Synthesis and field evaluation of synthetic blends of the sex pheromone of crocidosema aporema (lepidoptera: Tortricidae) in soybean

Article abstract of DOI:10.1590/S0103-50532012005000072

Crocidosema (= Epinotia) aporema (Walsingham) (Lepidoptera: Tortricidae) is a bud borer that feeds on soybean and forage legumes. Its economic importance is restricted to South America, where it can alternate throughout the year between forage and grain legumes. The sex pheromone of C. aporema females is composed of a 15:1 mixture of (7Z,9Z)-dodeca-7,9-dien-1-ol and (7Z,9Z)- dodeca-7,9-dienyl acetate. Aiming at the development of a monitoring tool, it was synthesized both components of the pheromone and evaluated male captures in pheromone traps baited with different blends of synthetic pheromone, in an experimental soybean field in Uruguay. The conjugated dienes were obtained from 2-pentyn-1-ol and 1,7-heptanediol, by oxidation of the former, Wittig coupling and Zn-catalyzed reduction of the triple bond. The 1:1 mixture was the most efficient in capturing males. The pheromone traps were attractive for up to 40 days, even with small septum loads (0.1 mg) and low population levels.

Full text of DOI:10.1590/S0103-50532012005000072

J. Braz. Chem. Soc., Vol. 23, No. 11, 1997-2002, 2012.
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Article
A
Synthesis and Field Evaluation of Synthetic Blends of the Sex Pheromone of
Crocidosema aporema (Lepidoptera: Tortricidae) in Soybean
Andrés González,*,a,b Paula Altesor,a Leticia Alves,c Paola Liberati,a Horacio Silva,d
Juan Ramos,c Ignacio Carrera,c David González,c Gustavo Seoane,c Carmen Rossini,a
Enrique Castiglionid and Daniela Gamenarac
aLaboratorio de Ecología Química and cDepartamento de Química Orgánica, Facultad de Química,
Universidad de la República, Gral. Flores, 2124, CP 11800, Montevideo, Uruguay
bInstituto de Investigaciones Biológicas Clemente Estable, Av. Italia, 3318, CP 11600, Montevideo, Uruguay
dDepartamento de Protección Vegetal, Facultad de Agronomía, EEMAC,
Universidad de la República, Ruta 3, km 363, CP 60000, Paysandú, Uruguay
Crocidosema (= Epinotia) aporema (Walsingham) (Lepidoptera: Tortricidae) é uma broca das
axilas que se alimenta de soja e leguminosas forrageiras. Sua importância econômica é restrita
à América do Sul onde pode alternar, ao longo do ano, entre leguminosas forrageiras e grãos.
O feromônio sexual das fêmeas de C. aporema é composto de uma mistura 15:1 de (7Z,9Z)-
dodeca-7,9-dieno-1-ol e acetato de (7Z,9Z)-dodeca-7,9-dienila. Com o intuito de desenvolver
uma ferramenta para monitoramento deste inseto praga, os dois componentes do feromônio foram
sintetizados e avaliou-se, em um experimento de campo no Uruguai, a captura de machos em
armadilhas iscadas com diferentes misturas do feromônio sintético. Os dienos conjugados foram
obtidos a partir de 2-pentin-1-ol e 1,7-heptanodiol, através da oxidação do primeiro, seguido pelo
acoplamento de Wittig e redução da ligação tripla catalisada com Zn. A mistura 1:1 testada no
campo foi a mais eficiente na captura dos machos. As armadilhas de feromônio foram atrativas
por mais de 40 dias, mesmo com pequenas quantidades dos compostos (0,1 mg) impregnadas nos
septos, e com baixas densidades populacionais dos insetos no campo.
Crocidosema (= Epinotia) aporema (Walsingham) (Lepidoptera: Tortricidae) is a bud borer
that feeds on soybean and forage legumes. Its economic importance is restricted to SouthAmerica,
where it can alternate throughout the year between forage and grain legumes. The sex pheromone
of C. aporema females is composed of a 15:1 mixture of (7Z,9Z)-dodeca-7,9-dien-1-ol and (7Z,9Z)-
dodeca-7,9-dienyl acetate.Aiming at the development of a monitoring tool, it was synthesized both
components of the pheromone and evaluated male captures in pheromone traps baited with different
blends of synthetic pheromone, in an experimental soybean field in Uruguay. The conjugated
dienes were obtained from 2-pentyn-1-ol and 1,7-heptanediol, by oxidation of the former, Wittig
coupling and Zn-catalyzed reduction of the triple bond. The 1:1 mixture was the most efficient in
capturing males. The pheromone traps were attractive for up to 40 days, even with small septum
loads (0.1 mg) and low population levels.
