Antagonism of pheromone response of Ostrinia nubilalis males and implications on behavior in the laboratory and in the field

Article abstract of DOI:10.1021/jf048994q

The antagonistic effect on the pheromone response and catabolism of male European corn borers, Ostrinia nubilalis, by several trifluoromethyl ketones is reported. (Z)-11-Tetradecenyl trifluoromethyl ketone (Z11-14:TFMK), the most closely related analogue

Full text of DOI:10.1021/jf048994q

1158 J. Agric. Food Chem. 2005, 53, 1158−1165
Antagonism of Pheromone Response of Ostrinia nubilalis
Males and Implications on Behavior in the Laboratory and in
the Field
MAGI RIBA,*,† ALBERT SANS,† JOAN SOLEÄ ,† LOURDES MUN˜ OZ,‡
M. PILAR BOSCH,‡ GLORIA ROSELL,§ AND A. GUERRERO*,‡
Universitat de Lleida, Centre UdL-IRTA, Rovira Roure 177, 25198 Lleida, Spain, Department of
Biological Organic Chemistry, Institute of Chemical and Environmental Research (CSIC),
Jordi Girona 18-26, 08034 Barcelona, Spain, and Department of Pharmacology and Therapeutic
Chemistry, Unity Associated to CSIC, Faculty of Pharmacy, University of Barcelona,
Avda. Diagonal s/n, 08028 Barcelona, Spain
The antagonistic effect on the pheromone response and catabolism of male European corn borers,
Ostrinia nubilalis, by several trifluoromethyl ketones is reported. (Z)-11-Tetradecenyl trifluoromethyl
ketone (Z11-14:TFMK), the most closely related analogue of the main component of the pheromone,
elicits a remarkable disruptive effect on close approach and source contact of males flying to a source
baited with mixtures of the pheromone and the antagonist in 5:1 and 10:1 ratios. In this experiment,
the male displayed an erratic flight track with frequent counter turns and intersections with the plume.
In the field, the TFMK significantly lowered the number of males caught when mixed with the
pheromone in a 10:1 ratio in comparison with the natural attractant. The compound was also a good
inhibitor of the antennal esterase of the insect with a IC50 value of 0.28 µM. The homologous (Z)-
10-tridecenyl trifluoromethyl ketone, with one carbon less in the chain, also elicited an antagonistic
effect in the wind tunnel, but in the field, the results were not conclusive. The effect induced was
lower than the one displayed by Z11-14:TFMK including the activity as the esterase inhibitor (IC50
value of 7.55 µM). The saturated tetradecyl trifluoromethyl ketone, tetradecyltrifluoropyruvamide, and
(Z)-11-2-thiatetradecenyl trifluoromethyl ketone resulted completely inactive. The results obtained in
conjunction to the previously shown low toxicity to mice by related trifluoromethyl ketones provide
new important data for the putative utilization of these chemicals as new pest control agents.
KEYWORDS: Pheromone antagonism; esterase inhibition; Ostrinia nubilalis; trifluoromethyl ketones;
European corn borer; wind tunnel; field tests
INTRODUCTION
fertile hybrids (2). Hybridization also takes place readily in the
laboratory, and a great effort has been devoted to study the
genetic basis of pheromone production, perception, and response
(2-5). The Z strain uses a blend of (Z)-11-tetradecenyl acetate
(Z11-14:Ac) and (E)-11-tetradecenyl acetate (E11-14:Ac) in a
97:3 ratio (6), whereas the E strain utilizes the same compounds
in blends ranging from 1:99 to 4:96 ratios (7, 8).
The European corn borer (ECB), Ostrinia nubilalis (Hu¨bner)
(Lepidoptera: Pyralidae), is a major pest of maize and other
crops, such as potato, green pepper, and winter wheat, in Europe,
North America, North of Africa, the Philippines, and Japan (1).
Control of this species is particularly difficult because insecticide
sprays are only effective during the short period that elapses
between egg hatching and young larvae boring into the stems.
The species displays polymorphism in the pheromone com-
munication system, i.e., different populations utilize different
compounds or different proportions of the same compound.
Despite the two different populations, ECB shows a successful
species isolation, and in areas where both strains are present,
there is enough genetic compatibility between them to produce
Trifluoromethyl ketones (TFMKs) are known to inhibit a
number of esterases and proteases, such as acetylcholinesterase,
chymotrypsin, or human liver carboxylesterases (9, 10), or
particularly the antennal esterases present in insect olfactory
tissues (9, 11-13). These are key enzymes for a rapid
degradation of pheromone esters, thus maintaining a low
stimulus noise level in sensory hairs (14, 15). The mode of
action of TFMKs has been explained in terms of the formation
of a stable hemiacetal of tetrahedral geometry with the serine
residue of the enzyme (16, 17). These chemicals have elicited
significant reduction of the EAG pheromone responses on
* To whom correspondence should be addressed. (A.G.) E-mail:
agpqob@iiqab.csic.es. (M.R.) E-mail: mriba@quimica.udl.es.
† Universitat de Lleida.
‡ Institute of Chemical and Environmental Research (CSIC).
§ University of Barcelona.
10.1021/jf048994q CCC: $30.25
© 2005 American Chemical Society
Published on Web 01/26/2005

Pheromone Response of Ostrinia nubilalis Males
J. Agric. Food Chem., Vol. 53, No. 4, 2005 1159
1
provide the corresponding carboxylic acid (369 mg, 86% yield). H
Chart 1. List of Chemicals Tested as Putative Inhibitors of the
NMR (300 MHz, CDCl3): δ 5.32 (m, 2H), 2.3 (t, J ) 7.2 Hz, 2H), 2.0
(m, 4H), 1.63 (m, 4H), 1.26 (s, 10H), 0.93 (t, J ) 7.4 Hz, 3H) ppm.
