Development and biological activity of a new antagonist of the pheromone of the codling moth cydia pomonella

Article abstract of DOI:10.1021/jf901979k

A new pheromone antagonist of the codling moth Cydla pomonella is reported. Presaturation of the antennae of the insects with vapors of the antagonist (E,E)-8,10-dodecadienyl trifluoromethyl ketone, analogue of the main component of the pheromone (codlemo

Full text of DOI:10.1021/jf901979k

8514 J. Agric. Food Chem. 2009, 57, 8514–8519
DOI:10.1021/jf901979k
Development and Biological Activity of a New Antagonist of the
Pheromone of the Codling Moth Cydia pomonella
§
M
ARTA
G
INER,† ALBERT
S
ANS,† MAGI
R
,
IBA,† DOLORS
AND NGEL
B
G
OSCH,‡ RAFAEL
,§
G
AGO
,
J
OSEP
R
AYO,§ GLORIA
R
OSELL
A
UERRERO
*
†University of Lleida and ‡Institute of Agri-Food Research and Technology (IRTA), Centre UdL-IRTA,
Rovira Roure, 191, 25198 Lleida, Spain, §Department of Biological Chemistry and Molecular Modelling,
Institute of Advanced Chemistry of Catalonia (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain, and
Department of Pharmacology and Therapeutic Chemistry, Unit Associated with CSIC, Faculty of
Pharmacy, University of Barcelona, Avda. Diagonal s/n., 08028 Barcelona, Spain
A new pheromone antagonist of the codling moth Cydia pomonella is reported. Presaturation of the
antennae of the insects with vapors of the antagonist (E,E)-8,10-dodecadienyl trifluoromethyl
ketone, analogue of the main component of the pheromone (codlemone), resulted in lower
electrophysiological responses to the pheromone relative to untreated insects. In the wind tunnel,
the compound elicited a remarkable disruptive effect on all types of behavior of males flying toward
a source baited with a pheromone/antagonist blend in 1:1, 1:5, and 1:10 ratios. The insects
displayed erratic flights in the presence of the antagonist, as shown by their flight parameters in
comparison to insects attracted to the pheromone alone. In the field, traps baited with mixtures of
1:10 codlemone/antagonist attracted considerably lower numbers of males than the natural
attractant. The antagonist, however, did not inhibit the pheromone-degrading enzymes present in
male antennae, suggesting that trifluoromethyl ketones are not sufficiently electrophilic to produce a
stable intermediate adduct with a cysteine residue of the enzyme, a mechanism previously proposed
for oxidase inhibition in insects. Overall and taking into account our previous reports and,
particularly, the reduction in damage induced in maize fields by a trifluoromethyl ketone analogue
´
of the pheromone of Sesamia nonagrioides (Sole, J.; Sans, A.; Riba, M.; Rosa, E.; Bosch, M. P.;
ꢀ
Barrot, M.; Palencia, J.; Castella, J.; Guerrero, A. Reduction of damage by the Mediterranean corn
borer, Sesamia nonagrioides, and the European corn borer, Ostrinia nubilalis, in maize fields by a
trifluoromethyl ketone pheromone analog. Entomol. Exp. Appl. 2008, 126, 28-39), the antagonist
might be a new candidate to consider in future strategies to control the codling moth.
KEYWORDS: Pheromone antagonist; codling moth; Cydia pomonella; trifluoromethyl ketones; elec-
trophysiology; wind tunnel; field tests
INTRODUCTION
of gravid females is prevented (6). Therefore, new approaches to
control the codling moth are needed.
The codling moth, Cydia pomonella (L.) (Lepidoptera:
Tortricidae), is one of the most important pests of apples, pears,
and walnuts worldwide. It is widely distributed across the pome
fruit-producing regions and can cause a high level of damage if it
is not properly controlled (2). Broad-spectrum pesticides have
been widely used to control the pest, but their secondary effects on
human health, natural enemies, and the environment along with
induced resistance to a variety of them preclude their use in the
future. The main sex pheromone component of the codling moth
was reported by Roelofs et al. (3) as (E,E)-8,10-dodecadienol
(codlemone), and several minor components of the pheromone
were later identified (4). The pheromone has been extensively
used to monitor and control pest populations via mating disrup-
tion techniques (5). However, control by mating disruption alone
is only effective at low population levels and where immigration
Pheromone disruptants of chemical communication are new
biorational approaches for insect control (7). Trifluoromethyl
ketones have been shown to be potent pheromone antagonists of
Spodoptera littoralis, Sesamia nonagrioides (8), and Ostrinia
nubilalis (9) when mixed with the pheromone in different ratios.
In these insects, structurally similar trifluoromethyl ketones to the
major component of the pheromone disrupted male orientation
flights to pheromone sources in a wind tunnel. In the field, these
chemicals elicited a significant decrease in the number of males
caught in traps baited with mixtures of the pheromone and the
antagonist compared to the pheromone alone. In addition, these
compounds reduced the electrophysiological responses to the
pheromone in S. littoralis, Mamestra brassicae, and Heliothis
zea (10). Trifluoromethyl ketones are also able to inhibit a
large number of esterases and proteases (11), including phero-
mone-degrading enzymes (PDEs) present in insect olfactory
tissues (8,10,12). These are key enzymes for the rapid degradation
*To whom correspondence should be addressed. Telephone: þ34-
93-4006120. Fax: þ34-93-2045904. E-mail: agpqob@iiqab.csic.es.
pubs.acs.org/JAFC
Published on Web 08/24/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8515
ofpheromone esters, thus maintaining a lowstimulusnoiselevelin
sensory hairs. The high potency of trifluoromethyl ketones is due
to the high polarization of the carbonyl by the trifluoromethyl
group, thereby increasing the electrophilicity of the carbonyl
carbon and making it more susceptible to nucleophilic attack by
a catalytic residue in the enzyme active site.
