, (ii) pure (þ)-1 (100 ng), (iii) (þ)-1 (100 ng) and plant odorant
(1 μg for methyleugenol and 100 μg for 1-hexanol), (iv) (þ)-1 and plant
odorant as in (iii) þ antagonist (100 μg), and (v) same as in (iii).
Depolarizations were corrected for the air response as in eq 1, and the
short-term effects were calculated for puff iii relative to puff ii and for puff
iv relative to puff ii. Long-term effects were calculated for puff v relative to
puff iii (for LTI of the mixed pheromone/plant odorant plume) and for
puff v relative to puff ii (to determine whether long-term effects of the plant
odorant and of the antagonist cancel).
Structure-Activity Modeling of Pure Compounds onto a Three-
Point Site. The compounds chosen for this exercise were pure compounds
(not “minilibraries”) and DEET. DEET was the reference compound, as it
was among the compounds with highest LTI and a molecular target site for
this compound has been proposed (28). Compounds were drawn using
ChemDraw (CambridgeSoft, 2007, version 11.0) and copied into Chem
3D (CambridgeSoft, 2007, version 11.0). Each compound was minimized
first using molecular mechanics and the MM2 force field, then using a
semiempirical method via the MOPAC interface (method = PM3; wave
function = closed shell; optimizer = EF; solvent = H2O). All minimiza-
tions were run to an rms of e0.1. Compounds were then compared to the
minimum DEET conformer by superposition. The comparison is based on
the hypothesis that an active compound will bind to a cognate site only if
the recognition groups on the compound are predisposed in the correct
orientation at or close to a stable conformation of the compound. The
more closely the recognition groups are predisposed to the active site in a
stable conformer, the more active the compound. Conversely, the less
stable the conformer of a compound that matches the active site, the less
active the compound. Details of the site and data are shown in the
Supporting Information.
usually took ∼5-7 s. The short-term inhibition STI(compound,
was
dose)
calculated for each mixed plume. The long-term inhibition LTI(dose, time) was
calculated for each dose and for each of the three puffs following the mixed
pheromone-inhibitor plume.
Experiment 3. The objective of this experiment was to determine
whether the long-term or short-term inhibitors have any effect on the
pheromone signal if puffed in pure form and well separated from the phero-
mone and whether these compounds elicit net EAG signals by themselves.
This experiment was also done with sets 3c{3,1-5} and 5b{1,1}, tested at 1,
10, and 100 μg, and with (þ)-1 cartridges containing 100 ng of
the pheromone. The following stimuli were given: (i) clean air, (ii) (þ)-1
(100 ng), (iii) 5b{1,1} (variable dose) or 3c{3,1-5} (variable dose), and
(iv-vi) (þ)-1 (100 ng). Again, the latter puffs were given to test for any time
decay of repeated disparlure signals following the pure inhibitor stimulus.
Experiment 4. (1) The first objective of this experiment was to
determine whether the strongest long-term inhibitors (antagonists, see
Results, 3c{2,3} and DEET) could also alter the EAG responses to odorants
other than (þ)-1. These included (-)-1, racemic 1, dispar alkene (the alkene
corresponding to the carbon framework of 1), methyleugenol (a character-
istic oak wood odorant), and 1-hexanol (a green leaf volatile, shown
previously to be detected by gypsy moth antennae). For the pure odorant
puffs, (-)-1 and racemic 1 were administered at 100 ng/cartridge, the alkene,
methyleugenol, was administered at 1 μg/cartridge, and 1-hexanol was
administered at 100 μg/cartridge. The antagonists were both administered at
100 μg/cartridge, mixed with the appropriate dose of the odorant. The puff
order was (i) air, (ii) pure odorant, (iii) odorant þ antagonist, and (iv) pure
odorant. All puffs were corrected for the air response as in eq 1. STI values
were obtained from the depolarization in response to puff ii relative to the
depolarization seen with puff i. LTI values were obtained by comparing the
depolarization of puff iv relative to that of puff ii.
