sintesis

1. Isolation, Identification, Synthesis, and Field Evaluation
of the Sex Pheromone of the Brazilian Population
of Spodoptera frugiperda
Luciane G. Batista-Pereira & Kathrin Stein & Andre´ F. de Paula &
Jardel A. Moreira & Ivan Cruz & Maria de Lourdes C. Figueiredo &
Jose´ Perri, Jr. & Arlene G. Correˆa
Received: 12 December 2003 / Revised: 17 April 2005 /
Accepted: 22 November 2005 / Published online: 20 May 2006
# Springer Science + Business Media, Inc. 2006
Abstract Several studies have shown intraspecific geographical variation in the
composition of sex pheromones. Pheromone lures from North America and Europe
were not effective against the fall armyworm Spodoptera frugiperda (Smith, 1797)
(Lepidoptera: Noctuidae) in Brazil, so we examined the composition of the sex
pheromone produced by females from Brazilian populations. Virgin female gland
extracts contained (Z)-7-dodecenyl acetate (Z7-12:Ac), (E)-7-dodecenyl acetate
(E7-12:Ac), dodecyl acetate, (Z)-9-dodecenyl acetate, (Z)-9-tetradecenyl acetate
(Z9-14:Ac), (Z)-10-tetradecenyl acetate, tetradecyl acetate/(Z)-11-tetradecenyl
acetate (Z11-16:Ac), and (Z)-11-hexadecenyl acetate. The relative proportions of
each acetate were 0.8:1.2:0.6:traces:82.8:0.3:1.5:12.9, respectively. This is the first
time that E7-12:Ac has been reported from the pheromone gland of S. frugiperda.
Only three compounds, Z9-14:Ac, Z7-12:Ac, and E7-12:Ac, elicited antennal
responses, and there were no differences in catch between traps baited with either
Z7-12:Ac + Z9-14:Ac or Z7-12:Ac + Z9-14:Ac + Z11-16:Ac blends. However, the
Z7-12:Ac + Z9-14:Ac + E7-12:Ac blend was significantly better than Z7-12:Ac + Z9-
14:Ac, indicating that E7-12:Ac is an active component in the sex pheromone of the
Brazilian populations of S. frugiperda.
J Chem Ecol (2006) 32: 1085–1099
DOI 10.1007/s10886-006-9048-5
L. G. Batista-Pereira :K. Stein :A. F. Paula :J. A. Moreira :A. G. Correˆa (*)
Departamento de Quı´mica, Universidade Federal de Sa˜o Carlos,
13565-905, Sa˜o Carlos, S.P., Brazil
e-mail: agcorrea@power.ufscar.br
I. Cruz I M. d. L. C. Figueiredo
Embrapa Milho e Sorgo,
CP 151, 35700-970, Sete Lagoas, M.G., Brazil
J. Perri, Jr.
Dow AgroScience, 14.680-0000, Jardino´polis, S.P., Brazil

2. Keywords Spodoptera frugiperda . Sex pheromone . EAG . (Z)-9-Tetradecenyl
acetate . (Z)-7-Dodecenyl acetate . (E)-7-Dodecenyl acetate . Field trapping
Introduction
The fall armyworm Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) is an
important economic pest of maize and other grass crops in North America, Central
America, and parts of South America (Andrews, 1988). In Brazil, this species causes
substantial damage in various crops, such as sugarcane and sorghum, and is the most
important pest in maize causing yield reduction up to 34% (Cruz et al., 1996). At
present, despite the negative environmental impact, as well as negative human and
animal health effects, pesticides are the preferred means of controlling this pest.
However, success is limited because of the location of larvae when feeding on the
host plant. More effective and ecologically acceptable means of control are
required.
The sex pheromone of S. frugiperda has been studied extensively with (Z)-9-
tetradecenyl acetate (Z9-14:Ac) being the first pheromone component reported
(Sekul and Sparks, 1967), whereas later studies have reported additional compo-
nents (Jones and Sparks, 1979; Mitchell et al., 1985; Tumlinson et al., 1986; Descoins
et al., 1988). However, blends that proved successful in trapping fall armyworm
males in North America and Europe performed poorly when tested in Brazil (Cruz
et al., unpublished results), Costa Rica (Andrade et al., 2000), and Mexico (Malo
et al., 2001).