We have reported that antennal esterase extracts of S. littoralis
and S. nonagrioides were inhibited in a slow-binding manner by a
number of trifluoromethyl ketones and other fluorinated deriva-
tives (12, 13). More recently, (Z)-11-tetradecenyl trifluoromethyl
ketone and its homologue (Z)-10-tridecenyl trifluoromethyl
ketone were found to be potent inhibitors of male antennal
esterase preparations of O. nubilalis, with an IC50 value of 0.28
and 7.5 μM, respectively (9). As noticed, all insects considered
thus far have an ester (acetate) as the main pheromone structural
motif, and therefore, at this point, it would be important to know
whether these fluorinated chemicals also act as pheromone
antagonists of insects lacking the ester moiety in their pheromone
composition and at the same time inhibit other enzymes
(oxidases, dehydrogenases, etc.) that are responsible for phero-
mone degradation. In this study, we investigated for the first time
the activity of (E,E)-8,10-dodecadienyl trifluoromethyl ketone
(Figure 1) as a putative antagonist of the pheromone of C.
pomonella in the laboratory and in the field and disclose its effect
on male antennal degrading enzymes.
Figure 1. Chemical structures of codlemone (1) and the antagonist (E,E)-
8,10-dodecadienyl trifluoromethyl ketone (2).
(300 mL/min) through a Pasteur pipet for 100 ms using a stimulus
controller TC-05 (Syntech). The pipet contained a small piece of filter
paper (2 ꢀ 1 cm) on which the stimulus compounds, diluted in hexane to
the appropriate concentrations, had been deposited. The solvent was
allowed to evaporate before the tests. For the intrinsic activity, five puffs
over 100 ng of each compound were applied to one male antenna at 45 s
intervals. Antennae of 12 insects were used for the experiments. Control
puffs with a piece of paper containing only solvent (hexane) were also
intercalated between two consecutive stimuli to determine the baseline
depolarization of the antenna. The signals were amplified (100ꢀ) and
filtered (DC to 1 kHz) with an ID-2 interface (Syntech), digitized on a PC,
and analyzed with the EAG2000 program. Depolarization means were
compared for significance using the least significant difference (LSD) test
(p<0.05).
For the inhibition assays, 2-3-day-old males (N = 20) were placed in
Petri dishes (9 cm in diameter) containing a small piece of filter paper (2 ꢀ
2 cm) on which varying amounts of the antagonist (1, 10, 50, and 100 μg)
dissolved in hexane had been deposited. The hexane was allowed to
evaporate before the assays. Males were exposed for 2 h in the dark to
vapors of the compound. Then, they were taken to another clean contain-
er, conditioned to light for 5 min, and their antennae were excised for EAG
recordings. The same protocol as for the intrinsic activity assay was
followed, and the depolarization means were compared for significance
(LSD test, p < 0.05) to those obtained with control insects (N=20), which
had been treated under the same conditions in the absence of the
antagonist.
MATERIALS AND METHODS
Chemicals. Codlemone (99.5 and 99.8% chemical and isomeric purity,
respectively) was purchased from Pherobank (The Netherlands). (E,E)-1-
Iodo-8,10-dodecadiene was obtained from (E,E)-8,10-dodecadienol as
previously described (14). The solvents were purchased from Fluka
(Sigma-Aldrich, Schweiz) and dried according to established procedures.
Other commercial products [tert-BuLi, n-decanol, nicotinamide adenine
dinucleotide (NAD), and ethyl trifluoroacetate] were obtained from
Sigma-Aldrich.
(E,E)-8,10-Dodecadienyl Trifluoromethyl Ketone. A solution of
(E,E)-1-iodo-8,10-dodecadiene (196 mg, 0.67 mmol) in a mixture of
anhydrous 3:2 pentane/ether (6 mL) was cooled to -78 °C under Ar.
Then, a 1.7 M tert-BuLi solution in hexane (0.6 mL, 1 mmol) was added,
and the mixture was stirred for 5 min. A solution of ethyl trifluoroacetate
(0.47 mL, 4.02 mmol) was then added, and the mixture stirred for 1 h at
-78 °C. The system was allowed to warm to room temperature and
quenched with NH4Cl saturated solution, and the organic phase was
washed with brine. After drying with anhydrous MgSO4 and evaporation
of the solvent, the crude was purified by flash chromatography on silica
gel, eluting with hexane to provide the expected trifluoromethyl ketone as
an oil (146 mg, 81%). IR (film) ν: 3016, 2929, 2856, 1765, 1456, 1404, 1377,
1295, 1208, 1150, 1042 cm-1. 1H nuclear magnetic resonance (NMR) (300
MHz, CDCl3) δ: 5.95-6.05 (m, 2H), 5.5-5.6 (m, 2H), 2.7 (t, J=8 Hz, 2H),
2.03 (broad t, J=8 Hz, 2H), 1.73 (d, J=8 Hz, 3H), 1.60-1.68 (b, 2H),
1.25-1.4 (b, 8H) ppm. 13C NMR (100 MHz, CDCl3) δ: 191.9 (q, J=
35 Hz, CO), 132.16 (CH), 131.85 (CH), 130.57 (CH), 127.07 (CH), 115.79
(q, J=292.8 Hz, CF3), 36.58 (CH2), 32.70 (CH2), 29.50 (CH2), 29.26
(CH2), 29.08 (CH2), 28.90 (CH2), 22.57 (CH2), 18.25 (CH3) ppm. 19F
NMR (282 MHz, CDCl3) δ: 79.87 (s) ppm. High-resolution mass spectro-
metry (HRMS) Calcd. for C14H20F3O (Mþ), 261.1466; found, 261.1459.