RESULTS
The compounds we tested gave weak or no olfactory responses
by themselves in electroantennogram (EAG) experiments
(Supporting Information). The EAG trace reflects the change
in the potential across the antenna when an air puff with an

3712 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Plettner and Gries
odorant is passed over the antenna. To understand the agonistic
or antagonistic effect of the compounds on pheromone responses
by male moth antennae, four types of EAG experiments were
conducted. First, the response of the antenna elicited by a
stimulus of pure (þ)-1 was compared to the responses elicited
by blends of (þ)-1 with the test compound (Materials and
Methods, Figure 1). The mixed plumes often gave a significantly
different response, compared to the pure (þ)-1 stimuli. This effect
was termed short-term inhibition (STI). A pure (þ)-1 stimulus,
given after the mixed stimuli, was sometimes significantly inhib-
ited, compared to the initial pure (þ)-1 stimulus, and this was
termed long-term inhibition (LTI). Second, the time decay and
dose responses for LTI activities were studied. Third, the stron-
gest inhibitors were tested for their ability to cause LTI by
themselves. Fourth, the strongest long-term inhibitors were tested
(1) against other host plant and pheromone odorants and (2)
against mixtures of (þ)-1 and host plant odorants.
Experiment 2. Decay of the Long-Term Inhibitory Effect and
Dose Responses. The LTI was strongest 10 s after the mixed
antagonist/pheromone stimulus, and the inhibition decayed to
e20% within 30 s (Figure 3A) The same decay pattern was seen
for pheromone doses of 10-1000 ng. This indicates that the
antenna can fully recover from the LTI caused by a mixed
antagonist/pheromone plume. The highest LTI was seen for the
lowest competing doses of pheromone (þ)-1, but even at very
high doses of pheromone (1000 ng), there still was significant LTI
(Figure 3B). This dose-response pattern suggests that both the
inhibitor and the pheromone are necessary, in a particular ratio,
to cause LTI.
The dose response of male gypsy moth antennae to pure
pheromone (þ)-1 is shown in Figure 3C. The LTI dose responses
with respect to (þ)-1 and antagonists 5b{1,1} or 3c{3,1-5} are
shown in Figure 3D. As noted previously, set 5b{1,1} (30 ( 7%
LTI) was a less effective long-term inhibitor than set 3c{3,1-5}
(71 ( 13% LTI). This was apparent in the dose response of the
first pure pheromone puff after the mixed antagonist/pheromone
plume. For example, at 100 ng of pure pheromone the depolar-
ization was at its maximal value (∼20 mV on average), but after
the mixed plume the depolarization for 100 ng of pheromone was
only 10 mV for 5b{1,1} and 7 mV for 3c{3,1-5}. The difference
between the two antagonists became magnified at higher doses
(Figure 3D). This suggests that LTI results from allosteric effects,
caused by the antagonist and the pheromone in a particular ratio.
Experiment 3. Long-Term Inhibition after Pure Antagonist
Plumes. Sets 5b{1,1} and 3c{3,1-5} showed no significant
depolarization by themselves, indicating that these compounds
do not function as odorants (Supporting Information). Pure
pheromone (þ)-1 puffs (given at 10 s intervals, starting 10 s after
the antagonist puff) showed no significant LTI for either set or for
the mixture of 5b{1,1} and 3c{3,1-5} (Figure S2 of the Support-
ing Information). There was no significant change in the depo-
larization of the puffs that followed the antagonist, compared to
the puff preceding the antagonist. This shows that the antenna
does not exhaust itself with repeated puffs at 10 s intervals and
that the LTI seen in experiments 1 and 2 is due to the mixed
pheromone/antagonist plumes and not due to antennal exhaus-
tion. The data obtained in this experiment also indicate that LTI
activity requires the exposure of the antenna to a mixed plume,
consisting of the odorant being inhibited (pheromone (þ)-1 in
this case) and the antagonist. The long-term inhibitors are not
odorants themselves, but interfere with the pheromone response
in a mixed plume, leading to a reversible long-term effect. A
possible mechanism for this is discussed later.
Experiment 4. Long-Term Inhibition of Host Plant Odor
Responses. To determine whether the long-term inhibitory effects
of 3c{2,3} and DEET are specific to (þ)-1 or also apply to other
odorants, compound 3c{2,3} and DEET were tested further with
other odorants: (-)-1, racemic 1, dispar alkene, methyleugenol,
and 1-hexanol. Compound 3c{2,3} and DEET differed in their
short-term and long-term effects on the other odorants tested
(Table 1). With (-)-1 or racemic 1, 3c{2,3} had a highly variable
short-term enhancing effect, whereas DEET had a more consis-
tent effect. Compound 3c{2,3} had a moderate LTI activity, and
DEET was not active. With dispar alkene, both 3c{2,3} and
DEET showed similar short-term enhancements and LTI activ-
ities. Methyleugenol gave the same responses as clean air, yet the
weak signal was enhanced short-term by both 3c{2,3} and DEET,
an activity not seen with the test compounds by themselves.