Keywords: sex pheromone synthesis, Crocidosema (= Epinotia) aporema, pest monitoring,
pheromone traps
Introduction
(= Epinotia) aporema (Walsingham) (Lepidoptera:
Tortricidae). This trend correlates with a significant increase
in the use of insecticides, which has risen up to three-fold
between 2001 and 2011.2
C. aporema is a stem and bud borer that has become a
major pest of legumes in Uruguay, Argentina, Chile and
Southern Brazil.3-6 Originally from Costa Rica, this species
Soybean production in Uruguay has increased
dramatically in the past decade,1 resulting in an expansion
of legume-specialized insects such as Crocidosema
*e-mail: agonzal@fq.edu.uy

1998
Synthesis and Field Evaluation of Synthetic Blends of the Sex Pheromone of Crocidosema aporema
J. Braz. Chem. Soc.
is now widely distributed across SouthAmerica. The insect
is active throughout the year, going through 5-6 generations
per year concentrated between November and April.7
The economic importance of C. aporema is restricted
to the Southern Cone due to the year-round availability
of hosts (soybean, alfalfa, lotus, peas, flax, red clover).8-11
Soon after hatching, the larvae bore inside leaves, stems and
buds, decreasing the effectiveness of contact insecticides.12
In soybean, the larvae feed on vegetative plant parts,
affecting the normal growth of the plant. In other legumes,
larval feeding can severely affect the flower and prevent
the production of seeds, an important commodity in some
forage legumes such as alfalfa and lotus.8,11
C. aporema populations are monitored by direct
observation of the larvae, a highly inefficient method with
poor predictive capacity. Chemical control is therefore
prophylactic, accounting, at least in part, for the large
amounts of insecticides used in legume crops in the region.
As it is the case for many tortricid moths, population
monitoring with pheromone traps would result in a more
informed use of insecticides, reducing both environmental
damage and production costs for soybean and other
legumes. To develop a pheromone-based monitoring tool
for the management of C. aporema in soybean, our group
has previously studied the reproductive behavior of virgin
females13 and characterized (7Z,9Z)-dodeca-7,9-dien-1-ol
and (7Z,9Z)-dodeca-7,9-dienyl acetate (hereafter referred
to as Z7,Z9-12:OH and Z7,Z9-12:Ac) as the sex pheromone
components in female gland extracts.14 Since field studies
are an essential step to develop a monitoring strategy,
it is now reported the synthesis of Z7,Z9-12:OH and
Z7,Z9-12:Ac in preparative scale, as well as male captures
in a soybean field with four synthetic pheromone blends.
phosphonium salt 3 in 15 mL of dry toluene. The reaction
mixture was refluxed for 2 h when aldehyde 2 was added.
After 72 h, the reaction suspension was filtered using
silica-gel and the solvent removed under reduced pressure.
The residue was purified by column chromatography
(hexane:AcOEt 8:2). Overall yield: 92%, Z/E ratio 85:15.
1H NMR (400 MHz, CDCl3): d/ppm 1.17 (t, 3H,
J 7.5 Hz, 7E); 1.19 (t, 3H, J 7.5 Hz, 7Z); 1.30-1.47 (m, 6H,
7Z + 6H, 7E); 1.59-1.69 (m, 2H, 7Z + 2H, 7E); 2.06 (s,
3H, 7E + 3H, 7Z); 2.27-2.33 (m, 2H, 7Z, + 2H, 7E); 2.37
(dq, J1 7.5 Hz, J2 2.1 Hz, 2H, 7Z + 2H, 7E); 4.06 (t, 2H,
J 6.7 Hz, 7E); 4.07 (t, 2H, J 6.7 Hz, 7Z); 5.43-5.49 (m, 6H,
7E + 6H, 7Z); 5.80 (dt, 1H, J1 10.7 Hz, J2 7.3 Hz, 7Z); 6.05
(dt, 1H, J1 15.8 Hz, J2 7.1 Hz, 7E); 13C NMR (400 MHz,
CDCl3): d/ppm 13.2; 14.1; 21.1; 25.7; 28.5; 28.7; 28.8;
29.7; 29.8; 64.5; 64.6; 109.5; 109.9; 142.3; 143.1; 171.3;
EI-MS (70 eV) m/z (%) 222 (2.5), 162 (5.5), 161 (2.2),
147 (6.1), 135 (3.5), 134 (13.9), 133 (28.4), 123 (2.3),
121 (9.1), 120 (10.7), 119 (31.2), 117 (3.9), 108 (4.2), 107
(11.1), 106 (16.3), 105 (44.9), 103 (2.7), 96 (2.3), 95 (9.5),
94 (13.4), 93 (25.8), 92 (22.3), 91 (100.0), 83 (2.8), 82
(8.3), 81 (12.3), 80 (23.2), 79 (79.4), 78 (20.0), 77 (37.1),
69 (8.1), 68 (2.9), 67 (26.0), 66 (5.3), 65 (12.8), 63 (2.2),
61 (4.0), 56 (2.0), 55 (14.6), 54 (3.2), 53 (7.0), 52 (3.5),
51 (4.9), 43 (62.8), 42 (2.9), 41 (24.9) (Figures S1-S3 in
Supplementary Information (SI) section).