Insects. Insects were collected from an infested apple orchard in Lleida
(northeast Spain) and maintained on a semi-synthetic diet (15) under a
16:8 h light/dark photoperiod at 25 ( 5 °C. Insects were reared in small
groups (3-4 individuals) in small cylindrical plastic boxes. The last instar
larvae were sexed, and males were maintained on corrugated cardboard in
plastic boxes (15 cm diameter ꢀ 7 cm high) at 23 ( 2 °C until emergence.
Males of 2-3 days old were used for the experiments.
Wind Tunnel Experiments. Assays were conducted in a glass tunnel
of 200 ꢀ 50 ꢀ 50 cm, as previously described (8). Illumination (10-20 lux,
simulating crepuscular conditions) was obtained by two red light bulbs
covered with a white sheet and located 2 m above the tunnel. The average
temperature inside the tunnel was 23 ( 1 °C, and the relative humidity was
65 ( 5%. The air flow in the tunnel was regulated to 0.15 m/s. The odor
source consisted of a rubber septum containing 10 μg of codlemone alone
or mixed with varying amounts of antagonist (10, 50, and 100 μg). The
septa were hung at 25 cm high and 30 cm distance of the far end of
the tunnel. Before the tests, a single insect in the first hour of scotophase
was placed into a glass cylinder (2.5 cm in diameter and 15 cm in height),
and this was placed on a holder into the tunnel at 20 cm high and 125 cm
from the emission source. After a 30 s acclimatization period, the cover of
the cylinder was removed and the behavior of the male was recorded for
3 min. For each responding insect, the following types of behavior were
considered: wing fanning (WF), upwind flight onto the pheromone plume
until 1/3 (F1), 2/3 (F2), and 3/3 (F3) of the total length of the tunnel, and
source contact (SC). For each treatment, a minimum of 45 males were used
and each insect was tested only once. The experiments were conducted in
blocks with exposed and control insects, and statistical analysis was
performed within each block (χ2 homogeneity test, p < 0.05).
Tracks of flying insects to an attractant source containing pheromone
(10 μg) and a 2:1 mixture of codlemone (10 μg) and the trifluoromethyl
ketone (5 μg) were recorded with a video camera CCD 2400JB Presentco
(Rister, Barcelona, Spain) equipped with a 12 mm wide-angle lens. The
camera was mounted at a height of 140 cm above the tunnel in a
perpendicular position to record the insects flight with minimal optical
distortion. The camera covered a 130 ꢀ 45 cm section of the tunnel, and the
recorded tracks were sent to a monitor for visualization. The tracks were
converted into computer files at a rate of 25 frames/s with the aid of digital
video software (Pinnacle Systems 5.1, Mountain View, CA). The succes-
sive insect positions were converted into XY coordinates using an in-house
software, and only uninterrupted flights were considered. The following
Electrophysiology Tests. The electroantennogram (EAG) apparatus
was purchased from Syntech (Hilversum, The Netherlands). In brief, a
flow of humidified pure air (1000 mL/min) was continuously directed over
the male antenna through the main branch of a glass tube (7 cm long ꢀ
5 mm diameter). Test stimulations were carried out by giving puffs of air

8516 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Giner et al.
flight parameters were recorded: flight duration (s), flight distance (cm),
ground speed (cm/s), turning frequency (turns/s), track angle (degrees),
drift angle (degrees), and course angle (degrees) (for the significance of
´
these parameters, see Kuenen and Carde (16)) and analyzed for signifi-
cance (LSD test, p < 0.05).