In terms of LTI, only 3c{2,3} was active, giving peaks that were
smaller on average than the response to air. Leaf volatile
1-hexanol is a weak odorant whose response was not significantly
affected short-term by either antagonist. There was weak LTI
Experiment 1: Short- and Long-Term Inhibitors of Pheromone
Signaling. The complete set of STI and LTI, obtained in experi-
ment 1, is shown in Tables S1-S3 of the Supporting Information.
STI showed structure-activity patterns for some compounds
or sets (Tables S1-S3 and Figure S1 of the Supporting
Information), but for many compounds it varied between diffe-
rent batches of moths. The strongest, most robust short-term
inhibitor was set 5b{1,1} (a mixture of 1-allyl-2,4-dimethoxy-
benzene and 2-allyl-1,3-dimethoxybenzene). The eugenols
showed consistent negative STI values, signifying that they
enhanced the antennal responses to the pheromone.
Long-term inhibition (LTI) showed robust structure-activity
patterns that could be reproduced from year to year and between
different sources and lots of moths. The alkoxyphenols showed
less activity than the dialkoxybenzenes (Figure 2A-D). For
o-alkoxyphenols the LTI increased from methyl to propyl and
then decreased for butyl and isopentyl. The dialkoxybenzenes
with R1 = R2 gave higher LTI and showed structure-activity
patterns (Figure 2B): ortho compounds showed increasing LTI
with increasing group size from methyl to butyl, and a loss of
activity for isopentyl; meta compounds had highest activity for
midsized groups; and para compounds had moderate and vari-
able activity. The dialkoxybenzene sets (Figure 2C) showed
moderate activity for all ortho and meta sets, with the methyl
sets being highest. The para sets showed a structure-activity
pattern: the propyl set had the highest LTI activity. Individual
compounds from the propyl set tested (Figure 2D) and compound
3c{2,3} (1-ethoxy-4-propoxybenzene) were the strongest long-
term inhibitors. The meta allyl series of compounds (3b{n,6}) was
also tested (Figure 2E), and 3b{3,6} (1-allyloxy-3-propoxy-
benzene) was the most active long-term inhibitor.
The ortho and para 1-allyloxy, 2- or 4-alkoxybenzene sets
(3a{6,1-5} and 3c{6,1-5}) were subjected to thermal Claisen
rearrangement to provide sets 4a{1-5} and 4c{1-5}, respec-
tively. These sets of phenols were divided and alkylated to give six
new sets of compounds: 5a{1, 1-5} to 5a{6,1-5} and 5c{1,1-5}
to 5c{6,1-5}. In the meta case, the 1-allyloxy-3-alkoxybenzene
precursors were kept in smaller groups (methyl by itself, ethyl and
propyl, butyl and isopentyl) (Figure 2H), because each precursor
could form two products (23). These Claisen rearranged products
or the eugenols showed little LTI compared to the most active
dialkoxybenzenes. Among the 5a compounds, the ones with a
midsized second alkyl group (R2) were most active (Figure 2G),
and among the 5b compounds the ones with the methyl group
were most active (Figure 2H).
The strongest long-term inhibitors were DEET and set 3c-
{3,1-5} (1-alkoxy-4-propoxybenzene), of which compound
3c{2,3} (1-ethoxy-4-propoxybenzene) was the most active.

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J. Agric. Food Chem., Vol. 58, No. 6, 2010 3713
Figure 2. SAR for long-term inhibition (LTI) of EAG responses toward (þ)-1: (A) alkoxyphenols; (B) dialkoxybenzenes with equal substituents; (C)
dialkoxybenzene sets; (D) individual o-dialkoxybenzenes (3c{3,n},1-propoxy-4-alkoxybenzenes) [the dashed line indicates the LTI value obtained for DEET
((SE, shown with the solid lines)]; (E) individual m-dialkoxybenzenes (3b{n,6},1-allyloxy-3-alkoxybenzenes); (F) eugenol and derivatives; (G) allyl
dialkoxybenzenesetsobtainedfrom Claisen rearrangementof1-allyloxy-2-alkoxybenzenesand 1-allyloxy-4-alkoxybenzenes, followedbyalkylation of the new
phenol group (Scheme 1); (H) allyl dialkoxybenzene sets obtained from Claisen rearrangement of 1-allyloxy-3-alkoxybenzenes(Scheme 2). In Figure 1, the
depolarization for puff vi (second pure (þ)-1 puff) is much smaller than for puff ii (first pure (þ)-1 puff). The percentage difference between these two puffs is
the LTI (see Materials and Methods for calculations). Positive values denote inhibition, negative ones enhancement.
against 1-hexanol by 3c{2,3} but not by DEET. Compared to
DEET, compound 3c{2,3} was the more broadly tuned long-term
inhibitor. This broader inhibition of olfaction suggests that
compound 3c{2,3} targets a component of several different
populations of sensory hairs (sensilla) on the antenna.