(Z)-10-Tridecenyl Trifluoromethyl Ketone. To a solution of (Z)-11-
tetradecenoic acid (0.36 g, 1.61 mmol) in anhydrous CH2Cl2 (5.5 mL),
cooled to 0 °C, a 2 M solution of oxalyl chloride in CH2Cl2 (2.42 mL,
4.84 mmol) (25) was added under Ar. The mixture was stirred at room
temperature for 3 h. The solvent was stripped off under anhydrous
conditions, the acid chloride was taken up in anhydrous ether (5 mL),
and the solution was cooled again to 0 °C. Then, trifluoroacetic acid
(1.34 mL, 9.67 mmol) and anhydrous pyridine (1.04 mL, 12.9 mmol)
were added. The reaction mixture was stirred at room temperature for
1.5 h and cooled to 0 °C, and water (30 mL) was slowly added so that
the temperature was kept below 10 °C. The organic phase was separated,
and the aqueous layer was extracted with CH2Cl2 (3 × 15 mL). The
organic phase was washed with water, dried (MgSO4), and concentrated
under vacuum to leave a residue, which was purified by flash column
chromatography on SiO2 to afford the expected ketone (234 mg, 52%
yield). IR (film): ν 3005, 1764 cm-1. 1H NMR (300 MHz, CDCl3): δ
5.27 (m, 2H), 2.64 (t, J ) 7.2 Hz, 2H), 1.94 (m, 4H), 1.61 (m, 2H),
1.22 (s, 14H), 0.89 (t, J ) 7.5 Hz, 3H) ppm. 13C NMR (75 MHz,
CDCl3): δ 191.6 (q, J ) 35 Hz), 131.5, 129.2, 115.6 (q, J ) 290 Hz),
36.3, 29.7, 29.4, 29.3, 29.2, 29.1, 28.7, 14.3 ppm. 19F NMR (282 MHz,
CDCl3): δ -79.87 (s) ppm. MS m/z (%): 278 (M+, 13), 209 (10), 97
(34), 69 (100).
Pheromone Response and Catabolism of O. nubilalis
Spodoptera littoralis (SL), Mamestra brassicae, and Heliothis
zea (18) and of single sensillum responses to the pheromone of
SL (18) and Antheraea polyphemus (19). In a wind tunnel,
TFMKs have been found to disrupt the orientation flight of SL
and Sesamia nonagrioides (SN) males to pheromone sources
(20). In the field, Z11-16:TFMK, a closely related analogue of
the pheromone, elicited on SN males a significant decrease in
the number of catches in traps baited with mixtures of the
inhibitor and the pheromone in comparison with the pheromone
alone (21, 22). To investigate the effect of this type of chemical
on other economically important pests, we present herein the
activity of (Z)-11-tetradecenyl trifluoromethyl ketone (Z11-14:
TFMK), (Z)-10-tridecenyl trifluoromethyl ketone (Z10-13:
TFMK), tetradecyl trifluoromethyl ketone (14:TFMK), tetra-
decyltrifluoropyruvamide (14:TFPAm), and (Z)-11-2-thiatetra-
decenyl trifluoromethyl ketone (Z11-2S-14:TFMK) (Chart 1)
in wind tunnel bioassays and in the field, as well as on the
antennal esterases present in extracts of the Z strain of the insect.
N-Tetradecyltrifluoropyruvamide. This compound was prepared
in a two-step process from N-tetradecylamine (26).
N-Tetradecylisonitrile. To a solution of NaOH (0.726 g) in water
(0.9 mL) was slowly added a solution of tetraethylammonium bromide
(0.042 g, 0.2 mmol) and N-tetradecylamine (0.92 g, 2.343 mmol) in
chloroform (10 mL). The reaction was stirred at room temperature
overnight. Water was then added, the organic phase was decanted, and
the aqueous layer was extracted with chloroform (5 × 15 mL). The
combined organic phases were washed with water, dried (MgSO4), and
concentrated to leave a residue, which was chromatographed in neutral
alumina (act. II) eluting with hexane:ethyl acetate 97:3. The expected
isonitrile was obtained in pure form (0.247 g, 47% yield). IR (film):
MATERIALS AND METHODS
Chemicals. Z11-14:Ac was obtained by acetylation of (Z)-11-
tetradecenol (Aldrich Chemical Co., 95% purity), and E11-14:Ac (97%
purity) was purchased from-Sigma Chemicals Ltd. and used directly
as received. The solvents (trace analysis quality) were from Fluka-
Riedel-de Hae¨n.
1
ν 2925, 2854, 2147, 1463, 1376, 1353 cm-1. H NMR (CDCl3): δ
3.37 (tt, J1 ) 6.6 Hz, J2 ) 1.9 Hz, 2H), 1.63 (m, 2H), 1.25 (br, 22H),
0.87 (t, J ) 6.5 Hz, 3H) ppm. 13C NMR (CDCl3): δ 155.85, 41.54,
31.88, 29.63, 29.60, 29.55, 29.46, 29.32, 29.07, 28.66, 26.27, 22.64,
14.06 ppm. Elemental analysis: calcd for C15H29N: C, 80.65; H, 13.08;
N, 6.27. Found: C, 80.70; H, 13.15; N, 6.27.