PDE Inhibition. Antennae of 1-2-day-old males were excised and
stored at -80 °C until use. To minimize individual variations in the
extracts, a single large extract was prepared, from which the required
aliquots were taken out for the assays. Several (3-4) Eppendorf tubes
containing a total of ca. 600 antennae were immersed in liquid nitrogen
and powdered with a plastic homogenizer. The tubes were then placed on
ice, and to them, a total of 1.8 mL of 100 mM sodium phosphate buffer
(pH 6) was added. The suspensions were combined, and the resulting
extract was sonicated at 40 W in an ultrasonic bath at 4 °C for 10 s. The
suspension was centrifuged at 12000g and 4 °C for 10 min. The super-
natant was removed, distributed in several Eppendorf tubes, and stored at
-80 °C until analysis. Toeach tubea solution of NAD inbuffer (pH 6) was
added, so that the final concentration in the tube was 1 mM. A total of
100 μL of these solutions (equivalent to 10 antennae) was placed in
borosilicate tubes with the corresponding amount of the possible inhibitor
(2 μL of a 250-1500 μM solution in DMSO or MeCN). The solution was
vortexed for 30 s and preincubated in a thermostatic bath at 25 °C for 10,
30, and 60 min. Then, 2 μL of a 15 μM solution of codlemone in DMSO or
MeCN was added, and incubation was continued for a further 60 or
90 min. Then, 180 μL of 9:1 hexane/ether containing n-decanol as the
internal standard was added. The mixture was shaken, and the organic
phase was carefully decanted and concentrated to 20-30 μL for gas
chromatography (GC) analysis. No inhibitor was present in control
experiments. Chromatographic analyses were carried out in a Trace
2000 gas chromatograph, equipped with a split-splitless injector system,
flame ionization detector (FID), and a 25 m ꢀ 0.2 mm inner diameter ꢀ
0.25 μm HP-5 fused silica capillary column (Agilent Technologies,
Madrid, Spain). The chromatographic conditions were as follows: injec-
tion at 80 °C for 1 min and program of 10 °C/min up to 150 °C, 3 °C/min
up to 180 °C, and 5 °C/min up to 250 °C, which was maintained for 5 min
more. The carrier gas was helium at a flow of 1 mL/min. Under these
conditions, codlemone, (E,E)-8,10-dodecadienal, and (E,E)-8,10-dodeca-
dienoic acid showed retention times of 14.8, 13.2, and 16.3 min, respec-
tively. GC-mass spectrometry (MS) analyses were carried out on a Fisons
MD 800 instrument under EI conditions using a 30 m ꢀ 0.25 mm inner
diameter ꢀ 0.25 μm HP-5MS fused silica capillary column (Agilent
Technologies). The analyses were performed in 2-3 replicates.
Field Tests. Delta traps were deployed in commercial apple orchards
cv. Golden in the fruit-growing area of the Lleida province from May to
August 2006-2008. Solutions of codlemone and (E,E)-8,10-12:trifluoro-
methyl ketone were prepared in hexane, and the required volume contain-
ing 100 μg of the pheromone and the corresponding amount of the
antagonist was applied on 9-10 mm red rubber septa (Aldrich, Milwau-
kee, WI). The traps were hung in the canopy of apple trees at a height of
2.5 m and spaced a minimum of 20 m from each other. Four trials using
blends of codlemone/trifluoromethyl ketone in 1:0, 1:0.5, 1:1 (this mixture
in two different fields), and 1:10 ratios were carried out in 2006 with 3-6
replicates per formulation and no rotation of traps. New experiments were
conducted in 2007 and 2008, with blends in 1:0, 0:1, 1:5, and 1:10 ratios
(5 replicates per formulation). In these trials, a fully randomized block was
considered and the traps were rotated once or twice a week according to
the population density. Trials finished after two complete trap position
Figure 2. Mean [(standard deviation (SD)] EAG responses of C.
pomonella male antennae (N = 20) to codlemone after 2 h of exposure
in a Petri dish to vapors of different amounts of (E,E)-8,10-dodecadienyl
trifluoromethyl ketone absorbed on a filter paper relative to the control
(N = 20). Bars with the same letter are not significantly different (LSD test,
p < 0.05).
control agents (7, 17). After having discovered new disruptants
of pheromone communication in insect pests, such as S. littor-
alis (8, 10), S. nonagrioides (1, 8, 18), and O. nubilalis (1, 9), we
were intrigued to know whether the same family of chemicals
(trifluoromethyl ketones) could also affect the chemical commu-
nication of other important pests, such as the codling moth,
whose major pheromone component does not contain an
ester moiety. Trifluoromethyl ketones are known to inhibit the
antennal esterase responsible for the catabolism of pheromone
acetates (12,13,19), but there are no reports on the effect on other
PDEs.
Electrophysiology. (E,E)-8,10-12:Trifluoromethyl ketone eli-
cited on male antennae an electrophysiological response of 42 (
11% in comparison to codlemone. This is in line with the lower
but significant activity displayed by other antagonists of the
pheromone, such as codlemone acetate (20). In presaturation
experiments using several amounts of the fluorinated ketone,
antennae of treated males displayed lower EAG depolarization
responses to the pheromone than those of untreated insects, with
the effect being dose-dependent. Vapors of 1 and 10 μg of the
antagonist induced only a small, non-significant inhibition of the
pheromone response. However, doses of 50 and 100 μg of the
chemical elicited a significant 92-93% lower EAG response to
the pheromone (Figure 2). For comparison purposes, control
insects that had been handled similarly to treated males in the
absence of ketone displayed virtually identical EAG responses to
regular insects (2.1-2.7 ( 0.6 versus 2.4 ( 0.6 mV, respectively).
Similar results were previously reported by Renou et al. (10), who
found that S. littoralis males that had been exposed to air loaded
with 3-octylthiotrifluoropropan-2-one displayed a decreased
EAG amplitude and an increased repolarization time in response
to the pheromone. The authors also found that 3-octylthiotri-
fluoropropan-2-one reduced the amplitude of the EAG responses
to (Z)-11-hexadecenol, a known inhibitor of the main pheromone
compound of M. brassicae. In all cases, the effects were reversible.