Because insect pheromone responses are altered by host plant
odors and both methyleugenol and 1-hexanol are odorants of
oak, we compared the ability of compound 3c{2,3} and DEET to
alter the response of the male moth antennae to mixtures of
pheromone and the plant odorant (Table 2). Methyleugenol, at 10

3714 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Plettner and Gries
Figure 3. (A) Dose and time decay properties of the long-term inhibition (LTI) activity by set 3c{3,1-5}: LTI dependence on the delay time of the pure (þ)-1
puffs (v-vii, experiment2) after themixed(þ)-1/inhibitor puff. The amount of (þ)-1was varied from 10 ng to 1 μg, and theamount ofinhibitor wasconstantat
100 μg. (B) Doseresponseof LTIwith respect to variable(þ)-1 responses and time responses for thestrongest long-term inhibition ofEAG responses toward
(þ)-1. (C) Dose response of pure pheromone (þ)-1. (D) Dose response of (þ)-1 mixed with 100 mg of either compound 5b{1,1} (the strongest short-term
inhibitor) or set 3c{3,1-5} (the strongest long-term inhibitor).
times excess relative to (þ)-1, enhanced the response to pher-
omone weakly in a few cases, consistent with the previous data
with three different methyleugenol doses (see Table S3 of the
Supporting Information). Interestingly, the ternary mixture of
(þ)-1, methyleugenol, and the antagonist gave a significantly
enhanced short-term response, relative to the (þ)-1/plant odorant
mixture. Also, there was no significant LTI for either antagonist
on the (þ)-1/plant odorant mixture or on a pure (þ)-1 puff
following the mixed puff. A similar picture was obtained with
1-hexanol, except that DEET was weakly long-term enhancing,
whereas 3c{2,3} had no LTI activity. The similarities in activity
between DEET and 3c{2,3} suggest that they may act on a similar
target site, but the differences in activity also suggest that there are
additional modes of action that differ between DEET and
3c{2,3}.
Correlation between Recovery from the Mixed Antagonist/
Pheromone Plume and LTI from Experiment 1. The recovery
phase (hyperpolarization) of a pheromone stimulus is propor-
tional to the magnitude of the preceding depolarization
(Figure 1). Analysis of the correlation between the inhibition of
the recovery phase, RI (see Materials and Methods), and the LTI
revealed a strong positive correlation between these two para-
meters for all of the compounds (Supporting Information Figure
S3). Groups of compounds with similar structures also showed
correlation between LTI and RI, for example, the ortho com-
pounds (Figure S3-B of the Supporting Information, R2 = 0.9).
This suggests that the recovery phase from the antagonist/
pheromone plume can determine the ability of the antenna to
respond to a pure pheromone plume: the shallower the hyperpo-
larization of the mixed plume, the stronger the inhibition
of a following pure pheromone plume. This indicates that the
dialkoxybenzene long-term inhibitors can interfere with recovery
processes in the antenna (see below). In contrast, DEET did not
show significant RI activity (9 ( 9%, N = 6), but had strong LTI
activity. This suggests that the dialkoxybenzenes cause LTI via at
least two modes: one similar to DEET and a second mode that
affects RI and is not affected by DEET.
Correlation of LTI in Adult Male Gypsy Moths with Larval
Feeding Deterrence in Trichoplusia ni, the Cabbage Looper, and in
Gypsy Moth. Most of the compounds that were screened in this
work have also been assessed against T. ni as either feeding
deterrents or oviposition deterrents (Akhtar, Isman, Plettner,
unpublished data). There was a positive correlation between LTI
of pheromone stimulation in gypsy moth and feeding deterrence
in cabbage looper larvae for a set of 39 active compounds/sets
(R2 = 0.66). Preliminary tests with gypsy moth larvae also
suggest a correlation between LTI of pheromone stimulation in
adult males and feeding deterrence of the larvae for some of the
active compounds. This suggests that the antagonists identified in
that group of compounds target a component of the olfactory
system that is conserved between these two species of Lepidoptera
and between different developmental stages.