(7Z,9Z)-Dodeca-7,9-dienyl acetate (Z7,Z9-12:Ac)
30 mg (0.15 mmol) of 4 and TMSCl (0.15 mL) were
added to a suspension of Zn in MeOH:H2O 1:1 (Zn was
previously activated by stirring for 30 min in HCl 10%).
The reaction was stirred 24 h at room temperature, and
monitored by gas chromatography. The crude was filtered
with silica-gel and washed with methanol, which was
removed afterwards under reduced pressure. The residue
was re-suspended in AcOEt, washed with brine (3 × 10
mL) and dried with Na2SO4, then filtered and concentrated
under vacuum. The residue was purified by column
chromatography using hex:AcOEt (1:1). Yield: 66% (oil).
Z/E ratio 85:15.
1H NMR (400 MHz, CDCl3): d/ppm 1.01 (t, 3H, J 7.6,
7E,9Z isomer); 1.02 (t, 3H, J 7.6, 7Z,9Z isomer); 1.32-1.46
(m, 4H, 7Z,9Z isomer + 4H, 7E,9Z isomer); 1.59-1.67
(m, 4H, 7Z,9Z isomer + 4H, 7E,9Z isomer); 2.07 (s, 3H,
7Z,9Z isomer + 3H, 7E,9Z isomer); 2.16-2.24 (m, 4H,
7Z,9Z isomer + 4H, 7E,9Z isomer); 4.07 (t, 2H, J 6, 9 Hz,
7Z,9Z isomer + 2H, 7E,9Z isomer); 5.29-5.36 (m, 1H, 7E,9Z
isomer ); 5.42-5.50 (m, 2H, 7Z,9Z isomer); 5.63-5.70 (m,
1H, 7E,9Z isomer); 5.90-5.96 (m, 1H, 7E,9Z isomer); 6.19-
6.36 (m, 2H, 7Z,9Z isomer + 1H, 7E,9Z isomer); 13C NMR
(400 MHz, CDCl3): d/ppm 14.2; 14.3; 21.0; 21.1; 25.8;
Experimental
Synthesis
(Z)-Dodec-7-en-9-ynyl acetate (4)
An amount of 1.16 g (9.8 mmol) of freshly prepared
MnO2 was added to a solution of 100 mg (1.19 mmol) of
2-pentyn-1-ol (1) in dry CH2Cl2 under nitrogen atmosphere.
The mixture was stirred at room temperature during
24 h. The course of the reaction was monitored by gas
chromatography: Rt (1) = 5.7 min; Rt (2) = 3.4 min. Crude
reaction was filtered using silica-gel and washed with
CH2Cl2. The solvent was distilled under reduced pressure
and the resulting crude was used in the following reaction
without further purification. Under nitrogen atmosphere,
0.5 g (3.6 mmol) of K2CO3 and 18-crown-6 in catalytic
amount were added to a solution of 0.4 g (0.8 mmol) of the

Vol. 23, No. 11, 2012
González et al.
1999
27.3; 28.6; 28.9; 29.3; 29.5; 32.8; 64.6; 122.9; 123.6; 125.6;
127.9; 131.8; 133.5; 133.8; 134.4; 171.3. EI-MS (70 eV)
m/z (%) 224 (8.6), 165 (3.6), 164 (25.7), 136 (6.7), 135
(24.6), 123 (2.6), 122 (9.3), 121 (31.4), 110 (2.2), 109 (5.8),
108 (15.7), 107 (19.2), 105 (2.1), 97 (3.9), 96 (20.8), 95
(34.7), 94 (22.5), 93 (41.2), 92 (2.7), 91 (10.2), 83 (7.6), 82
(47.6), 81 (36.6), 80 (24.4), 79 (73.6), 78 (7.6), 77 (11.7),
69 (8.9), 68 (26.1), 67 (100.0), 66 (9.0), 65 (5.9), 61 (2.5),
57 (3.6), 55 (28.3), 54 (8.2), 53 (9.6), 43 (57.7), 42 (4.0),
41 (31.0), 40 (2.1) (Figures S4-S6, in the SI section).