Moth sex pheromones are species-specific communication systems for mate
location and would be under intense stabilizing selection when selection pressures
are similar for all populations (Hansson et al., 1990). However, significant in-
traspecific geographic variation in the pheromone composition of insects, especially
moths, has been reported (Anglade et al., 1984; Lo¨fstedt et al., 1986; Hansson et al.,
1990; Lo¨fstedt, 1990; To´th et al., 1992; Miller et al., 1997; Andrade et al., 2000;
El-Sayed et al., 2003). These are probably the result of different selective pressures
acting on allopatric populations of the same species. Thus, the failure to effectively
capture Brazilian fall armyworm males with European and North American lures
may be caused by geographic variability in the sex pheromone. Furthermore, in the
case of S. frugiperda, there are at least two morphologically indistinguishable host-
plant-specific strains (Pashley et al., 1985, 1992; Pashley, 1986; Levy et al., 2002) that
are also present in Brazil (Busato et al., 2002). Therefore, we undertook a study of
the sex pheromone of Brazilian S. frugiperda, in both laboratory and field
experiments, and developed a short and efficient synthetic route for the preparation
of the pheromone components.
Methods and Materials
Insect Rearing
The colony of S. frugiperda was established at the Insect Bioassay Laboratory of the
Universidade Federal de Sa˜o Carlos, Brazil, using pupae collected in maize plan-
tations at Piracicaba, in Sa˜o Paulo State, Brazil. All specimens were from the second
1086 J Chem Ecol (2006) 32: 1085–1099

3. generation that had been reared on a pinto bean-based artificial diet (Parra, 1986).
The pupae were sexed, placed in individual plastic vials (6 Â 6 cm diam), and then
held in an incubation chamber under a reversed 12:12 hr light/dark photoperiodic
cycle, at 25 T 1-C, 70 T 5% relative humidity, until the adults emerged.
Pheromone Extraction
During peak calling activity, about the 4th hour of the second scotophase (Tumlinson
et al., 1986), the abdominal tips, with pheromone glands, were excised from virgin
females after emergence and extracted for 24 hr in hexane. The supernatant was
collected and stored in dark microvials with Teflon
-lined screw caps at j20-C until
needed. Five extracts of 24, 30, 30, 31, and 36 pheromone glands were prepared.
Chromatographic Analysis
Gland extracts and synthetic solutions were analyzed on a Shimadzu 17-A
chromatograph, equipped with a DB-1 column (30 m  0.25 mm, i.d. 0.25 mm;
J&W Scientific) coupled to Shimadzu QP 5000 mass spectrometer using helium as
carrier gas. Split (synthetic standards) and splitless (gland extracts) injections of 2
ml of the sample and standard solution [(Z)-7-dodecenyl acetate (Z7-12:Ac), (E)-
7-dodecenyl acetate (E7-12:Ac), dodecyl acetate (12:Ac), Z9-14:Ac, and (Z)-11-
hexadecenyl acetate (Z11-16:Ac)] were performed at 250-C. The initial oven
temperature was 50-C for 1 min, then increased to 230-C at a rate of 5-C/min,
and finally to 280-C at a rate of 25-C/min. The final temperature was retained
for 21 min. Electron impact mass spectra were monitored at 70 eV in the m/z
range of 33–250.
The geometric isomers were separated on a Shimadzu 17-A chromatograph with a
flame ionization detector (FID) equipped with a 30 Â 0.25 mm, i.d. 0.25 mm DB-5
column (J&W Science) using H2 as carrier gas. The oven temperature program was
50-C for 1 min up to 145-C at a rate of 3-C/min, then to 230-C at 5-C/min, and finally
to 280-C at 30-C/min, where it was maintained for 21 min. The coinjection (isothermal
analyses at 100-C) was carried out by injecting simultaneously 2 ml of the gland extract
and 1 ml of a diluted standard solution containing E7-12:Ac and Z7-12:Ac.
Coupled Gas Chromatography–Electroantennographic Detection
Pheromone extracts were subjected to gas chromatography–electroantennographic
detection (GC-EAD) analyses, using a Shimadzu 17-A gas chromatograph, equipped
with a DB-5 column (30 m  0.25 mm, i.d. 0.25 mm; J&W Scientific) and a Syntech
electroantennography system (Hilversum, The Netherlands). The same chromato-
graphic conditions as for the GC–mass spectrometry (MS) analyses were used for the
chromatographic separation. The FID was kept at 280-C, whereas the temperature of
the transfer capillary was maintained at 290-C to avoid condensation.
Electrophysiology
Electroantennogram (EAG) recordings of Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac
alone and in various combinations were tested at 0.01, 0.10, 0.25, 0.50, 0.75, 1.00,
10.00, and 100.00 mg/ml, using antennae of 1- to 2-d-old S. frugiperda males. The
J Chem Ecol (2006) 32: 1085–1099 1087

4. antenna was stimulated with 0.3-sec puffs of purified and humidified air (1.2 l/min)
delivered through a Pasteur pipette, containing a filter paper strip (ca. 0.8 cm),
impregnated with 5 ml of the test solution. Puffs from a filter paper plus solvent were
used as control stimulus. The test compounds (Z7-12:Ac, Z9-14:Ac, and Z11-16)
alone were used at increasing concentrations, i.e., a range of doses starting from the
lowest to the highest, and blends were applied randomly. All test compounds were
applied at intervals of 30 sec. Hexane stimulation was made at the beginning and at
the end of every series of EAG experiments. The Syntech EAG software calculated
the normalized values automatically.