Modeling of Long-Term Inhibitors onto a Three-Point Site. The
single-compound antagonists with the strongest LTI activity were
modeled onto a three-point recognition site for DEET or DDT
(Figure S4-A ofthe Supporting Information), because DEET and
DDT target sites are known, and both compounds cause deter-
rence of insects by interference with olfactory cues (27, 28). Also,
DEET was one of the best LTI-causing compounds in this study
(Table S1 of the Supporting Information; Figure 2C), so it was
used as a reference compound. We assumed that three odotopes,
arranged around a benzene ring, exist for the repellent activity of

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J. Agric. Food Chem., Vol. 58, No. 6, 2010 3715
DEET or DDT: (1) a hydrocarbon chain end (CH3) or group of
equivalent size (Cl in DDT), (2) a polar moiety (amide carbonyl
group in DEET or CCl3 in DDT), and (3) a hydrocarbon chain
end (CH3 group) or π-system (aryl or vinyl) nearthe polar moiety.
These three odotopes are arranged in DEET as follows: (1)-(2)
6.1 A; (2)-(3) 3.6 A; and (3)-(1) 6.0 A. For the compounds
described here, we assumed that one of the oxygen atoms serves as
the polar group (Supporting Information Figure S4-A). For each
compound modeled, the most stable conformer was compared to
the three-point site with respect to the position of the three
odotopes. If the conformer did not fit into the site, then individual
bonds were rotated, until the distance between the odotope was
close. The distances were then constrained, and the compound
was minimized again. The heat of formation of the conformer
constrained to the three-point site was compared to the heat of
formation of the most stable conformer.
There was a negative correlation between the log of enthalpy
penalty (the enthalpy a particular compound had to invest to fit
into the three-point site relative to its minimum conformation)
and the LTI (Supporting Information Figure S4-B,C). This
suggests that, to cause LTI through the same target site as DEET,
a compound needs to fit into the three-point site at or close to its
minimum conformation. To have a stable conformation that fits
the site, the alkyl groups of the dialkoxybenzenes needed to be
within a particular size range for a particular geometry. For
example, methyl was too small overall; ethyl was too small for
ortho and meta, but reasonable for para; one propyl group along
with a smaller group was appropriate for para; butyl was
appropriate for ortho but too large for meta and para; isopentyl
was too large overall.
DISCUSSION
Peripheral (on the Antenna) versus Central (in the Brain)
Odorant Agonism and Antagonism. Short-term (blend) effects in
insect olfaction have been described before, for various naturally
occurring and synthetic blends of odorants. For example, host
plant odors are able to modulate both the EAG (15, 17, 18, 25,
29-34) and behavioral responses of insects to their pheromone
(15-20,25,31,33,35,36). Synthetic compounds can alsoalter the
peripheral responses of insects. For example, trifluoromethyl
ketone mimics of pheromones interfere with the EAG respon-
ses (21, 22, 37, 38) and with the behavior of the insects toward
their pheromone (21, 22, 38). Another example is DEET: it
caused either a decrease or an enhancement in the response to
food odors of selected olfactory neurons in Drosophila (28).
Peripheral enhancements of pheromone responses by plant
volatiles appear to be interpreted as higher pheromone doses in
the brain. For example, synergistic pheromone and leaf volatile
mixtures elicited enhanced responses by selected projection neu-
rons in the macroglomerular complex in silk moths, similar to
higher doses of pheromone (39). In the same study, the moths
showed greater behavioral sensitivity to the pheromone/plant
volatile mixtures than to the pheromone alone (39).
Direct effects of agonists or antagonists on the EAG are
mediated peripherally, through the antenna. In contrast, known
synergy/antagonism between pheromone components is medi-
ated not at the antennal level but in the brain. On the antenna,
different neurons (housed in distinct populations of sensilla)
respond to various pheromone components highly selectively
(see, e.g., ref 40). The information corresponding to individual
pheromone components is relayed from the primary sensory
neurons on the antenna to the macroglomerular complex (41)
and to higher brain centers. Because primary neurons respond in
a dose-dependent manner, even pheromone blend ratios can be
interpreted by specialized interneurons in the brain (42). Such
pheromone component mixtures can also elicit enhancement or
inhibition of a characteristic behavior triggered by the phero-
mone. An example is the pheromone synergy and antagonism in
the gypsy moth/nun moth presented in the introduction. Social
insects are another example, in which synergy and antagonism
between large multicomponent pheromone blends control the
social fabric of the colony (43, 44).