Macherey-Nagel, Germany) and visualized with UV light
(254 nm) and/or p-anisaldehyde in acidic ethanolic solution.
Further analyses were performed by gas chromatography
(GC) in a Shimadzu 2014 equipment (Japan), with
FID detector and a Carbowax 20M MEGA column
(30 m × 0.25 mm × 0.25 mm). The temperature program
was 80-15 ºC per min, 120 ºC (5 min). TSPLIT: 220 ºC, TFID
:
250 ºC.
GC-MS (GC-mass chromatograph) analyses were done
using a QP-2010 Shimadzu GC-MS (Japan), equipped with
a AT-5 MS column (Alltech) (30 m × 0.25 mm, 0.25 mm),
operated with a constant carrier flow of 1 mL per min (He).
The temperature program was 70 ºC (1 min), 7 ºC per min,
250 ºC. The injector temperature, 250 ºC; the interphase
temperature, 300 ºC. Injection (1 mL) was in the split mode.
EI-MS (electron ionization-MS) were obtained in scan
mode from m/z 40 to 350.
Column chromatography was performed using silica
gel flash (Kieselgel 60, EM reagent, 230-240 mesh.) from
Macherey-Nagel.
NMR (nuclear magnetic resonance) spectra (1H and
13C) were carried out in a Bruker Avance DPX 400 MHz
(Germany) equipment.All experiments were taken at 30 ºC,
CDCl3 was used as solvent and TMS as internal standard
was purchased to SIgma-Aldrich Inc. (USA).
(7Z,9Z)-Dodeca7,9-dien-1-ol (Z7,Z9-12:OH)
30 mg of Lipase B from Candida antarctica (CaL B,
Novozym 435) were added to a solution of Z7,Z9-12:Ac
(0.2 g, 1 mmol) in 2 mL of methanol. The mixture was
stirred for 2 h in an orbital shaker at 30 ºC. The enzyme was
filtered, the solvent was distilled under reduced pressure and
the crude was purified by column chromatography using
hex:AcOEt 1:1. Yield 98% (oil). Z/E ratio 85:15.
1H NMR (400 MHz, CDCl3): d/ppm 1.01 (t, 3H, J 7.1 Hz,
7E,9Z isomer); 1.02 (t, 3H, J 7.1 Hz, 7Z,9Z isomer);
1.34-1.46 (m, 2H, 7Z,9Z isomer + 2H, 7E,9Z isomer);
1.56-1.65 (m, 2H, 7Z,9Z isomer + 2H, 7E,9Z isomer);
2.16-2.25 (m, 2H, 7Z,9Z isomer + 2H, 7E,9Z isomer); 3.66
(t, 2H, J 5.4 Hz 7Z,9Z isomer + 2H, 7E,9Z isomer); 5.28-5.36
(m, 1H, 7E,9Z isomer); 5.43-5.50 (m, 2H, 7Z,9Z isomer);
5.64-5.71 (m, 1H, 7E,9Z isomer); 5.90-5.96 (m, 1H, 7E,9Z
isomer); 6.20-6.36 (m, 2H, 7Z,9Z isomer + 1H, 7E,9Z
Field experiments
13
isomer); C NMR (400 MHz, CDCl3): d/ppm 14.2; 14.3;
Pheromone blends were evaluated in red rubber septa
placed in the center of plastic Delta traps (15 × 15 × 25 cm).
The traps were distributed within an experimental soybean
field (2 Ha) situated in an agricultural experimental station
(Estación Experimental Mario A. Cassinoni, Facultad de
Agronomía, 32.5° South, 58.0° West). The experiment was
conducted for six weeks, during March and April of 2009.
The treatments were blends (15:1, 1:1, 1:0, 0:1) of the
synthetic pheromone (Z7,Z9-12:OH : Z7,Z9-12:Ac), in
all cases with a total septum load of 0.1 mg in 10 µL of
hexane. Septa loaded with hexane were used as controls.
The experiment was arranged in a randomized block design,
with 8 blocks separated by at least 30 m, and 15 m trap
separation within a block. The traps were hang just above
the height of the foliage, and rotated randomly twice a
week upon recording male captures. During the experiment,
C. aporema larvae were sampled from 10 random plants
within each experimental block.
21.0; 21.1; 25.6; 27.4; 29.0; 29.3; 29.6; 32.7; 32.8; 60.44;
63.0; 122.9; 123.6; 125.6; 127.9; 131.8; 131.9; 133.8; 134.5.