Z11-14:TFMK. This compound was obtained by reaction of the
corresponding iodide (23) with tert-butyllithium and ethyl trifluoro-
acetate, as previously described by us (24). IR (film): υ 3010, 2932,
N-TetradecyltrifluoropyruVamide. To a solution of N-tetradecyl-
isonitrile (100 mg, 0.44 mmol) in CH2Cl2 (1.5 mL) was added, under
Ar at -78 °C, trifluoroacetic anhydride (74 µL, 0.53 mmol) freshly
distilled. The mixture was stirred at this temperature for 90 min, water
was added (5 mL), and the mixture was left to warm to room
temperature. The organic phase was decanted, and the aqueous layer
was extracted with CH2Cl2 (4 × 10 mL). The combined organic phases
were washed with water, dried (MgSO4), and concentrated to leave a
residue, which was purified by recrystallization in hexane to give the
expected product as a white solid. The compound was a mixture of
the keto and hydrate forms in 4:96 ratio; mp 105-108 °C. IR (film):
1
1771, 1541, 1203, 1142 cm-1. H NMR (300 MHz, CDCl3): δ 5.34
(m, 2H), 2.70 (t, J ) 7.2 Hz, 2H), 2.03 (m, 4H), 1.67 (m, 2H), 1.27
(br, 14H), 0.95 (t, J ) 7.5 Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3):
δ 191.65 (q, J ) 35 Hz), 131.53, 129.28, 115.57 (q, J ) 290 Hz),
36.36, 29.74, 29.48, 29.33, 29.24, 29.16, 28.72, 27.07, 22.36, 20.50,
14.39 ppm. 19F NMR (CDCl3): δ -79.91 (s) ppm. MS m/z (%): 292
(M+, 18), 223 (20), 97 (60), 83 (71), 69 (92), 55 (100).
14:TFMK. This compound was also obtained as previously de-
scribed (24). IR (film): υ 2928, 1762, 1210, 1154 cm-1. 1H NMR (300
MHz, CDCl3): δ 2.70 (t, J ) 7.2 Hz, 2H), 1.67 (m, 2H), 1.25 (br,
22H), 0.87 (t, J ) 8.7 Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ
191.68 (q, J ) 34 Hz), 115.58 (q, J ) 291 Hz), 36.36, 31.91, 29.66,
1
ν 3313, 2920, 2850, 1681, 1644, 1557, 1475, 1204, 1182 cm-1. H
NMR (CDCl3): δ 4.10 (br, 3H), 3.33 (dd, J1 ) 13.1 Hz, J2 ) 7.0 Hz,
2H), 1.55 (m, 2H), 1.25 (br, 22H), 0.88 (t, J ) 6.8 Hz, 3H) ppm. 13C
NMR (CDCl3): δ 165.65, 121.69 (q, JC-F ) 285.7 Hz), 90.49 (q, JC-F
) 33.1 Hz), 40.69, 31.92, 29.64, 29.52, 29.44, 29.34, 29.12, 29.06,
26.60, 22.67, 14.06 ppm. 19F NMR (CDCl3): δ -76.9 (s), -84.5 (s)
ppm. MS m/z (%): 318 (23), 240 (31), 155 (18), 85 (65), 71 (85), 57
(100). Exact mass: calcd for C19H34F3NO2, 337.2199; found, 337.2229.
Z11-2S-14:TFMK. This compound was prepared from 9-dodecyn-
1-thiol in a three-step process.
9-Dodecyn-1-thiol. This compound was obtained following a similar
procedure to that previously described (27). Thus, a solution of
9-dodecyn-1-ol (0.941 g, 5.16 mmol), tosyl chloride (2.947 g, 15.5
mmol), and anhydrous pyridine (12 mL) was placed in a round-
bottomed flask and left in the refrigerator for 12 h. The solution was
acidified with 0.1 N HCl and extracted with hexane. The combined
organic phases were washed with brine and dried (MgSO4), and the
29.63, 29.60, 29.52, 29.34, 29.16, 28.73, 22.68, 22.37, 14.10 ppm. 19
F
NMR (CDCl3): δ -79.87 (s) ppm. MS m/z (%): 294 (M+, 0.4), 225
(24), 97 (65), 83 (70), 57 (100), 69 (80).
(Z)-10-Tridecenyl Trifluoromethyl Ketone. This compound was
obtained starting from (Z)-11-tetradecenol after oxidation to the
corresponding carboxylic acid.
(Z)-11-Tetradecenoic Acid. In a round-bottomed flask containing (Z)-
11-tetradecenol (400 mg, 1.88 mmol), pyridinium dichromate (4.96 g,
13.2 mmol) in anhydrous dimethyl formamide (30 mL) was added at
0 °C. The mixture was magnetically stirred overnight at room
temperature, cooled, and quenched by the addition of water (75 mL).
The organic material was extracted with ether (5 × 30 mL), the organic
phase was washed with water (6 × 50 mL) and dried (MgSO4), and
the solvent was stripped off. The residue was purified by column
chromatography on silica gel eluting with hexane-ether mixtures to

1160 J. Agric. Food Chem., Vol. 53, No. 4, 2005
Riba et al.
solvent was removed to afford the corresponding tosylate (1.562 g,
90% yield), which was used directly in the next step without further
purification. The tosylate (0.828 g, 2.46 mmol) was added to a solution
of potassium ethyl xanthogenate (0.610 g, 3.69 mmol) in acetone (9
mL), and the mixture was refluxed for 30 min. The solution was then
allowed to cool to room temperature, the precipitated potassium salt
was filtered out, and the solvent was evaporated. The residue was taken
up in chloroform, and the organic phase was washed with brine, dried
(MgSO4), and evaporated at reduced pressure. The crude ester was
decomposed at room temperature to the corresponding thiol by stirring
for 30 min in the presence of ethylenediamine (4 mL). The solution
was acidified with 0.1 N HCl and extracted with hexane. The combined
organic phases were washed with brine and dried (MgSO4), and the
residue was chromatographed on silica gel eluting with hexane to obtain
pure thiol (0.371 g, 76% yield). IR (film): υ 3421, 2929, 1457, 1217
cm-1. 1H NMR (300 MHz, CDCl3): δ 2.50 (q, J ) 7.4 Hz, 2H), 2.13
(m, 4H), 1.59 (m, 2H), 1.29 (br, 10H), 1.09 (t, J ) 7.4 Hz, 3H) ppm.