Table 1. Interaction of Several Pheromone and Plant Odorants Relevant to
the Gypsy Moth with DEET or Compound 3c{2,3} in the EAG
short-term
activityb,d (%)
long-term
activityc,d (%)
odorant
antagonist
N
(-)-disparlure
3c{2,3}
DEET
5
5
-1226 ( 1056
-322 ( 66
43 ( 12
-10 ( 29
racemic disparlure
dispar alkenea
methyleugenol
1-hexanol
3c{2,3}
DEET
6
6
-154 ( 45
-468 ( 231
69 ( 5
-126 ( 133
Potential Target Sites for Peripherally Active Compounds.
Sensillatrichodea are responsible for pheromone detection. These
are single-walled sensilla with pores, innervated by two or three
sensory neurons (13). The neurons project a dendrite into the
hollow space of the hair, the sensillar lymph space (Figure 4), and
their axon projects to the macroglomerular complex. Three
accessory cells support the neurons physiologically. First, the
thecogen cell surrounds the neuronal cell body and the inner
dendritic segment. Tight junctions between the membranes of the
neuron and the thecogen create a diffusion barrier for many
solutes and isolate the sensillar lymph space (45). Second, the
thecogen cell is surrounded by the tricogen cell, and this is
surrounded by the third cell, the tormogen. The tricogen and
3c{2,3}
DEET
4
4
-244 ( 15
-955 ( 647
39 ( 12
52 ( 22
3c{2,3}
DEET
4
4
-1158 ( 834
-598 ( 506
46 ( 24
8 ( 8
3c{2,3}
DEET
4
4
-34 ( 28
6 ( 30
29 ( 18
7 ( 18
a This compound is (Z) 2-methyloctadec-7-ene. b This refers to the short-term
inhibition (positive) or enhancement (negative) of the mixed odorant/antagonist puff,
relative to the first pure odorant puff. c This refers to the long-term inhibition (positive)
or enhancement (negative) of the pure odorant puff that followed the mixed odorant/
antagonist puff, relative to the first pure odorant puff (see Materials and Methods).
d Mean ( SE of N replicates.
Table 2. Effect of Plant Odors and DEET or Compound 3c{2,3} on the EAG Responses of Male Gypsy Moth Antennae to Pheromone (þ)-1a
short-term effect
with plant
long-term
effect on the mixed
(þ)-1/plant odor plumes
long-term
effect on
plant
synergist
short-term effect
with the plant synergistd
antagonist
synergist þ antagonist
(þ)-1 response
methyleugenolb
1-hexanolc
3c{2,3}
DEET
-39 ( 37 (9)
-19 ( 24 (10)
-125 ( 95 (4)
-210 ( 120 (5)
-99 ( 60 (5)
11 ( 47 (4)
30 ( 27 (5)
5 ( 24 (5)
12 ( 33 (4)
27 ( 29 (5)
20 ( 18 (5)
-53 ( 48 (5)
3c{2,3}
DEET
-339 ( 206 (5)
-24 ( 19 (5)
a Pheromone (þ)-1 was applied at 100 ng/cartridge. b Applied at 1 μg/cartridge. c Applied at 100 μg/cartridge. d Number of replicates given in parentheses; means ( SE.

3716 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Plettner and Gries
and the axon (58). Whether other voltage-gated channels are
involved in the sensillar responses to odorants in insects is not
known, but likely: in lobsters several IP3, one IP4, and one cAMP-
activated channel have been identified (59). Once the pheromone
stimulus has ended, the neuron ceases to depolarize and spike,
and the recovery process begins with an arrestin, which has been
mapped to sensilla of insects (60). The resting potential can then
regenerate via the Naþ/Kþ-ATPase and the support cell vacuolar
ATPase (45). The spiking onset and termination to background
levels is also controlled by two perireceptor proteins that bind
odorants and that have opposing effects on neuronal spiking: the
PBP and the sensory-neuron membrane protein (SNMP). The
SNMP appears to inhibit background spiking (61, 62), and the
PBP appears to stimulate it (63). When PBP is missing from a
sensillum, the neuron shows no background spiking and is
unresponsive, and when SNMP is missing, the neuron is perma-
nently activated. Neither mutant responds to the pheromone, and
a double mutant appears like a SNMP mutant, suggesting that
SNMP is upstream of the PBP in the control of the CR (61, 62)
and participates in signal termination (62). Genetic experiments
with fluorescent proteins have suggested that SNMP, CR, and
OR interact in close proximity (55).