Wind Tunnel. In the presence of codlemone, over 90% of males
took flight and 61% of them contacted the source. However,
when males were allowed to fly toward a lure containing mixtures
of codlemone and the fluorinated ketone, they experienced a clear
disruptive effect on all types of behavior. Thus, in the presence of
a 1:1 mixture of codlemone/trifluoromethyl ketone only 53% of
males displayed wing fanning, 45% flew 1/3 of the length of the
tunnel, and 20% contacted the source. This disruptive effect was
directly correlated with the amount of antagonist present in the
rotations in 2007 and three complete rotations in 2008. The total number
√
of catches per trap was transformed ( x þ 0.5) prior to analysis. Data
from the 2006 trials were analyzed by a Student’s t test (p < 0.05). Data
from the 2007 and 2008 trials were combined in a 2-way ANOVA, and the
mean number of catches was compared by a Tukey’s test (p < 0.05)
because the interaction treatment-year was not significant. Analyses were
carried out with GenSat 11th ed. for Windows. Details of the field trials are
shown in the Supporting Information.
RESULTS AND DISCUSSION
Disruption of chemical communication in insects is a biora-
tional approach to interfere location and mating of insects, and
therefore, it may lead to development of new species-selective pest

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8517
lure, and thus, in the presence of 1:5 and 1:10 codlemone/
antagonist mixtures, only 7-11% of males passed 2/3 the length
of the tunnel and 2% successfully contacted the source (Figure 3).
A similar type of effect in a wind tunnel has also been induced by
trifluoromethyl ketones on other insects, such as S. littoralis (8),
S. nonagrioides (8, 18), and O. nubilalis (9).
through hydrolysis to the corresponding alcohol followed by
oxidation to the acid probably through the action of an alcohol
oxidase (23).
To develop a reliable procedure for inhibition of the PDE,
incubation of antennal extracts with the antagonist was tested
under different experimental conditions (concentration of the
substrate, time and temperature of incubation, co-solvent, type of
buffer, and pH). The optimal conditions were use of MeCN as a
co-solvent in amounts e4%, the presence of NAD and phosphate
buffer at pH 6, preincubation of the ketone for 30 min, and
incubation of the antennal extracts for 90 min. Degradation of
codlemone against incubation time followed an exponential
decaying curve, with the following relative amounts of phero-
mone remaining: 72.9% of pheromone at 0 min, 80.2% of
pheromone at 10 min, 45.5% of pheromone at 30 min, 25.1%
of pheromone at 60 min, 11.1% of pheromone at 90 min, and
2.2% of pheromone at 180 min (Figure 4). It is interesting to note
that immediately after the addition of the substrate ca. 27% of
pheromone was not extracted from the system, whereas in a blank
experiment in the absence of enzyme extraction of the pheromone
was almost complete. A fast binding of the substrate (bombykol)
with the pheromone-binding protein (PBP) of Bombyx mori has
been noted by Leal et al. (24), who determined the half-life for the
uptake of pheromone in vivo as ca. 1 ms in base to the high
concentration of PBP in the sensillar lymph (10 mM) (Figure 4).
The presence of the possible catabolic products of the pheromone
[(E,E)-8,10-dodecadienal and (E,E)-8,10-dodecadienoic acid; see
above] was checked by GC and GC-MS in comparison to
authentic samples. However, because these compounds were only
detected in very minute amounts (ca. 1% relative to the nonde-
graded pheromone) at incubation times up to 180 min, the extent
of the inhibition was determined in base to the amount of the
A number of flight tracks of males flying toward the phero-
mone and 2:1 pheromone/antagonist were also recorded. Tracks
in the presence of the antagonist showed profound differences
compared to those of males flying toward codlemone alone
(Table 1). Track and course angles were remarkably higher in
insects flying in plumes containing the antagonist than in codle-
mone alone (24.2 ( 6° versus 87.2 ( 4.6°, respectively, for the
track angle and 8.2 ( 3.3° versus 32 ( 12°, respectively, for
the course angle), indicating a much more directed flight toward
the source in the absence of antagonist. The drift angle (difference
between the course and the track angle) was also significantly
higher. In the presence of antagonist, males flew 1.5-fold longer
distances than control insects, although they did not take longer
to reach the source. Consequently, insects increased their ground
speed (121 ( 15 versus 97 ( 8 cm/s of control insects) to
compensate for the casting movement induced by the antagonist
(Table 1).
PDE Inhibition. In base to previous reports, we assumed that
the most likely metabolic products of codlemone could be the
aldehyde and the acid. Thus, Prestwich and co-workers (21, 22)
detected the corresponding carboxylic acid in metabolism studies
of aldehyde pheromones of Heliothis sp. In addition, degradation
of (Z)-11-tetradecenyl acetate, the major component of the
pheromone of the Z strain of O. nubilalis, was reported to occur
Figure 3. Behavioral responses of C. pomonella males flying toward a
source baited with mixtures of codlemone and (E,E)-8,10-dodecadienyl
trifluoromethyl ketonein 1:1 (N = 51), 1:5(N = 46), and 1:10 (N = 45) ratios
with regard to codlemone alone (N = 79) in the wind tunnel. Means within a
specific behavior followed by the same letter are not significantly different
(2 ꢀ 2 χ2 homogeneity test, p < 0.05). Behavioral responses are wind
fanning (WF), flying upwind over 1/3 (F1), 2/3 (F2), or 3/3 (F3) the length of
the tunnel, and source contact (SC).