EI-MS (70 eV) m/z (%) 182 (6.1), 164 (6.3), 135 (6.7), 122
(4.0), 121 (11.3), 111 (3.6), 109 (5.2), 108 (6.0), 107 (8.2),
98 (6.7), 97 (5.2), 96 (12.7), 95 (27.3), 94 (9.2), 93 (25.7),
91 (8.4), 84 (3.0), 83 (7.7), 82 (47.3), 81 (31.8), 80 (15.6),
79 (47.0), 78 (4.7), 77 (11.6), 71 (2.9), 70 (3.8), 69 (9.1),
68 (27.3), 67 (100.0), 66 (6.2), 65 (6.4), 57 (4.4), 56 (2.5),
55 (27.7), 54 (9.8), 53 (9.9), 51 (2.0), 43 (6.8), 42 (2.4), 41
(32.2), 40 (2.0) (Figures S7-S9, in the SI section).
General experimental procedures
Solventswerepurifiedanddriedbyconventionalmethods.
Commercial reactants were purchased from Sigma-Aldrich
Inc. (Germany) Lipase Novozym 435 (C. antarctica B, CaL
B) was obtained from Novozymes (USA). The phosphonium
salt 3 [(7-acetoxyheptyl)triphenylphosphonium bromide]
was prepared according to published procedures.15
The degree of advance of the reactions and the purity of
reactants were preliminary monitored using analytical (thin
layer chromatograph) on silica gel (Kieselgel HF254 from
Missing data resulting from traps that fell to the ground
between measurements were estimated by an iterative
method.16 Male captures, after log (x + 1) transformation,
were analyzed using a factorial ANOVA (analysis of
variance) to compare means among treatments, with blocks

2000
Synthesis and Field Evaluation of Synthetic Blends of the Sex Pheromone of Crocidosema aporema
J. Braz. Chem. Soc.
AcO
7 PPh3+Br−
3
MnO2
AcO
OH
K2CO3, 18-crown-6
toluene, reflux
CH2Cl2, rt.
CHO
1
2
4, 85:15, E:Z
Zn* (HCl), TMSCl
MeOH:H2O 1:1, rt
92%
66%
CaL B, MeOH
HO
AcO
30 ºC
98%
Z7,Z9-12:OH; E7,Z9-12:OH (85:15)
Z7,Z9-12:Ac; E7,Z9-12:Ac (85:15)
Figure 1. Synthetic route to Z7,Z9-12:OH and Z7,Z9-12:Ac, components of the sex pheromone of Crocidosema aporema.
and time as factors, using the Tukey’s test for pairwise
Further hydrolysis of the mixture of Z7,Z9-12:Ac
comparisons among treatments (p < 0.05).
and E7,Z9-12:Ac was performed through enzymatic
transesterification with lipase B from Candida antarctica
(CaL B) as biocatalyst, and methanol as nucleophile-
solvent, yielding Z7,Z9-12:OH in 98% yield. This
biocatalytic hydrolysis is an efficient and clean strategy
for acetate removal, requiring virtually no workup other
than filtering the immobilized enzyme.
Overall, the desired pheromone components were
prepared in a stereoselective fashion, using a concise 3 step
synthesis with a 60% overall yield, therefore constituting a
more efficient approach than those previously reported.28-32
Traps loaded with hexane (control) and the proportion
0:1 (OH:Ac) did not catch any males and were therefore
not included in the statistical analysis. All the remaining
treatments captured males, showing a population peak toward
the end of the experiment (Figure 2A). Male captures were
significantly affected by the three factors: time F10,140 = 8.78,
Results and Discussion
Both pheromone components were prepared through
a concise chemoenzymatic synthetic design based on a
Wittig olefination and stereoselective Z alkyne reduction.
This approach differs from previous reports starting from
diynols17 since it avoids the use of protecting groups and
does not afford complex mixtures of stereoisomers. Using
2-pentynol (1) as starting material, it was developed a
novel oxidation-olefination strategy to afford compound
4 in a rapid and efficient manner (Figure 1). A screening
of oxidants and bases was performed, and the best results
were obtained using activated MnO2 as the oxidating agent,
and a Wittig-Boden protocol18,19 with phosphonium salt
3, to yield 4 in an excellent yield (92%) and a good Z:E
stereoselectivity (85:15). The oxidation and alkylation
steps may also be achieved in situ, as previously shown.20,21
Phosphonium salt 3 was prepared from readily available
1,7-heptanediol as described elsewhere.22 The presence of a
J coupling of 10.7 Hz for the olefinic protons in the 1H NMR
of the major isomer of 4 confirmed the Z-stereochemical
assignment, in agreement with previous reports.17,23
For the reduction of the conjugated triple bond, the
traditional Lindlar hydrogenation protocol24,25 was not
stereoselective, resulting in a mixture of geometrical
isomers. Using instead activated Zn as the reducing
agent26 afforded the Z7,Z9-12:Ac isomer with complete
stereoselectivity and 66% yield. Our own previous attempts
to obtain the Z7,Z9-12 carbon skeleton were based on a
“one-pot” oxidation-olefination procedure of (Z)-2-penten-
1-ol (prepared from 2-pentyn-1-ol by Lindlar’s reduction).