Synthesis of Pheromone Components
Unless otherwise noted, all commercially available reagents were purchased from
Aldrich Chemical Co. and were purified, when necessary, according to the usual
procedures described in the literature. The infrared (IR) spectra refer to films and
were measured on a Bomem M102 spectrometer. 1
H and 13
C NMR spectra were
recorded on both a Bruker ARX-200 (200 and 50 MHz, respectively) and a DRX-
400 (400 and 100 MHz, respectively). Mass spectra were recorded on a Shimadzu
GCMS-QP5000. Analytical thin-layer chromatography was performed on a 0.25-mm
film of silica gel containing fluorescent indicator UV254 (Sigma-Aldrich). Flash
column chromatography was performed using silica gel (Kieselgel 60, 230–400 mesh,
E. Merck). Gas chromatography was performed in a Shimadzu GC-17A with H2 as
carrier and using a DB-5 column.
Preparation of bromoalcohols 2: The diol (65 mmol), HBr 48% (8.2 ml, 78
mmol), and benzene (180 ml) were mixed and refluxed for 12 hr in a 250-ml flask
with Dean-Stark and reflux condensers. Once the temperature reached rt, an ice
bath was added to precipitate out the excess diol. The solid was filtered and washed
with chilled benzene. The crude product was employed in the next step after
concentration in vacuo without further purification.
1-Bromo-8-octanol (2a): 83% yield. IR (film): nmax. (cmj1
) = 3352, 2930, 2857,
1464, 1249, 1053. 1
H NMR (400 MHz, CDCl3): d (ppm) = 3.69 (t, 2H, J = 6.6 Hz),
3.41 (t, 2H, J = 6.8 Hz), 1.90 (quint, 2H, J = 8.0 Hz), 1.60 (quint, 2H, J = 6.6 Hz), 1.5–
1.2 (m, 8H). 13
C NMR (50 MHz, CDCl3): d (ppm) = 25.51, 28.03, 28.70, 29.19,
32.76(2C), 34.02, 62.99. MS (70 eV): m/z (%) = 55 (100), 69, 83, 109, 111, 148, 150,
162, 164.
1-Bromo-10-decanol (2b): 78% yield. IR (film): nmax. (cmj1
) = 3367, 2927, 2856,
1460, 1366, 1245, 1041. 1
H NMR (200 MHz, CDCl3): d (ppm) = 3.63 (t, 2H, J = 6.4
Hz), 3.40 (t, 2H, J = 6.8 Hz), 1.85 (quint, 2H, J = 6.8 Hz), 1.50–1.70 (m, 2H), 1.20–
1.50 (m, 12H). 13
C NMR (50 MHz, CDCl3): d (ppm) = 62.97, 34.00, 32.79 (2C),
29.47, 29.35 (2C), 28.71, 28.13, 25.70. MS (70 eV): m/z 55 (100%), 69, 83, 97,135, 137,
148, 150, 162, 164.
1-Bromo-6-hexanol (2c): 75% yield after purification by column chromatog-
raphy in silica gel and hexane: ethyl acetate 20:1 as eluent. IR (film): nmax.
(cmj1
) = 3343, 2900, 1696, 1453, 1247, 1058. 1
H NMR (200 MHz, CDCl3): d (ppm) =
3.64 (t, 2H, J = 6.4 Hz), 3.42 (t, 2H, J = 6.6 Hz), 1.88 (quint, 2H, J = 6.6 Hz), 1.56
(quint, 2H, J = 6.7 Hz), 1.3–1.7 (m, 4H).13
C NMR (50 MHz, CDCl3): d (ppm) =
62.17, 33.90, 32.65, 32.43, 27.86, 24.87. MS (70 eV): m/z 55 (100%), 67, 69, 83, 92, 94,
106, 108, 134, 136.
1088 J Chem Ecol (2006) 32: 1085–1099

5. Alkylation of 1-hexyne with bromoalcohols 2: At j20-C, 1-hexyne (1 mmol) was
placed in a 25-ml flask containing THF (2 ml) under N2 atmosphere, and then an
n-BuLi solution in hexane (2.2 mmol) was added dropwise. The bath temperature
was allowed to rise to 0-C over 30 min, then returned to j30-C, at which time a solution
of the bromoalcohol 2 (1 mmol) in HMPA or DMI (2 ml) was added dropwise.
Water was added, and the mixture was extracted with ethyl ether (3 Â 15 ml). The
organic layer was washed with an aqueous solution of HCl 10% (3 Â 10 ml) and
brine (3 Â 10 ml), dried over NaSO4, and concentrated in vacuo. The crude product
was purified by flash chromatography in silica gel with hexane/ethyl acetate 6:1 as an
eluent (see Table 1).