Short-term agonists or antagonists may target the OR.
Agonists perhaps stabilize an active form of the OR, and
antagonists either compete with the activating odorant or
stabilize an inactive form. Similar blend effects have been
observed with vertebrate receptors. For example, the response
of one mouse OR that responds to eugenol was significantly
inhibited when mixtures of eugenol and methylisoeugenol or
isosafrole were given in the stimulus. The effect was short-term:
responses to pure eugenol following the mixed stimulus were
normal (64). In a second example, the mouse octanal receptor
was specifically short-term inhibited by citral, which did not
activate the OR by itself (65). In a third example, the human
sperm OR response to burgeonal was inhibited by the addition
of undecanal (66).
Figure 4. Schematic diagram of a sensillum trichodeum: 1, cuticle (the
cuticularwallofthesensillumhas pores thatendinporecanals);2, sensillar
lymph space [this compartment contains a solution rich in Kþ and
pheromone-binding protein (PBP, A in the top expansion window)]; 3,
sensory neuron (3a, outer dendritic segment; 3b, inner dendritic segment;
3c, cell body; 3d, proximal axon segment); 4-6, accessory cells (4,
tormogen; 5, tricogen; 6, thecogen) [thecogen, 6, envelopes the cell body
and the inner dendritic segment; the space between the cell body, 3c, and
thethecogen cell, 6, isseparated from spaces 2 and8 by tight junctionsthat
are diffusion barriers for ions and other solutes (black bars)]; 7, glial cells
(these cells envelope the axon); 8, perineurial space [this is found only
between the proximal part of the axon and the glia; the expansion windows
show known molecular components, relevant to this study, from the outer
dendrite and sensillar lymph (top), the apical membrane of the accessory
cells (middle), and the axonal/glial membranes (bottom)]; A, PBP; B,
conserved receptor; C, olfactory receptor; D, sensory neuron membrane
protein; E, ligand-activated monovalent cation channel; F, sodium/potas-
sium ATPase; G, voltage-gated ion channel; H, V-type proton/potassium
ATPase.
Long-term inhibitors may target a similar site as DEET, for
which one site has been mapped within this cascade to the CR:
mosquitoes lacking the CR did not respond to DEET behavio-
rally. In addition, DEET appears to either inhibit or enhance the
response of selected ORs to their cognate odorants (28). Because
DEET is a strong long-term inhibitor in this study, it is reason-
able to assume that the long-term inhibitors target a similar site
as DEET. This is also suggested by our modeling of the
compounds onto a three-point contact site that fits DEET or
DDT. It is interesting that long-term inhibition is seen only
when a mixed inhibitor/pheromone plume precedes the pure
pheromone stimulus that is inhibited. This observation is con-
sistent with observations made for DEET with Drosophila:
DEET affected the responses to food odorants only when it
was applied simultaneously with the odorants (28). It is also
important to note that the long-term inhibition in this study
decays within ca. 30 s (Figure 3A) and that the inhibitor by itself,
even when given alternating with pheromone every 10 s, does
not cause long-term inhibition of pheromone signals. One
interpretation of these three observations is that the three-point
site for the inhibitor is at a protein/protein interface that
transiently arises when pheromone activates the system. This
site could be between the CR/OR or CR/SNMP pairs, and this
needs to be investigated further.
tormogen have highly folded membranes on their apical sides,
facing the sensillar lymph space. These cells secrete pheromone-
binding proteins (PBPs) and Kþ ions. The ion secretion is
mediated by a vacuolar-type Hþ/Kþ-ATPase, found only on
the apical membranes of the three support cells (46). The Kþ ions
appear to be important in the maintenance of the resting receptor
neuron potential, partly through a Naþ/Kþ-ATPase located on
the neuronal plasma membrane (45).