According to previous reports,27 this procedure would
minimize the isomerization of (Z)-2-pentenal to the
thermodynamically more stable (E)- isomer, prior to the
olefination reaction with phosphonium salt 3. Nevertheless,
complete isomerization took place, and the decomposition
of the Wittig product to a terminal alkene was also detected.
Figure 2. Mean (A) and cumulative (B) captures of Crocidosema aporema
males with different blends of Z7,Z9-12:OH and Z7,Z9-12:Ac. The 1:1
blend (black squares) captured significantly more males than the major
compound (1:0, open triangles) and the blend in female glands (15:1, open
circles) (Tukey’s test, p < 0.05). Error bars represent SE.

Vol. 23, No. 11, 2012
González et al.
2001
blocks F7,140 = 6.63, and treatments F2,140 = 30.86; p < 0.0001.
Pairwise comparisons among treatments showed that the
1:1 mixture (OH:Ac) was more attractive than either the
alcohol alone or the proportion in the gland extracts (15:1)
(Tukey’s test, p < 0.05) (Figure 2). Cumulative captures
showed a clear difference between the 1:1 mixture and the
other treatments, trapping an average of 48 12 males during
the whole experiment (mean SE; n = 8 blocks; 11 capture
recordings throughout 41 days) (Figure 2B).
The field experiment shows that a small amount of
pheromone (0.1 mg initial load per septum) is enough for
capturing males for several weeks, and that the presence
of the E,Z (about 15%) isomers is not inhibitory for
male attraction. Moreover, the synthetic pheromone is
attractive enough to capture males even in low population
densities. Indeed, only one C. aporema larva was found
in the 2900 buds monitored during the experiment, which
made it impossible to correlate male captures with larval
population density. The low population of C. aporema
during this experiment is probably explained by climatic
effects, and contrasts with the 2007/2008 season, during
which more than 650 males were captured in a preliminary
field experiment with the 1:1 pheromone blend (0.1 mg
per septa, 6 weeks, data not shown). Such oscillation in
population levels among seasons underlines the importance
of an efficient monitoring tool for determining the need of
chemical treatments against C. aporema.
It is worth to note that 25 days into the experiment
there was an increase in male captures in the 1:0 and 15:1
treatments, reaching the capture levels of the 1:1 blend
(Figure 2A). This probably responds to a peak in the adult
population, and shows that the alcohol alone is sufficient
for attracting males. This increase in male captures also
indicates that a small initial pheromone load (0.1 mg per
septa) does not evaporate completely for at least one month,
highlighting the monitoring power of the pheromone.
Differing from the wind tunnel experiments, in which
more males responded to the pheromone blend found in
female glands (15:1; OH:Ac),14 more males were captured
in the field with the 1:1 mixture. This result shows the
importance of conducting behavioral experiments both in the
laboratory and the field. Wind tunnel and field experiments
with semiochemicals are indeed not redundant, inasmuch
as they differ both in their spatial and temporal scales.
Considering the later, wind tunnel experiments are conducted
with freshly-loaded dispensers, which emit the pheromone
blend only during the duration of a given replicate (usually
10 min), and under controlled temperature conditions. To the
contrary, dispensers in the field emit pheromone for several
days or weeks, depending on the rate of septum replacement,
and under more variable field conditions. When taking into
account the spatial component of both experiments, it is
also clear that different results may be obtained from them.
The behavior of the male in the wind tunnel is studied in
close proximity to the dispenser, about 1 or 2 m depending
on the tunnel dimensions. In the field, however, male
catches depend on several other factors, among them, the
long-range attraction of the males. A blend that performs
better in close proximity to the trap may not be the best for
long-range attraction because the relative proportions of the
blend vary with the distance from the trap, depending on the
relative volatility of the compounds. It is therefore likely that
changes in the pheromone blend in time and space explain
the difference between wind tunnel and field experiments.