Figure 4. Effectof the incubation time on the pheromonecatabolismofthe
codling moth C. pomonella.
Table 1. Selected Parameters from Tracks of C. pomonella Males Flying Upwind Toward an Attractant Source Containing a 2:1 Mixture of Codlemone and
(E,E)-8,10-Dodecadienyl Trifluoromethyl Ketone Relative to Pheromone Alonea
parameter
codlemone (N = 5)
2:1 codlemone/trifluoromethyl ketone (N = 4)
total flight distance (cm)
total flight duration (s)
ground speed (cm/s)
turning frequency (turns/s)
track angle (degrees)
drift angle (degrees)
227 (17.6) a
7.1 (0.4) a
97 (8) a
340.5 (63.5) b
8.05 (1.1) a
121 (15) b
4.75 (0.5) b
87.2 (4.6) b
55.2 (8.5) b
32 (12) b
11.6 (2.45) a
24.2 (6) a
16 (4.5) a
8.2 (3.3) a
course angle (degrees)
a Means [(standard error of the mean (SEM)] within a row followed by different letters are significantly different (LSD test, p < 0.05).

8518 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Giner et al.
Figure 5. PDE inhibition by (E,E)-8,10-dodecadienyl trifluoromethyl ke-
tone (average ( SD of 2-3 replicates) in acetonitrile at pH 6 and in the
presence of NAD as a co-factor. Blank experiment, no enzyme; control,
enzyme present but no antagonist. Preincubation period of the antagonist,
30 min; incubation period with the enzyme extract, 90 min.
Figure 7. Mean ((SD) number of catches of C. pomonella males per trap
baited with mixtures of codlemone and (E,E)-8,10-dodecadienyl trifluoro-
methyl ketone in 1:0, 1:5, 1:10, and 0:1 ratios in field trials conducted in
2007 and 2008 (5 replicates per formulation each year). The amount of
codlemone in each trap was 100 μg. The results of the 2 years are
combined because the interaction treatment-year was not significant.
Bars with the same letter are not significantly different (Tukey’s test,
p < 0.05).
(Z,Z)-3,6-dodecadienol, the trail pheromone of Reticulitermes
flavipes, which did not degrade to the corresponding carboxylic
acid but underwent a ω-oxidation to the 1,12 diol, probably
through the action of a monooxygenase (27).
Field Tests. The activity of the antagonist in the field was
evaluated by comparing the number of insects caught with blends
of codlemone/trifluoromethyl ketone relative to those trapped
with codlemone alone. In 2006, although fewer catches than the
pheromone were caught with 1:0.5 and 1:1 (this formulation
tested in two different fields) blends, the difference was only
significant when the baits contained the 1:10 pheromone/antago-
nist mixture (22.0 ( 0.4.6 insects/trap caught with the pheromone
versus 2.0 ( 1.2 with the blend) (Figure 6). New experiments
with 1:0, 1:5, 1:10, and 0:1 codlemone/antagonist formulations
were also implemented in 2007 and 2008. In these trials, the
number of catches were quite low, and because the interaction
treatment-year was not significant (F=1.3; df=3.28; p=0.29),
the number of catches per trap and year were combined. Here,
again, the 1:10 blend caught a significantly lower number of
moths (6.3 ( 1.3) than codlemone alone (12.0 ( 1.5). The
antagonist was not attractive to codling moth males when used
alone (1.6 ( 0.3 insects/trap) (Figure 7).
Pheromone antagonists have been used to control pests in
mating disruption experiments (17). In general, these chemicals
are analogues of the natural pheromones that prevent hetero-
specific attraction, and in some cases, they have resulted in more
potentcommunicationdisruptantsthanthepheromoneitself(28).
In previouspapers, we have reported that trifluoromethyl ketones
can be effective in vivo pheromone antagonists in the field for a
number of insect pests (1, 9, 18). Particularly remarkable is the
reduction in damage caused in maize fields by the Mediterranean
corn borer and the European corn borer by (Z)-11-hexadecenyl
trifluoromethyl ketone, an analogue of the major component of
the Mediterranean corn borer pheromone (1). We have shown
here that (E,E)-8,10-dodecadienyl trifluoromethyl ketone can be
an effective pheromone antagonist of the codling moth in the
laboratory and in the field and, therefore, a putative candidate in
future strategies to control the pest. In this context, it is worth
noting the low toxicity displayed by trifluoromethyl ketones.
Administration of (Z)-11-hexadecenyl trifluoromethyl ketone
and 3-octylthiotrifluoropropan-2-one, a general esterase inhibi-
tor, on Swiss mice elicited a LD50 of 1 g/kg body weight after
Figure 6. Mean ((SD) number of catches of C. pomonella males per trap
baited with codlemone and mixtures of codlemone and (E,E)-8,10-
dodecadienyl trifluoromethyl ketone in 1:10, 1:1, and 1:0.5 ratios in field
trials conducted in 2006 (3-6 replicates per formulation). The amount of
codlemone in each trap was 100 μg. Within the same trial, bars with the
same letter are not significantly different (Student’s t test, p < 0.05).
remaining nondegraded pheromone relative to the internal stan-
dard (n-decanol).