9-Tetradecyn-1-ol (3a): IR (film): nmax. (cmj1
) = 3338, 2930, 2857, 1462, 1331,
1056. 1
H NMR (200 MHz, CDCl3): d (ppm) = 3.6 (t, 2H, J = 6.7 Hz), 2.52 (s, 1H),
2.13 (t, 4H, J = 5.8 Hz), 1.32–1.55 (m, 16H), 0.9 (t, 3H, J = 7.1 Hz). 13
C NMR (50
MHz, CDCl3): d (ppm) = 80.07 (2C), 62.68, 32.63, 31.19, 29.28, 29.06 (2C), 28.71,
25.66, 21.84, 18.65, 18.35, 13.53. MS (70 eV): m/z 54, 67, 81, 96 (100%), 110, 121, 135,
167.
11-Hexadecyn-1-ol (3b): IR (film): nmax. (cmj1
) = 3347, 2924, 2857, 1460, 1227,
1056, 745. 1
H NMR (200 MHz, CDCl3): d (ppm) = 3.6 (t, 2H, J = 0.5 Hz), 2.13 (t, 4H,
J = 6.0 Hz), 1.29–1.55 (m, 20H), 0.90 (t, 3H, J = 6.9 Hz). 13
C NMR (50 MHz, CDCl3):
d (ppm) = 80.15 (2C), 62.8, 32.68, 31.23, 29.35 (4C), 28.48, 25.90, 25.71, 21.88, 18.71,
18.40, 13.6. MS (70 eV): m/z 55 (100%), 69, 83, 102, 116, 123, 147, 152, 194.
7-Dodecyn-1-ol (3c): IR (film): nmax. (cmj1
) = 3376, 2932, 2863, 1455, 1364, 1225,
1053, 913, 733. 1
H NMR (200 MHz, CDCl3): d (ppm) = 3.53 (t, 2H, J = 6.5 Hz), 2.72
(s, 1H), 2.07 (t, 4H, J = 6.7 Hz), 1.28–1.52 (m, 12H), 0.9 (t, 3H, J = 6.8 Hz). 13
C NMR
(50 MHz, CDCl3): d (ppm) = 80.15 (2C), 62.48, 32.50, 31.15, 29.00, 28.53, 25.23,
21.81, 18.57, 18.31, 13.49. MS (70 eV): m/z 54, 67(100%), 81, 93, 110, 121, 153.
Hydrogenation of alkynols 3: Quinoline (0.6 ml) and Lindlar reagent (0.3 g) were
added to a solution of alkynol 3 (14.3 mmol) in methanol (30 ml), and the resulting
suspension was hydrogenated at rt under hydrogen atmosphere for 6 hr. The
mixture was filtered, washed with ethyl ether (3 Â 10 ml), and the filtrate was then
washed with a 1 M aqueous solution of HCl (3 Â 10 ml), saturated solution of
CuSO4 (3 Â 10 ml), and brine (2 Â 10 ml). The solvent was evaporated at reduced
pressure to provide the desired product.
(Z)-9-Tetradecen-1-ol (4a): 85% yield. IR (film): nmax. (cmj1
) = 3338, 2926, 2857,
1459, 1046, 715. 1
H NMR (200 MHz, CDCl3): d (ppm) = 5.16–5.32 (m, 2H), 3.50
(t, 2H, J = 6.5 Hz), 2.85 (s, 1H), 1.8–2.0 (m, 4H), 1.45 (quint, 2H, J = 6.3 Hz), 1.1–1.3
(m, 14H), 0.89 (t, 3H, J = 7.0 Hz). 13
C NMR (50 MHz, CDCl3): d (ppm) = 129.7
(2C), 62.46, 31.84, 30.65, 29.63, 29.36 (2C), 29.13, 27.05, 26.77, 25.70, 22.20, 13.83. MS
(70 eV): m/z 55(100%), 67, 82, 96, 109, 123, 138, 166, 194.
Entry Bromoalcohol 2 (n) Yield (%)
HMPA DMI
1 5 90 87
2 7 90 91
3 9 83 88
Table 1 Alkylation reaction of
1-hexyne with bromoalcohols 2
(Fig. 2) in different solventsa
a
Bromoalcohol 2 was added in
dry HMPA or DMI and 1-hex-
yne in dry THF.
J Chem Ecol (2006) 32: 1085–1099 1089

6. (Z)-11-Hexadecen-1-ol (4b) and (Z)-7-Dodecen-1-ol (4c) were obtained as de-
scribed above and employed in the acetylation reaction without further purification
or characterization.