13C NMR (75 MHz, CDCl3): δ 81.59, 79.45, 33.98, 29.06, 28.98, 28.91,
28.72, 28.28, 24.60, 18.68, 14.36, 12.38 ppm. MS m/z (%): 81 (36),
68 (84), 67 (100).
2-Thiatetradec-11-ynyl Trifluoromethyl Ketone. To a solution of
9-dodecyn-1-thiol (0.200 g, 1.01 mmol) in anhydrous CH2Cl2 (10 mL)
was added diisopropylethylamine (0.17 mL, 1.01 mmol) and 3-bromo-
1,1,1-trifluoropropan-2-one (0.955 g, 5.04 mmol) (13). The mixture
was stirred at room temperature for 4 h. The solvent was eliminated at
reduced pressure, and the residue was directly purified by column
chromatography on silica gel eluting with hexane:ether 90:10 to afford
the expected acetylenic trifluoromethyl ketone (0.239 g, 77% yield) as
a mixture of ketone and hydrate in a 30:70 ratio. IR (film): υ 3438,
2933, 1746, 1185 cm-1. 1H NMR (300 MHz, CDCl3): δ 4.13 (s, 2H),
3.46 (s, 2H), 2.87 (s, 2H), 2.69 (t, J ) 7.5 Hz, 2H), 2.49 (t, J ) 7.5
Hz, 2H), 2.12 (m, 4H), 1.57 (m, 2H), 1.35 (br, 10H), 1.09 (t, J ) 7.5
Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ 185.03 (q, J ) 34 Hz),
122.9 (q, J ) 285 Hz), 115.49 (q, J ) 291 Hz), 92.33 (q, J ) 32 Hz),
81.65, 79.46, 36.41, 34.73, 33.54, 31.89, 29.25, 29.00, 28.94, 28.91,
28.67, 28.53, 28.50, 28.47, 18.63, 14.30, 12.35 ppm. 19F NMR
(CDCl3): δ -76.27 (s), -85.92 (s) ppm. MS (CI, NH3) m/z (%): 326
[(M + 18)+, 100], 309 [(M + 1)+, 4], 197 (49).
raphy (GC) analysis. Analyses were carried out in a Thermo Quest
GC Trace 2000 gas chromatograph, fitted with a splitless sample
injector, a flame ionization detector (FID), and a SP-2300 (60 m ×
0.25 mm i.d.) fused silica capillary column (Supelco, Sigma-Aldrich
Co.). The chromatographic conditions were as follows: column flow,
0.5 mL He/min; column temperature, 80 °C for 2 min followed by a
program of 5 °C/min up to 180 °C, which was maintained for 10 min.
These conditions led to a full resolution of the Z and E isomers
(retention times 18.4 and 18.9 min for E11-14:Ac and Z11-14:Ac,
respectively). Out of 120 females analyzed, 118 belonged to the Z strain,
one to the E strain, and one appeared to be hybrid. The progenies of
these two females were discarded.
The pure Z strain was reared following a typical artificial diet for
Noctuidae (28) to which the following chemicals were added, nipagin
(methyl-4-hydroxybenzoate) (Fluka Biochemika) (0.12%), flumidil
(potassium o-oxyquinolinsulfonate + sulfonylamidotiazol) (Kessler
Ibe´rica, S. L.) (0.12%), and aureomicin (chlortetracycline hydrochloride)
(Fluka Biochemika) (0.04%). Pupae were sexed and placed in cylindri-
cal boxes (12 cm height × 17 cm diameter) in a climatic chamber
with a 16:8 L:D photoperiod at 25 ( 1 °C and 65 ( 10% relative
humidity until emergence. Adults were provided with a 10% sucrose
solution soaked on a cotton pad, separated daily by age, and kept on a
filter paper in plastic containers until use. For mating, couples were
transferred to mating cages (17 cm diameter × 11.5 cm height)
containing a waxed paper around the walls to allow females to lay
eggs.