The potential antagonist was not a good inhibitor of the PDE
because only 20.3% inhibition was recorded at 250 μM, 30.6% at
500 μM, etc. and only at exceedingly high concentrations (over 1
mM) did the amount of nondegraded pheromone approach 50%
(Figure 5). These results suggest that oxidase activity in the
antenna is not the only factor involved in the behavioral response
of the male. The lackof invitro activity ofthe chemicalmay be due
to the fact that the trifluoromethyl ketone moiety is not suffi-
ciently electrophilic to produce a stable tetrahedral adduct with a
cysteine residue of the active enzyme. This mechanism has been
postulated to occur with highly reactive cyclopropanones as
inhibitors of the aldehyde dehydrogenase of H. virescens (21).
The metabolism of pheromone esters and pheromone alde-
hydes has been studied in a variety of insects (7, 25). However,
metabolism of pheromone alcohols has received little attention.
Using [3H]-labeled bombykol and homologous fatty alcohols
applied over male antennae, Kasang (26) discovered that the
major component of the silkworm moth pheromone was con-
verted to long-chain fatty acids and fatty acid esters. The
degradation was sensitive to temperature, suggesting catalysis
by a dehydrogenase. Similarly, degradation of the tritiated
pheromone of the European corn borer (Z)-11-tetradecenyl
acetate occurred through hydrolysis to the alcohol followed
by conversion to tetradecenoic acid, possibly by an alcohol
oxidase, which in turn was degraded via β-oxidation to carbon
dioxide and water (23). An interesting case was the catabolism of

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8519
6 days of treatment, whereas the parent pheromone (Z)-11-
hexadecenyl acetate induced a LD50 of 5 g/kg body weight (18).
These data agree withthe report ofAshourand Hammock (29), in
which doses of up to 250 mg/kg body weight of some fluorinated
ketones caused no mortality on Swiss-Webster mice over a period
of 3 months. In addition and in aquatic ecotoxicity studies, we
have found that thetoxicity of (Z)-11-hexadecenyl trifluoromethyl
ketone was only moderate, with EC50 values ranging from 3.11 to
103.7 mg/L in algae growth and from 0.07 to 1.2 mg/L in Daphnia
survival (30). The low toxicity of these chemicals is likely
due to their reversible inhibition mechanism in contrast to other
much more toxic irreversible inhibitors of carboxylesterases and
proteases.
(13) Quero, C.; Rosell, G.; Jimenez, O.; Rodriguez, S.; Bosch, M. P.;
´
Guerrero, A. New fluorinated derivatives as esterase inhibitors.
Synthesis, hydration and crossed specificity studies. Bioorg. Med.
Chem. 2003, 11, 1047–1055.
(14) Camps, F.; Gasol, V.; Guerrero, A. A new and efficient one-pot
preparation of alkyl halides from alcohols. Synthesis 1987, 511–512.
ꢁ
(15) Pons, S.; Eizaguirre, M.; Sarasua, M. J.; Avilla, J. Influencia del
fotoperiodo sobre la induccion de diapausa de Cydia pomonella
´
(Lepidoptera: Tortricidae) en laboratorio y campo. Invest. Agric.
Prod. Veg. 1994, 9, 477–491.
(16) Kuenen, L. P. S.; Carde, R. T. Strategies for recontacting a lost
´
pheromone plume: Casting and upwind flight in the male gypsy
moth. Physiol. Entomol. 1994, 19, 15–29.
(17) Renou, M.; Guerrero, A. Insect parapheromones in olfaction
research and semiochemical-based pest control strategies. Annu.
Rev. Entomol. 2000, 48, 605–630.
(18) Riba, M.; Sans, A.; Bau, P.; Grolleau, G.; Renou, M.; Guerrero, A.
Pheromone response inhibitors of the corn stalk borer Sesamia
nonagrioides. Biological evaluation and toxicology. J. Chem. Ecol.
2001, 27, 1879–1897.
ACKNOWLEDGMENT
We gratefully acknowledge Catalina Perello for preliminary
´
ꢁ
inhibition assays and Dr. Jesus Avilla for technical advice.
(19) Vogt, R. G.; Riddiford, L. M.; Prestwich, G. D. Kinetic properties of
a pheromone-degrading enzyme: The sensillar esterase of Antheraea
polyphemus. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8827–8831.
(20) Backman, A.-C.; Anderson, P.; Bengtsson, M.; Lofqvist, J.; Unelius,
C. R.; Witzgall, P. Antennal response of codling moth males, Cydia
pomonella L. (Lepidoptera: Tortricidae), to the geometric isomers of
codlemone and codlemone acetate. J. Comp. Physiol., A 2000, 186,
513–519.
(21) Ding, Y.-S.; Prestwich, G. D. Chemical studies of proteins that
degrade pheromones: Cyclopropanated, fluorinated and electrophi-
lic analogs of unsaturated aldehyde pheromones. J. Chem. Ecol.
1988, 14, 2033–2046.
(22) Tasayco, M. L.; Prestwich, G. D. Aldehyde-oxidizing enzymes in
adult moth: In vitro study of aldehyde metabolism in Heliothis
virescens. Arch. Biochem. Biophys. 1990, 278, 444–451.
(23) Klun, J. A.; Scharwz, M.; Uebel, E. C. Biological activity and in vivo
degradation of tritiated female sex pheromone in the male European
corn borer. J. Chem. Ecol. 1992, 18, 283–298.