(E)-7-Dodecen-1-ol: Small pieces of sodium metal (0.35 g, 15 mmol) were added
to liquid ammonia (20 ml) held at j70-C. This was briskly stirred until all the
sodium had dissolved to give a blue-colored solution, at which time a solution of
alcohol 3c (0.14 g, 0.75 mmol) in THF (0.5 ml) was added. The reaction was
followed by GC analyses. The ammonia was allowed to evaporate, saturated
ammonium chloride solution was cautiously added, and then the reaction mixture
was extracted with ether (3 Â 15 ml). The organic layer was washed with brine
(30 ml), dried over MgSO4, filtered, and evaporated in vacuo. The residue was
chromatographed on silica gel (hexane/ethyl acetate, 95:5), and 0.127 g of the (E)-7-
dodecen-1-ol was obtained (90% yield). IR (film): nmax. (cmj1
) = 3320, 2920, 2860,
1465, 1040. 1
H NMR (200 MHz, CDCl3): d (ppm) = 5.28–5.47 (m, 2H), 3.64 (t, 2H,
J = 6.4 Hz), 1.85–2.05 (m, 4H), 1.1–1.7 (m, 13H), 0.88 (t, 3H, J = 6.6 Hz). 13
C
NMR (50 MHz, CDCl3): d (ppm) = 130.46, 130.13, 63.03, 32.75, 32.49, 32.25, 31.82,
29.57, 28.89, 25.59, 22.18, 13.94.
Acetylation of alcohols 4: Alcohol 5 (7 mmol), acetic anhydride (2.0 ml, 21
mmol), pyridine (2.8 ml), and hexane (35 ml) were mixed in a 125-ml flask, stirred at
rt for 8 hr, before adding ethyl ether (50 ml) and then washing the organic layer with
a 1 M aqueous solution of HCl (3 Â 50 ml) and 1 M NaOH (3 Â 50 ml). The solvent
was removed under reduced pressure, and the residue was purified by flash
chromatography on silica gel with hexane/ethyl acetate 9:1 as the eluent.
(Z)-9-Tetradecenyl acetate (5a): 90% yield, isomeric purity >99% by GC. IR
(film): nmax. (cmj1
) = 3002, 2928, 2857, 1743, 1461, 1367, 1239, 1040, 721. 1
H NMR
(200 MHz, CDCl3): d (ppm) = 5.26–5.42 (m, 2H), 4.05 (t, 2H, J = 6.7 Hz), 1.9–2.1 (m,
7H), 1.5–1.7 (m, 2H), 1.15–1.45 (m, 14H), 0.89 (t, 3H, J = 7.0 Hz). 13
C NMR (50
MHz, CDCl3): d (ppm) = 171.2, 129.8 (2C), 64.6, 31.9, 29.7, 29.4, 29.2 (2C), 28.6,
27.14, 26.9, 25.9, 22.32, 20.96, 13.96. MS (70 eV): m/z 55(100), 61, 67, 82, 96, 110, 124,
151, 194.
(Z)-11-Hexadecenyl acetate (5b): 91% yield in two steps, isomeric purity >99%
by GC. IR (film): nmax. (cmj1
) = 3002, 2927, 2856, 1743, 1461, 1366, 1239, 1040, 721.
1
H NMR (200 MHz, CDCl3): d (ppm) = 5.25–5.50 (m, 2H), 4.04 (t, 2H, J = 6.7 Hz),
1.85–2.1 (m, 7H), 1.60 (quint, 2H, J = 6.7 Hz), 1.1–1.4 (m, 18H), 0.89 (t, 3H, J = 6.9
Hz). 13
C NMR (50 MHz, CDCl3): d (ppm) = 171.05, 129.77 (2C), 64.56, 31.93, 29.72,
29.48 (3C), 29.23 (2C), 28.58, 27.14, 26.87, 25.88, 22.30, 20.88, 13.93. MS (70 eV): m/z
55, 67, 82, 96 (100), 110, 124, 138, 152, 166, 180, 222.
(Z)-7-Dodecenyl acetate (5c): 85% yield in two steps, isomeric purity >99% by
GC. IR (film): nmax. (cmj1
) = 3003, 2930, 2859, 1742, 1460, 1367, 1239, 1041, 725.1
H
NMR (200 MHz, CDCl3): d (ppm) = 5.23–5.4 (m, 2H), 4.05 (t, 2H, J = 6.6 Hz), 1.95–
2.1 (m, 7H), 1.62 (quint, 2H, J = 6.5 Hz), 1.25–1.45 (m, 10H), 0.89 (t, 3H, J = 6.8 Hz).
13
C NMR (50 MHz, CDCl3): d (ppm) = 171.00, 129.92, 129.48, 64.48, 31.87, 29.52,
28.79, 28.52, 26.98, 26.83, 25.75, 22.25, 20.83, 13.87. MS (70 eV): m/z 55, 61, 67, 81
(100), 96, 123, 138, 152, 166.