In our experiment, it is proposed that the blend becomes
slowly enriched in the alcohol, given that it is less volatile
than the acetate33,34 (Vp = 4.37E-4 torr andVp = 6.35E-4 torr,
respectively),35 and it may therefore be concluded that soon
after the experiment begun, the 15:1 (OH:Ac) initial mixture
approximated to the 1:0, decreasing hence its attractivity,
and that the 1:1 was enriched in the alcohol, becoming more
similar to the blend that was most active in the wind tunnel.
In fact, the 15:1 and 1:0 initial mixtures performed similarly
throughout the experiment, and so did the 1:1 initial mixture
towards the end (Figure 2), possibly due to a complete
evaporation of the acetate. Similar results have been found for
other moth species such as Eucosma notanthes (Lepidoptera:
Tortricidae), whose pheromone is composed of Z8-12:Ac and
Z8-12:OH,36 and for Dioryctria abietivorella (Lepidoptera:
Pyralidae), which produces a mixture of Z9,E11-14:Ac and
Z3,Z6,Z9,Z12,Z15-25:Hy.37
Conclusions
The Z,Z conjugated dienes that compose the sex
pheromone of C. aporema can be synthesized in
milligram quantities from readily available 2-pentynol and
1,7-heptanediol, using a “one-pot” oxidation-olefination
strategy. Sub-milligram amounts of synthetic pheromone are
sufficient to load rubber septa, which under field conditions
remain attractive to C. aporema males for several weeks, even
with a small proportion of the E,Z stereoisomer stemming
from the Wittig reaction. Further work is needed in order
to finally develop a monitoring strategy for massive use.
Indeed, parameters such as septum load, septum duration,
trap height and color, as well as threshold capture levels,
need to be optimized for a final monitoring product. It was
shown, however, that the two key factors are possible to
achieve, namely, a convenient and scalable synthetic route,
and the capture of males in field traps baited with synthetic
pheromone. Therefore, population monitoring of C. aporema
in legume crops is practical and feasible, and may result in

2002
Synthesis and Field Evaluation of Synthetic Blends of the Sex Pheromone of Crocidosema aporema
J. Braz. Chem. Soc.
a significant reduction in insecticide use when chemical
control is employed preventively.
Económica - Reconocimiento, Biología y Daños de las Plagas
Agrícolas y Forestales; Agropecuaria Hemisferio Sur SRL:
Montevideo, Uruguay, 2006.
Supplementary Information
13. Altesor, P.; Horas, V. R.; Arcia, M. P.; Rossini, C.; Zarbin,
P. H. G.; González, A.; Neotrop. Entomol. 2010, 39, 324.
14. Altesor, P.; Rossini, C.; Zarbin, P. H. G.; González,A.; J. Chem.
Ecol. 2009, 35, 349.
Supplementary information (Figures S1-S9) is available
free of charge at http://jbcs.sbq.org.br as a PDF file.
15. Horiike, M.; Hirano, C.; Agric. Biol. Chem. 1980, 44, 2229.
16. Snedecor, G.; Cochran, W.; Statistical Methods, Iowa State
University Press: Ames, Iowa, USA, 1989.
Acknowledgments
The authors wish to thank the Agencia Nacional de
Investigación e Innovación (ANII) for the scholarship to
P.A. and the Comisión Sectorial de Investigación Científica
(UdelaR) for the scholarship to L. A. The Funding by
Instituto Nacional de Investigación Agropecuaria (INIA),
the International Foundation for Science-Organization
for the Prohibition of Chemical Weapons (IFS-OPCW)
and The Lindbergh Foundation are also acknowledged.
Abstract translation into portuguese was kindly provided
by Dr. Raul Laumann.
17. Chrelashvili, Z. G.; Mavrov, M. V.; Ugrak, B. I.; Kutin, A. A.;
Serebryakov, E. P.; Russ. Chem. Bull. 1993, 42, 1593.
18. Boden, R. M.; Synthesis 1975, 12, 784.
19. Pandolfi, E.; López, G.V.; Días, E.; Seoane, G.; Synth. Commun.
2003, 33, 2187.
20. Blackburn, L.; Wei, X.; Taylor, R. J. K.; Chem. Commun. 1999,
14, 1337.
21. Quesada, E.; Taylor, R. J. K.; Tetrahedron Lett. 2005, 46, 6473.
22. Horiike, M.; Hirano, C.; Agric. Biol. Chem. 1980, 44, 2229.
23. Ando, T.; Kurotsu, Y.; Kaiya, M.; Uchiyama, M.; Agric. Biol.
Chem. 1985, 49, 141.