Esterase Inhibition Assays. Inhibition bioassays were carried out
according to the methodology already described by us (17). Two day
old males O. nubilalis were anesthetized with CO2, and their antennae
were removed. The antennae were immediately frozen in liquid nitrogen
and kept at -80 °C until use. Crude antennal esterase preparations
were obtained by homogenizing batches of frozen antennae in 100 mM
phosphate buffer solution (pH 7.4) on a variable speed mixer (Heidolph
ZZR-2000) at 680 rpm for 5 min in an ice bath. The contents of the
tube were transferred to an Eppendorf tube, sonicated at 40 W for 10
s, and centrifuged at 3000 rpm for 5 min at 6 °C to remove the cuticular
debris. In borosilicate tubes, previously treated with a saturated solution
of 1-decanol in ethanol for 24 h and washed with distilled water (3×),
were placed 100 µL of the extract (equivalent to three antennae) and
the corresponding amount of inhibitor (2 µL of a 1.5 µM to 5 mM
solution in ethanol). The solution was vortexed for 30 s and preincu-
bated in a thermostatized bath at 28 °C for 10 min. Then, 2 µL of an
ethanol solution (1 µg/µL) of Z11-14:Ac was added and incubation
was continued for 1 h more under the same conditions. Previous studies
had shown that this incubation period was required for a good level of
hydrolysis (>75%) of Z11-14:Ac. Incubation was stopped by addition
of 180 µL of hexane, the mixture was shaken, and the organic phase
was separated and concentrated to 20-30 µL for GC analysis. No
inhibitor was added in control experiments. Chromatographic analyses
were carried out by injection of 1 µL of the above solution into a
Thermo Quest Trace 2000 gas chromatograph, equipped with a split-
splitless injector system, FID, and a HP-5 (25 m × 0.2 mm i.d.) fused
silica capillary column. The chromatographic conditions used were as
follows: injection at 80 °C for 2 min and program of 10 °C/min up to
280 °C, which was maintained for 5 min. The carrier gas was helium
at a flow of 1 mL/min. Under these conditions, Z11-14:Ac and Z11-
14:OH showed retention times of 15.00 and 13.57 min, respectively.
The inhibition degree of the chemicals was calculated as the percentage
of the relative decrease of hydrolysis in the presence of inhibitor in
relation to the mean values of hydrolysis in control experiments,
according to the formula:
(Z)-11-2-Thiatetradecenyl Trifluoromethyl Ketone. In a pressure flask
was placed a suspension of the previous acetylenic ketone (0.200 g,
0.65 mmol) and PtO2 (20 mg, 0.08 mmol) in ethanol (5 mL). The
mixture was stirred at room temperature under a 2.5 bar pressure of
hydrogen for 4 h. The catalyst was then filtered over Celite and washed
thoroughly with hexane, and the solvent was evaporated under vacuum
to give, after purification by column chromatography on silica gel
eluting with hexane:ether 90:10, the trifluoromethyl ketone (176 mg,
87% yield) as a mixture of ketone and hydrate in a 45:55 ratio. IR
1
(film): υ 3423, 3005, 2928, 2854, 1746, 1182 cm-1. H NMR (300
MHz, CDCl3): δ 5.34 (m, 2H), 3.87 (s, 2H), 3.48 (s, 2H), 2.90 (s,
2H), 2.71 (t, J ) 7.2 Hz, 2H), 2.51 (t, J ) 7.5 Hz, 2H), 2.03 (m, 4H),
1.59 (m, 2H), 1.29 (m, 10H), 0.95 (t, J ) 7.5 Hz, 3H) ppm. 13C NMR
(75 MHz, CDCl3): δ 185.06 (q, J ) 34 Hz); 131.58, 129.21, 122.90
(q, J ) 284 Hz), 115.52 (q, J ) 290 Hz), 92.36 (q, J ) 32 Hz), 36.39,
34.77, 33.60, 31.94, 29.69, 29.33, 29.30, 29.15, 29.08, 29.04, 28.61,
28.57, 28.52, 27.03, 20.48, 14.36 ppm. 19F NMR (CDCl3): δ -76.26
(s), -85.96 (s) ppm. MS m/z (%): 241 (1.8), 199 (79), 69 (71), 55
(100). Elemental analysis: calcd for C15H25F3OS: C, 58.04; H, 8.12;
F, 18.36; S, 10.33. Found: C, 58.11; H, 8.15; F, 18.34; S, 10.46.
Selection and Rearing of a Z Strain in the Laboratory. Because
preliminary analytical studies of gland extracts of wild females pointed
out the possible presence of hybrids, to establish a “pure” reliable Z
race in the laboratory, 12 wild mated females were allowed to hatch in
the laboratory. The resulting progenies were isolated, and between 6
and 12 females of each progeny were chosen to study the chemical
composition of the pheromone. Gland analysis was carried out
individually on 3 day old virgin females between the 3rd and 4th h
into the scotophase. The ovipositor was cut and transferred into a conical
glass vial containing 10 µL of hexane and 10 ng of dodecyl acetate as
internal standard. The vial was sealed, and after extraction for 30 min
at room temperature, the solid tissue was removed and the extract was
concentrated under nitrogen to a 2-5 µL volume for gas chromatog-
% hydrolysis with inhibitor
% hydrolysis in control
% inhibition ) 1 -
× 100
(
)
The IC50 of each compound was calculated by least squares regression
analyses in duplicate experiments considering the following doses of
inhibitor: 10, 100, 500, 1000, and 2000 ng for Z11-14:TFMK and 100,
1000, 2000, and 5000 ng for Z10-13:TFMK.
Wind Tunnel Experiments. Assays were conducted in a glass tunnel
of 180 cm × 50 cm × 50 cm as previously described (29). The wind

Pheromone Response of Ostrinia nubilalis Males
J. Agric. Food Chem., Vol. 53, No. 4, 2005 1161
was pushed through the tunnel by a 30 cm diameter fan at 30 cm/s.
The active space of the pheromone was viewed with the aid of a TiCl4
smoke dispenser to ensure that most of the insect flight took place
within the plume boundaries. The tunnel was illuminated with two red
light fluorescent tubes dimmed to 0.75 lux. The temperature was
maintained at 23 ( 1 °C, and the relative humidity was 65 ( 5%. A
video camera Pulnix B/W TM50 was installed at 135 cm above the
tunnel and in perpendicular position to the floor to minimize optical
distortion of the flight track. The camera covered a 130 cm × 45 cm
section of the tunnel, and flight tracks were recorded with a JVC-
SR306E video recorder and converted to computer files at a rate of 25
frames/s. With the aid of in-house computer software, insect positions
were arbitrarily converted to X,Y coordinates.