(E)-7-Dodecenyl acetate: isomeric purity >99% by GC. IR (film): nmax. (cmj1
) =
3010, 2935, 2860, 1740, 1453, 1368, 1045, 730. 1
H NMR (200 MHz, CDCl3): d (ppm) =
5.15–5.40 (m, 2H), 4.05 (t, 2H, J = 6.6 Hz), 2.04 (s, 3H), 1.50–2.00 (m, 4H), 1.05–1.50
(m, 12H), 0.89 (t, 3H, J = 6.8 Hz). 13
C NMR (50 MHz, CDCl3): d (ppm) = 171.40,
1090 J Chem Ecol (2006) 32: 1085–1099

7. 130.52, 130.05, 64.61, 32.43, 32.23, 31.80, 29.45, 28.70, 28.57, 25.76, 22.16, 20.95, 13.89.
MS (70 eV): m/z 55, 61, 67, 81(100), 96, 123, 138, 152, 166.
Field Experiments
All tests were conducted in Embrapa experimental maize plantations at Sete
Lagoas, Minas Gerais State, Brazil. Pherocon 1C traps were suspended at the top of
the plant canopy (100 cm above ground level) at 20-m intervals. In all trials, traps
were emptied every second day. They were baited with red rubber septa (Aldrich
Chemical Co.) impregnated with pheromone components in hexane. We ran the
following trials to compare the efficacy of:
(1) Z7-12:Ac + Z9-14 (0.01:1.00 mg) and Z7-12:Ac + Z9-14:Ac + Z11-16:Ac
(0.01:1.00:0.10 mg). There were three replicates run over 26 d.
(2) 10, 1, and 0.1 mg of Z7-12:Ac + Z9-14:Ac + Z11-16:Ac [at a 1:100:15 (m/m)
ratio]. There were five replicates run over 24 d.
(3) 10, 1, and 0.1 mg of Z7-12:Ac + Z9-14:Ac [at 1:100 (m/m) ratio]. There were
five replicates run over 24 d.
(4) Z7-12:Ac + Z9-14:Ac (0.01:1.00 mg), E7-12:Ac + Z9-14:Ac (0.01:1.00 mg), and
Z7-12:Ac + E7-12:Ac + Z9-14:Ac (0.01: 0.01:1.00 mg). There were 5 replicates
run over 28 d.
In the last three trials, traps baited with either rubber septa impregnated with
hexane or two virgin females, provided with 10% sucrose solution as food source,
were used as controls. The treatments were replicated in a Latin square design
(Perry et al., 1980).
Statistical Analysis
The mean values of the EAG responses calculated automatically by software EAG
for Windows, as well as the data from the first field trial, were submitted to one-way
analysis of variance (ANOVA) and compared using Tukey’s test (P < 0.05). The last
three field experiments were submitted to two-way ANOVA and compared using
Tukey’s test (P < 0.05).
Results
The virgin female gland extracts of Brazilian S. frugiperda showed seven peaks with
spectral characteristics of long-chain acetates (Fig. 1). The two largest peaks, 4 and
7, were identified, based on mass spectral analysis, retention times compared with
known synthetic standards, and index comparisons, as Z9-14:Ac and Z11-16:Ac,
respectively. The mass spectrum of peak 5 indicated an acetate with one double
bond based on the retention index data reported by Marques et al. (2000) and was
identified as (Z)-10-tetradecenyl acetate (Z10-14:Ac). Mass spectrum of peak 6
showed characteristics of saturated and unsaturated long-chain acetates (abundance
of the m/z 61, M-60), suggesting a coelution of (Z)-11-tetradecenyl acetate (Z11-
14:Ac) and tetradecyl-1-ol acetate (14:Ac). This assumption was supported by the
similarity of our results with the retention index data of these two compounds
J Chem Ecol (2006) 32: 1085–1099 1091

8. reported by Marques et al. (2000). The last three acetates present in minor
quantities were identified as Z7-12:Ac (peak 1), E7-12:Ac (peak 2), and 12:Ac (peak
3) using mass spectrum analysis and retention time comparisons and coinjection
with the synthetic compounds (Fig. 2). While confirming the presence of these
geometric isomers of the dodecenyl acetate by using a prolonged temperature
program during the GC separation, we found another substance in trace quantities
coeluting with Z9-12:Ac, but, given the small quantities, were unable to carry out
any further analyses. The relative proportions of Z7-12:Ac, E7-12:Ac, 12:Ac, Z9-
12:Ac, Z9-14:Ac, Z10-14:Ac, 14:Ac/Z11-14:Ac, and Z11-16:Ac in gland extracts of
virgin females were 0.8:1.2:0.6:traces:82.8:0.3:1.5:12.9, respectively.
The straight chain (C10–C16) alcohols or acetates with a double bond that are
found in many sex pheromones are typically prepared by a Wittig reaction or by
alkylation of an acetylene with an alkyl halide followed by selective reduction.