The outer dendritic membrane is the location of the signal
transduction pathway that interacts with the pheromone and
causes the slow depolarization seen in EAG traces. The main
components that interact with odorants are specialized odorant
receptors (OR), each type being expressed in different popula-
tions of neurons, housed in different populations of sensilla
(47-51). The insect ORs are seven-transmembrane G-protein
coupled proteins; a member of the Gq family has been mapped to
insect sensory neuron dendritic membranes on the intracellular
side (52) and has been shown to play a role in insect olfactory
transduction (53, 54). One protein that resembles an OR, but is
topologically reversed, is expressed in all olfactory sensilla and
appears to be a chaperone and dimerization partner for the
odorant-selective OR (55). This “conserved receptor” (CR) also
appears to form or interact with a monovalent cation channel,
upon odorant activation of the OR. This might be the conduit for
the initial slow depolarization of the sensory neuron (56, 57).
Once the dendrite has been depolarized, voltage-gated sodium
channels generate action potentials. Sodium channels have been
detected in cultured insect neurons on the outer dendritic segment
Unlike DEET, many of the long-term inhibitors from our
collection of dialkoxybenzenes also caused inhibition of the
hyperpolarization of the mixed puff preceding the pure phero-
mone test stimulus. This suggests that these compounds may bind
to a second site that is involved in the recovery phase. One

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J. Agric. Food Chem., Vol. 58, No. 6, 2010 3717
candidate for this second site is the Naþ/Kþ-ATPase. If this
enzyme is inhibited, then the potentials across the dendritic
membrane will not return to the optimal level, and the EAG
depolarization of a closely following signal will be less because the
ion gradients are smaller. DDT also affects olfactory responses in
insects, and it is known that DDT targets the mitochondrial
ATPase and a Ca2þ-ATPase, but can also weakly inhibit the
Naþ/Kþ-ATPase (67). It is unlikely that our compounds inhibit
the mitochondrial or vacuolar ATPases, because they do not
show acute toxicity toward moths (68). A second candidate for
the site of action for the recovery inhibitors is the voltage-gated
sodium channel. Insecticides that cause paralysis (knockdown)
and sensory disruption, such as DDT, are known to bind to the
insect voltage-gated Naþ channel (69). Our inhibitors did not
cause paralysis in the moths, which suggests that the voltage-
gated Naþ channel is an unlikely target site. However, odorant-
activated channels could be affected by the inhibitor þ phero-
mone mixtures. Finally, the compounds that inhibit the recovery
phase of EAG traces from mixed stimuli might interfere with the
recovery function of SNMP. The SNMP has been shown to
regulate the decay of action potential frequency in sensilla, after a
stimulus, to the background frequency (62). This recoveryprocess
may require the close interactions that have been demonstrated
between SNMP, CR, and OR (55). Our observation that com-
pound 3c{2,3} is inhibitory against other odorants and other
developmental stages of moths is consistent with the hypothesis
that it targets conserved components of the pheripheral olfactory
system.
Potential Practical Application of the Agonists and Antagonists.
Some of the compounds from this study have been field tested,
but more tests are required to develop a practical application.
Potential applications could include enhanced attraction of
insects to traps, masking of the pheromone emitted by the
females, or deterrence of the insects from certain areas. Attraction
could be usedfor monitoringor, ifattraction is sufficientlystrong,
sufficient traps are deployed, and the population density is
suitable, mass trapping of males. Masking of the natural pher-
omone plumes and deterrence could be used to protect valuable
stands of trees or to prevent an outbreak in certain areas.
Conclusion. We describe the activity of synthetic aromatic
compounds that modulate the electrophysiological responses of
male gypsy moth antennae to the pheromone. Compounds were
identified that cause short-term enhancements of inhibition of
olfactory responses. Other compounds caused inhibition of the
recovery phase of mixed pheromone þ compound plumes and,
finally, several compounds caused the inhibition of pure pher-
omone stimuli that followed the mixed plume. The best long-term
inhibitor was 1-ethoxy-4-propoxybenzene. On the basis of the
structure-activity relationships we propose that this long-term
inhibitor acts on a conserved component of the moth’s peripheral
olfactory system.
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We thank the Canadian Forest Service (Sault Ste. Marie) for
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Supporting Information Available: Tables S1-S6, Figures
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Received for review November 27, 2009. Revised manuscript received
February 12, 2010. Accepted February 17, 2010. This research was
funded by the Natural Sciences and Engineering Research Council
(NSERC) of Canada, Grants STPGP 307515-04 and STPGP 351045-
2007 to E.P., and in part (R.G.) by a NSERC Industrial Research Chair to
G. Gries, with Contech Enterprises Inc., SC Johnson Canada, and Global
Forest Science (GF-18-2007-226; GF-18-2007-227) as industrial sponsors.