Table 1. Percentage of Behavioral Responses of O. nubilalis Males
Flying toward a Source Baited with Mixtures of Pheromone and
Z11-14:TFMK in Several Ratios in a Wind Tunnel (N
)
40)a,b
pheromone
1:0.1
+ Z11-14:TFMK ratio (%)
pheromone
(%)
1:0.05
1:1
1:5
1:10
1:20
TF
77 a
77 a
65 a
60 a
80 a
77 a
57 ab
57 ab
82 a
75 a
47 ab
42 ab
82 a
62 a
45 ab
37 ab
85 a
67 a
30 bc
25 bc
75 a
50 a
22 bc
12 c
82 a
50 a
15 c
7 c
OF
CA
SC
a Means within a file followed by the same letter are not significantly different
× 2
ø2 homogeneity test, P < 0.05). b Behavioral responses are as follows: TF,
(2
O. nubilalis males were acclimatized to the experimental conditions
of the tunnel for 30 min and individually released into the tunnel
between the 3rd and 4th h of the 2nd or 3rd scotophase. Before the
tests, individual insects were introduced into a wire mesh cylinder (3
cm diameter, 8 cm height) and placed on a holder at 20 cm high and
125 cm distance from the emission source. After a further 20-30 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 four types of behavior were recorded: TF, taking
flight; OF, oriented flight (upwind flight onto the pheromone plume
and arrival to the middle of the tunnel); CA, close approach (arrival to
the proximity of the lure); and SC, source contact (landing, contact
with the source, and copulation attempts). In each treatment, 40 males
were used and each insect was tested only once.
As control, the attractant source consisted of a rubber septum loaded
with 30 µg of the pheromone blend (mixture of Z11-14:Ac and E11-
14:Ac in a 97/3 ratio), whereas for the antagonism experiments the
required amount of the antagonist was also added to the lure.
Experiments were conducted in blocks including exposed and control
insects, and statistical analyses (ø2 homogeneity test, P < 0.05) were
performed within every block.
Field Tests. Heliothis traps (Scentry, Ecogen Inc.) were deployed
in maize fields of the Lleida province (Catalonia, northeast Spain) from
July to October 2001-2003 to cover the most damaging 2nd and 3rd
generations of the pest. Baits were prepared by dissolving 100 µg of
the pheromone and the appropriate amount of the disruptant in 100 µL
of hexane to obtain the required pheromone:antagonist ratio and
transferring the blends to rubber septa (Sigma-Aldrich Co). The solvent
was allowed to evaporate, and septa containing pheromone alone (100
µg) were used as control. The field was divided into four independent
blocks, equally spaced around the field and separated ca. 50 m from
each other. In every block, the traps were hung at a height of ca. 1.5
m and spaced 25 m from each other. The traps were set up in a
randomized block design and revised and rotated every week. All of
the data were transformed (x× + 0.5) and analyzed for significance
(Student’s t-test, P < 0.05). The disruptant effect of the compounds
was calculated by the decrease in catches obtained with a specific
formulation relative to those obtained with pheromone alone.
taking flight; OF, oriented flight; CA, close approach; and SC, source contact.
Table 2. Percentage of Behavioral Responses of O. nubilalis Males
Flying toward a Source Baited with Mixtures of Pheromone and
Z10-13:TFMK in Several Ratios in a Wind Tunnel (N
)
40)a,b
pheromone
1:1
+ Z10-13:TFMK ratio (%)
pheromone
(%)
1:0.1
1:5
1:10
1:20
TF
77 a
77 a
65 a
60 a
80 a
77 a
60 a
52 a
77 a
60 a
40 ab
35 a
80 a
55 a
37 ab
30 a
85 a
45 a
17 bc
7 b
80 a
37 a
5 c
OF
CA
SC
5 b
a Means within a file followed by the same letter are not significantly different
× 2
ø2 homogeneity test, P < 0.05). b Behavioral responses are as follows: TF,
(2
taking flight; OF, oriented flight; CA, close approach; and SC, source contact.
TFMK in 1:0.05, 1:0.1, 1:1, 1:5, 1:10, and 1:20 ratios, no
apparent effect on the number of TF was observed, and about
75-80% of insects were able to orient their flights to the source
(Table 1). Nevertheless, CA and SC of males attracted to a
mixture of pheromone:disruptant 1:5 and higher were signifi-
cantly lower than the corresponding values for control males
approaching the pheromone alone (P < 0.05). Thus, only 15-
30% of males closely approached the source and 7-25%
successfully contacted the lure. The disruptive effect was dose-
dependent. The pheromone itself (control) induced activation
and oriented flights to 77% of males, attracted 65% to the
proximity of the lure, and led 60% of males to land (Table 1).
The same type of experiment was conducted with the one-
carbon shorter analogue Z10-13:TFMK (Table 2). This chemical
displayed a similar effect than the parent homologue, although
significant results were now obtained when Z10-13:TFMK was
mixed with the pheromone in 10:1 and 20:1 ratios. In the first
case, only 17% of males, out of the 85% that had initially taken
flight, were able to approach the lure and only 7% contacted
with the source, whereas in the second case the percentage of
males displaying both behaviors was only 5%. Here, also, the
effect was dose-dependent (Table 2).
The antagonistic effect of Z11-14:TFMK became evident
when a flight track of a moth flying to a source containing a
1:2 mixture of pheromone and antagonist was videorecorded.