However, the Wittig reaction is not as stereoselective as the alkylation route,
HO
OH
HO
Br
n n
2 eq.BuLi
solvent
HO n
HBr, Bz
reflux
HO n nAcO
Ac2O
py
H2, Lindlar
quinoline
60-63%
75-85% 90-95%
1 2 3
4 5
n = 5, Z7-12:Ac
n = 7, Z9-14:Ac
n = 9, Z11-16:Ac
3
Fig. 2 Synthetic route for the preparation of the sex pheromone components of S. frugiperda
Fig. 1 Chromatogram of the gland extract of Spodoptera frugiperda virgin females, analyzed on a
DB-1 column. Identification of peaks: (1) Z7-12:Ac, (2) E7-12:Ac, (3) 12:Ac, (4) Z9-14:Ac, (5) Z10-
14:Ac, (6) 14:Ac/Z11-14:Ac, and (7) Z11-16:Ac. (N = 30)
1092 J Chem Ecol (2006) 32: 1085–1099

9. Fig. 4 Coupled gas chromatogram–electroantennogram (GC-EAD) of a male S. frugiperda antenna,
stimulated by a gland extract of virgin female S. frugiperda (N = 36)
Fig. 3 Coinjection of gland extract of S. frugiperda virgin females and with standard solution
containing E7-12:Ac and Z7-12:Ac analyzed on a DB-1 column (30 Â 0.25 mm; 0.25 mm under
isothermal conditions at 100-C)
J Chem Ecol (2006) 32: 1085–1099 1093

10. whereas the alkylation route usually includes a protection of the bromoalcohol as
tetrahydropyranyl ether resulting in a decreased overall yield.
Mitra and Reddy (1989) synthesized Z9-14:Ac in ten steps starting from an
alkylation of the dianion of 4-butyn-1-ol, prepared with two equivalents of n-BuLi in
THF-HMPA, with 1-bromopentane that gave a 60% yield of 3-octyn-1ol. Lo and
Chao (1990) substituted HMPA, which is quite toxic, for DMI as solvent in the
alkylation step of w-tetrahydropyranyloxy-1-alkynes with bromoalkanes in the
synthesis of Z9-14:Ac and Z11-16:Ac.
We present an alternate four-step synthetic method for this type of compound
employing alkylation of alkynes with bromoalcohols in HMPA at j30-C. We also
investigated DMI as a solvent, and the alkylation occurred equally well (Fig. 3
and Table 1). Thus, using this methodology, Z9-14:Ac, Z7-12:Ac, and Z11-16:Ac,
pheromone components of S. frugiperda, have been efficiently prepared.
Fig. 5 Coupled GC-EAD of a male S. frugiperda antenna, stimulated by a mixture of synthetic
pheromone blends: (a) Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac; (b) E7-12:Ac, Z9-14:Ac, and Z11-16:Ac
1094 J Chem Ecol (2006) 32: 1085–1099

11. The strongest EAD response from the pheromone gland extract was elicited by
the major sex pheromone compound Z9-14:Ac (Fig. 4). We were unable to separate
the geometric isomers Z7-12:Ac and E7-12:Ac, so the second peak (Fig. 4) could
be the sum of the depolarizations from both compounds, as the two induced responses
when antennae were stimulated by mixtures of synthetic Z7-12:Ac, Z9-14:Ac, and
Z11-16:Ac (Fig. 5a) and E7-12:Ac, Z9-14, and Z11-16:Ac (Fig. 5b).
Over the range of concentrations from 0.01 to 100 mg/ml, Z11-16:Ac never
showed greater electrophysiological activity than hexane, whereas Z7-12:Ac and Z9-
14:Ac gave significantly higher responses than controls at concentrations of 0.5 mg/
ml and above (Fig. 6).