(24) Leal, W. S.; Chen, A. M.; Ishida, Y.; Chiang, V. P.; Erickson, M. L.;
Morgan, T. I.; Tsuruda, J. M. Kinetics and molecular properties of
pheromone binding and release. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 5386–5391.
(25) Tasayco, M. L.; Prestwich, G. D. Aldehyde oxidases and dehydro-
genases in antennae of five moth species. Insect Biochem. 1990, 20,
691–700.
(26) Kasang, G.; Nicholls, M.; von Proff, L. Sex pheromone conversion
and degradation in antennae of the silkworm moth Bombyx mori L.
Experientia 1989, 45, 81–87.
(27) Prestwich, G. D.; Yamaoka, R.; Carvalho, J. F. Metabolism of
tritiated ω-fluorofatty acids and alcohols in the termite Reticuli-
termes flavipes Kollar (Isoptera: Rhinotermitidae). Insect Biochem.
1985, 15, 205–209.
Supporting Information Available: Table containing details
of field trials conducted to test the antagonist effect of (E,E)-8,10-
dodecadienyl trifluoromethyl ketone. This material is available
free of charge via the Internet at http://pubs.acs.org.
::
::
LITERATURE CITED
(1) Sole, J.; Sans, A.; Riba, M.; Rosa, E.; Bosch, M. P.; Barrot, M.;
´
ꢀ
Palencia, J.; Castella, J.; Guerrero, A. Reduction of damage by the
Mediterranean corn borer, Sesamia nonagrioides, and the European
corn borer, Ostrinia nubilalis, in maize fields by a trifluoromethyl
ketone pheromone analog. Entomol. Exp. Appl. 2008, 126, 28–39.
(2) Alston, D.; Murray, M.; Reding, M. Codling moth (Cydia
pomonella), Utah Pests Fact Sheet. Utah State University and Utah
Plant Pest Diagnostic Laboratory, Logan, UT, 2006; p 7.
(3) Roelofs, W. L.; Comeau, A.; Hill, A.; Milicevic, G. Sex attractant of
the codling moth: Characterization with electroantennogram tech-
nique. Science 1971, 174, 297–299.
(4) http://www.pherobase.net/.
(5) Witzgall, P.; Stelinski, L.; Gut, L.; Thomson, D. Codling moth
management and chemical ecology. Annu. Rev. Entomol. 2008, 53,
503–522.
(6) Carde, R. T.; Minks, A. K. Control of moth pests by mating
´
disruption: Successes and constraints. Annu. Rev. Entomol. 1995,
40, 559–585.
(7) Plettner, E. Insect pheromone olfaction: New targets for the design
of species-selective pest control agents. Curr. Med. Chem. 2002, 9,
1075–1085.
(8) Bau, J.; Martınez, D.; Renou, M.; Guerrero, A. Pheromone-trig-
´
gered orientation flight of male moths can be disrupted by tri-
fluoromethyl ketones. Chem. Senses 1999, 24, 473–480.
´
(9) Riba, M.; Sans, A.; Sole, J.; Munoz, L.; Bosch, M. P.; Rosell, G.;
(28) McDonough, L. M.; Chapman, P. S.; Weissling, T. J.; Smithhisler,
C. L. Efficacy of nonpheromone communication disruptants of
codling moth (Cydia pomonella): Effect of pheromone isomers and
of distance between calling females and dispensers. J. Chem. Ecol.
1996, 22, 415–423.
(29) Ashour, M.-B. A.; Hammock, B. D. Substituted trifluoroketones as
potent selective inhibitors of mammalian carboxylesterases. Bio-
chem. Pharmacol. 1987, 36, 1869–1879.
Guerrero, A. Antagonism of pheromone response of Ostrinia
nubilalis males and implications on behavior in the laboratory and
in the field. J. Agric. Food Chem. 2005, 53, 1158–1165.
(10) Renou, M.; Lucas, P.; Malo, E.; Quero, C.; Guerrero, A. Effects of
trifluoromethyl ketones and related compounds on the EAG and
behavioural responses to pheromones in male moths. Chem. Senses
1997, 22, 407–416.
ꢁ ꢁ
(11) Szekacs, A.; Bordas, B.; Hammock, B. D. Transition state analog
´
ꢁ
(30) Rosa, E.; Barata, C.; Damasio, J.; Bosch, M. P.; Guerrero, A.
enzyme inhibitors: Structure activity relationships of trifluoromethyl
ketones. In Rational Approaches to Structure, Activity and Ecotox-
icology of Agrochemicals; Draber, W., Fujita, T., Eds.; CRC: Boca
Raton, FL, 1992; pp 219-245.
Aquatic ecotoxicity of a pheromonal antagonist in Daphnia magna
and Desmodesmus subspicatus. Aquat. Toxicol. 2006, 79, 296–303.
Received June 10, 2009. Revised manuscript received July 27, 2009.
Accepted July 29, 2009. We gratefully acknowledge CICYT
(AGL2006-13489-C02-01 and AGL2007-62366) for financial support.
ꢁ
(12) Duran, I.; Parrilla, A.; Feixas, J.; Guerrero, A. Inhibition of antennal
esterases of the Egyptian armyworm Spodoptera littoralis by tri
fluoromethyl ketones. Bioorg. Med. Chem. Lett. 1993, 3, 2593–2598.