As shown, the track showed profound differences as compared
with the one displayed when males were flying toward
pheromone alone (Figure 1). In the presence of antagonist,
males frequently exhibited erratic progress toward the plume,
flying across the wind and with multiple intersections with
plume boundaries. In addition, males took longer to contact with
the source flying longer distances than control insects (Figure
1).
RESULTS
Esterase Inhibition. Two representative compounds, Z11-
14:TFMK and Z10-13:TFMK, were chosen as putative esterase
inhibitors. Plots of inhibition percentage vs logarithm of dose
of each compound gave a straight line (r2 ) 0.97 for Z11-14:
TFMK, r2 ) 0.89 for Z10-13:TFMK) from which the IC50
values were determined. Z11-14:TFMK exhibited an IC50 value
of 0.28 µM, and Z10-13:TFMK displayed an IC50 value of 7.55
µM. For Z11-14:TFMK, the most similar analogue of the major
component of the pheromone, incubation of 1 ng of this
chemical on an extract equivalent to three male antennae was
sufficient to inhibit the total esterasic activity of the extract by
40%.
Wind Tunnel. In previous studies, we found that 3-4 day
old males were the most active and that the optimum peak of
activity was during the 2nd and 3rd h into the scotophase. When
males were attracted to mixtures of pheromone and Z11-14:
Field Tests. The activity of the antagonists in the field was
evaluated by comparing the number of males caught with
mixtures of the chemicals and the pheromone relative to those

1162 J. Agric. Food Chem., Vol. 53, No. 4, 2005
Riba et al.
Figure 1. Representative flight track of O. nubilalis males flying upwind toward a dispenser containing a 1:2 mixture of pheromone blend and Z11-14:
TFMK (B) relative to control (A). Black dots represent insect positions at 0.04 s intervals.
Figure 2. Number of catches of O. nubilalis males in traps baited with
mixtures of the trifluoromethyl ketones Z11-14:TFMK, Z10-13:TFMK, 14:
TFMK, and 14:NHCOCOCF3 and pheromone in a 10:1 ratio in comparison
with catches in traps containing pheromone alone. Rubber septa were
used as dispensers. The amount of pheromone in each trap was 0.1 mg.
Bars with the same letter are not significantly different (Student’s t-test,
P < 0.05). Tests were carried out on infested maize fields from July to
October 2001.
Figure 3. Number of catches of O. nubilalis males in traps baited with
mixtures of the trifluoromethyl ketones Z11-14:TFMK, Z10-13:TFMK, 14:
TFMK, and Z11-2S-14:TFMK and pheromone in 1:1 and 10:1 ratio in
comparison with catches in traps containing pheromone alone. Rubber
septa were used as dispensers. The amount of pheromone in each trap
was 0.1 mg. Bars with the same letter are not significantly different
(Student’s t-test, P < 0.05). Tests were carried out on infested maize
fields from July to October 2003.
trapped with the pheromone alone. In tests carried out in 2001,
Z11-14:TFMK, Z10-13:TFMK, 14:TFMK, and 14:NHCO-
COCF3 were tested in 10:1 blends with the pheromone but only
Z11-14:TFMK induced a significant reduction of catches (13.3
( 8.4) as compared to the number of males caught with the
pheromone (70.3 ( 11.6) (P < 0.05) (Figure 2). The remaining
compounds did not elicit any significant effect although baits
containing blends of Z10-13:TFMK and 14:TFMK with the
pheromone appeared to catch lower number of males (48.7 (
19.1 and 45.0 ( 20.9, respectively) than the reference attractant
alone.
New experiments were implemented in 2003 including blends
of Z11-14:TFMK:pheromone (1:1) and Z11-2S-14:TFMK:
pheromone (10:1) (Figure 3). In this case and although a
relatively low infestation was detected, Z11-14:TFMK again

Pheromone Response of Ostrinia nubilalis Males
J. Agric. Food Chem., Vol. 53, No. 4, 2005 1163
displayed a good disruptant activity, particularly in a 10:1
mixture with the pheromone (4.7 ( 1.5 vs 15.3 ( 5.5 males/
trap with the pheromone). Z10-13:TFMK also reduced the
number of males caught but to a lower extent than the
homologue (8.3 ( 1.5). Neither 14:TFMK nor the 2-thia
analogue elicited any disruptant effect (Figure 3).
15:TFMK and E12-15:TFMK did not disrupt upwind flight
behavior when coevaporated with the pheromone in a flight
tunnel, but the authors did not provide data on other key
behaviors, such as CA and SC (34).
In two different experiments in the field, Z11-14:TFMK
resulted in an effective antagonist of the pheromone action when
mixed with the natural attractant in a 10:1 ratio, the effect being
dose-dependent. The presence of the double bond with the right
location and stereochemistry as in the pheromone structure is a
determinant for the recognition and transduction processes (35).
It should be noted that the initial pheromone:antagonist ratio
present in the bait may not be the real active formulation because
the TFMKs considered in this study, with the exception of the
thia analogue Z11-2S-14:TFMK and pyruvamide 14:TFPAm,
are more volatile than the corresponding major component of
the pheromone Z11-14:Ac (21). In addition, the different
diffusion rate of the antagonist and the natural attractant,
provided they do not interact with the support, should also lead
to a different release rate of the compounds into the air. With
regard to Z10-13:TFMK, dissimilar results were obtained since
the 10:1 blend with the pheromone was active in only one of
the two experiments, which indicates that further experimenta-
tion is needed to clarify the effect of this chemical in the field.