Fig. 6 Mean values (TSD) of EAG responses of S. frugiperda males to individual compounds Z7-
12:Ac, Z9-14:Ac, and Z11-16:Ac in different concentrations (0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 10.0, and
100.0 mg/ml) and control (hexane). For any given compound, the mean values with the same letter
are not significantly different at P < 0.05 based on Tukey’s test (N = 10)
Fig. 7 Number (X T SD) of S. frugiperda males captured in Pherocon 1C traps baited with Z7-12:Ac +
Z9-14:Ac (0.01:1.00 mg), Z7-12:Ac + Z9-14:Ac + Z11-16:Ac (0.01:1.00:0.10 mg), or hexane solvent
(three replicates and 13 collections). Mean values with the same letter are not significantly different
(one-way ANOVA followed by Tukey’s test; P < 0.05)
J Chem Ecol (2006) 32: 1085–1099 1095

12. Fig. 8 Number (X T SD) of S. frugiperda males captured in Pherocon 1C traps baited with: (a) Z7-
12:Ac + Z9-14:Ac + Z11-16:Ac (1:100:15 ratio) at doses of 10, 1, and 0.1 mg, hexane solvent, and two
virgin females (five replicates and 12 collections); (b) Z7-12:Ac + Z9-14:Ac (1:100) at doses of 10, 1,
and 0.1 mg, hexane solvent, and two virgin females (five replicates and 12 collections); (c) Z7-12:Ac +
Z9-14:Ac + Z11-16:Ac (1:100:15 ratio) and Z7-12:Ac + Z9-14:Ac (1:100 ratio) at doses of 10 g, 1 g,
and 0.1 mg, respectively (five replicates and 12 collections). Mean values with the same letter are not
significantly different (two-way ANOVA followed by Tukey’s test; P < 0.05)
1096 J Chem Ecol (2006) 32: 1085–1099

13. Pherocon 1C traps, baited with either Z7-12:Ac + Z9-14:Ac (0.01:1.00 mg) or Z7-
12:Ac + Z9-14:Ac + Z11-16:Ac (0.01:1.00:0.10 mg), captured similar numbers of
males and, in both cases, were significantly higher than controls (Fig. 7). All
concentrations of Z7-12:Ac + Z9-14:Ac + Z11-16:Ac (1:100:15 ratio) captured
significantly more males than controls, and the two higher concentrations out-
competed virgin females. The 1-mg lure was more effective than either the 0.1- or
10-mg ones (Fig. 8a), in the latter case probably because of receptor saturation.
Similar patterns were observed with different concentrations of two- and three-
component blends (Fig. 8), with all concentrations tested catching more than
controls, and the higher doses performing better than virgin females (Fig. 8a,b).
The addition of Z11-16:Ac to Z7-12:Ac and Z9-14:Ac did not increase trap catches
(Fig. 8c), which is contrary to the results of Andrade et al. (2000) in Costa Rica,
where Z11-16:Ac did result in a slight increase in trap efficacy. However, the
addition of E7-12:Ac to the binary mixture resulted in a significant increase in the
number of males captured (Fig. 9).
Discussion
The pheromone glands of S. frugiperda from North America were found to contain
Z7-12:Ac, Z9-12:Ac, Z9-14:Ac, and Z11-16:Ac (Mitchell et al., 1985; Tumlinson et
al., 1986; Descoins et al., 1988), whereas in Guadeloupe (Caribbean), the main
components reported were Z9-12:Ac, Z9-14:Ac, and Z11-16:Ac (Andrade et al.,
2000). In the present study, we also found Z9-14:Ac and Z11-16:Ac but for the first
time report the presence of E7-12:Ac, present in higher quantities than Z7-12:Ac.
Malo et al. (2004) reported that Z9-14:Ac and Z9,E12-14:Ac evoked larger EAG
responses than Z7-12:Ac in male antennae of S. frugiperda, from Mexico, whereas
Z11-16:Ac and Z9,E11-14:Ac did not differ from the control hexane. The antennae
of S. frugiperda males from Costa Rica respond to Z11-16:Ac (cited as unpublished
Fig. 9 Number (X T SD) of S. frugiperda males captured in Pherocon 1C traps baited with Z7-12:Ac +
Z9-14:Ac at ratios of 0.01:1.00 mg, E7-12:Ac + Z9-14:Ac at ratios of 0.01:1.00 mg, Z7-12:Ac + E7-
12:Ac + Z9-14:Ac at ratios of 0.01:0.01:1.00 mg, hexane solvent, and two virgin females (five
replicates and 14 collections). Mean values with the same letter are not significantly different (two-
way ANOVA followed by Tukey’s test; P < 0.05)
J Chem Ecol (2006) 32: 1085–1099 1097

14. results of R. Gries in Andrade et al., 2000) but not in our studies, as significant EAG
responses were only observed to Z9-14:Ac, Z7-12:Ac, and E7-12:Ac (Figs. 4 and 5).
A comparison of our EAG data with those from North and Central America, together
with the results of our field trials, supports the idea that different geographic
pheromone races exist in S. frugiperda, as reported in other noctuids (Lo¨fstedt et al.,
1986; To´th et al., 1992; Wu et al., 1999; Gemeno et al., 2000). These findings are
important for the practical use of pheromones for monitoring of S. frugiperda
populations in Brazil, and the Z7-12:Ac, E7-12:Ac, and Z9-14:Ac (0.01:0.01:1.00 mg,
respectively) blend is currently being tested in integrated pest management of the
fall armyworm in Brazilian maize crop.
Acknowledgments We thank Dr. K. Ogawa, Shin-Etsu Chemical Co., for furnishing pheromone
samples, and G. C. R. Bernasconi for statistical analysis. This study was funded by FAPESP and
CNPq/RHAE (Brazil) and IFS/OPCW (Sweden).
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