References
24. Lindlar, H.; Dubuis, R.; Org. Synth. 1966, 46, 89.
25. Blaser, H.-U.; Indolese,A.; Schnyder,A.; Steiner, H.; Studer, M.;
J. Mol. Catal. A: Chem. 2001, 173, 3.
1. http://www.mgap.gub.uy/portal/hgxpp001.aspx?7,5,583,O,S,0,
MNU;E;27;7;MNU accessed in August 2012.
26. Boland, W.; Schroer, N.; Sieler, C.; Feigel, M.; Helv. Chim.
Acta 1987, 70, 1025.
2. http://www.mgap.gub.uy/dgssaa/DivAnalisisDiagnostico/
DAYD_PROFIT_ESTADISTICA.htm accessed inAugust 2012.
3. Alzugaray, R.; Zerbino, M. S.; Stewart, S.; Ribeiro, A.;
Eilenberg, J.; Rev. Soc. Entomol. Argent. 1999, 58, 307.
4. Iede, E. T.; Foerster, L.A.; An. Soc. Entomol. Bras. 1982, 11, 13.
5. Pereyra, P. C.; Sanchez, N. E.; Rev. Chil. Hist. Nat. 1998, 71, 269.
6. Sanchez, N. E.; Pereyra, P. C.; Gentile, M.V.; Rev. Soc. Entomol.
Argent. 1997, 56, 151.
27. Petroski, R. J.; Synth. Commun. 2003, 33, 3233.
28. Alexakis, A.; Gardette, M.; Colin, S.; Tetrahedron Lett. 1988,
29, 2951.
29. Ando, T.; Kurotsu, Y.; Kaiya, M.; Uchiyama, M.; Agric. Biol.
Chem. 1985, 49, 141.
30. Bestmann, H. J.; Süb, J.; Vostrowsky, O.; Liebigs Ann. Chem.
1981, 12, 2117.
7. Bentancourt, C.; Scatoni, I.; Lepidópteros de Importancia
Económica - Reconocimiento, Biología y Daños de las Plagas
Agrícolas y Forestales, vol. 1, 2nd ed.;Agropecuaria Hemisferio
Sur SRL: Montevideo, Uruguay, 2006.
31. Chrelashvili, Z. G.; Mavrov, M. V.; Ugrak, B. I.; Kutin, A. A.;
Serebryakov, E. P.; Russ. Chem. Bull. 1993, 42, 1593.
32. Daradics, L.; Oprean, I.; Hodosan, F.; J. Prakt. Chem. 1987,
329, 277.
8. Betancourt, C.; Scatoni, I.; Lepidópteros de Importancia
Económica - Reconocimiento, Biología y Daños de las Plagas
Agrícolas y Forestales, vol. 1; Agropecuaria Hemisferio Sur
SRL: Montevideo, Uruguay, 1995.
33. Butler, L. I.; Mc Donough, L. M.; J. Chem. Ecol. 1981, 7, 627.
34. Gut, L. J.; Stelinsky, L. L.; R., T. D.; Miller, J. R. In Integrated
Pest Management: Potential, Constraints and Challenges;
Koul, O.; Dhaliwlal, G. S.; Cuperus, G. W., eds.; CABI:
Cambridge, MA, USA, 2004, p. 73.
9. Morey, C.; Biología y Morfología Larval de Epinotia aporema
(Wals.) (Lepidoptera: Olethreutidae), vol. 123; 1-14; Bol. Univ.
Repub. Fac. Agron.; Montevideo, Uruguay, 1972.
35. Chemical Abstracts Service; American Chemical Society:
Columbus, Ohio, USA.
10. Biezanko, C.; Ruffinelli, A.; Carbonell, C.; Lepidoptera del
Uruguay - Lista Anotada de Especies, vol. 46; 1-152; Rev. Fac.
Agron.; Montevideo, Uruguay, 1957.
36. Hung, C. C.; Hwang, J. S.; Hung, M. D.;Yen,Y. P.; Hou, R. F.;
J. Chem. Ecol. 2001, 27, 1855.
37. Strong, W. B.; Millar, J. G.; Grant, G. G.; Moreira, J.A.; Chong,
J. M.; Rudolph, C.; Entomol. Exp. Appl. 2008, 126, 67.
11. Alzugaray, R.; Ribeiro, A. In INIA Serie Técnica: Manejo de
Plagas en Pasturas y Cultivos, vol. 112; Zerbino, S.; Ribeiro,
A., eds.; INIA: Montevideo, 2000, p. 13.
Submitted: February 9, 2012
12. Betancourt, C.; Scatoni, I.; Lepidópteros de Importancia
Published online: November 14, 2012