The 2-thia analogue Z11-2S-14:TFMK, resulting from replace-
ment of a methylene group by sulfur at position 2, was
disappointingly inactive. Presumably, the exceedingly high
content in hydrate form of the chemical, although desirable in
in vitro tests in which aqueous solutions are the reaction media,
may not be advisable in vivo where a sustained release from
the support is required. In this context, the general widely known
esterase inhibitor 2-octylthiotrifluoropropan-2-one (OTFP), a
â-thio-substituted TFMK similar to Z11-2S-14:TFMK but of
much shorter chain length, was also ineffective in the field (21).
This compound had previously displayed a remarkable anties-
terase activity in vitro with an IC50 value of 5.9 µM in SL and
16.3 µM in SN (30), decreased the EAG amplitude and increased
repolarization time in SL, and reduced the responses of males
to the pheromone after preexposure to vapors of the chemical
in a wind tunnel (18). The same assumption could be made for
the lack of activity of the trifluoropyruvamide 14:TFPAm (keto:
hydrate form 4:96). With regard to the lack of activity of the
saturated 14:TFMK, the structural deficiency of the analogue
makes it probably unable to interact properly with the receptor
site and to trigger adequate receptor cell responses to provoke
a successful behavioral output.
DISCUSSION
Following a series of putative esterase inhibitors (12, 17, 21,
30), we have selected the fluorinated compounds shown in
Chart 1 in base to the following. Z11-14:TFMK and Z10-13:
TFMK were considered because of the close structural analogy
with the major component of the pheromone Z11-14:Ac. 14:
TFMK was chosen to establish the role of the olefinic bond
upon the activity. 14:TFPAm was selected in base to the
enhanced electrophilic character of the carbonyl adjacent to the
CF3 group and, therefore, as the putative acceptor for nucleo-
philic residues, such as the serine hydroxyl or the cysteine thiol
of the enzyme. In addition, because this compound is highly
hydrated, it could provide a good model to relate the antagonistic
potency with the extent of hydration as already pointed out (30,
31). Z11-2S-14:TFMK was also considered by the enhanced
stability of the hydrate form due to the presence of a hydrogen
bond between the free electron pair of sulfur with an OH group
of the hydrate (32, 33).
The inhibitory potency of Z11-14:TFMK and Z10-13:TFMK
showed that both compounds were remarkable antennal esterase
inhibitors of ECB; Z11-14:TFMK, the most similar analogue
of the major component of the pheromone, was 10-fold more
active than the one-carbon shorter analogue. In addition and in
EAG studies, we have found that topical application on the
antennae of 10 pg of Z11-14:TFMK resulted in a significant
78% lower depolarization amplitude than the pheromone,
whereas Z10-13:TFMK required a 100 pg dose per antenna to
elicit a significant 60% lower EAG signal (Riba et al.
Unpublished results). The saturated 14:TFMK needed 10 ng,
i.e., a dose 3 orders of magnitude higher than the unsaturated
analogue, to exert a similar effect. The inhibitory potency results
confirm others previously obtained by us in that a stringent
structural similarity of the analogue is required for an optimum
level of inhibition (17, 30). The Z and E isomers of a one-
carbon longer TFMK analogue of the ECB pheromone (Z12-
15:TFMK and E12-15:TFMK) had been found by Klun et al.
(34) to be moderate inhibitors of the esterase activity in both
pheromone strains.
From the above results, it is inferred that in the ECB only
TFMKs very closely structurally related to the major component
of the natural attractant can be effective antennal esterase
inhibitors in vitro, and at the same time remarkable behavioral
antagonists in vivo of the male pheromone responses in the
laboratory and in the field. As far as the mechanism of action
of these TFMKs is concerned, whereas in in vitro assays the
effect of these chemicals can be attributed in principle to the
inhibition of the esterases present in the antenna, in vivo, this
may not be the only mechanism of action. In fact, TFMKs have
also elicited decreased responses to pheromones containing an
alcohol or aldehyde function in electrophysiological tests (18,
36). Moreover, TFMKs may be bound to the pheromone binding
proteins and transported to the sensillum lymph in competition
with pheromone molecules, facilitating interaction with the
pheromone catabolic enzymes (19, 37). However and because
of the structural similarity to the pheromone, these compounds
may also produce an overstimulation and adaptation of the
pheromone receptor cells (20).
When ECB males were allowed to fly toward a source baited
with mixtures of the pheromone and Z11-14:TFMK or Z10-
13:TFMK, both compounds elicited a disruptive effect only of
the CA and SC behaviors. Z11-14:TFMK exerted a significant
effect when mixed with the pheromone in a 5:1 ratio wherein
only 30% of the insects were able to approach to the vicinity
of the lure. Z10-13:TFMK, in turn, needed a double dose in
the lure to elicit a significant reduction in the number of
approaching males. In SC, the effect was similar with regard to
the minimum dose of chemical required to disrupt this behavior.
When either TFMK was present in the lure, males displayed a
highly erratic flight to the source in contrast to the much more
oriented flight onto the plume shown by control insects.
Therefore and again, a TFMK structurally similar to the
pheromone elicits a remarkable disruption of flight, as previously
described for SL and SN (20, 21). This effect was also noted
on SL males flying to cage-containing females after their
antennae were topically treated with some TFMKs (20). Z12-

1164 J. Agric. Food Chem., Vol. 53, No. 4, 2005
Riba et al.
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Received for review June 21, 2004. Revised manuscript received
November 23, 2004. Accepted December 3, 2004. We are also indebted
to CICYT (AGL2003-06599-C02-01, PTR1995-0656-OP, and AGL2003-
06599-C02-02) and Generalitat de Catalunya (2001SGR-00342) for
financial support.
JF048994Q